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In previous studies, monensin (M) and unsaturated plant oils independently increasedtrans fatty acid concentrations in cultures of mixed ruminal microorganisms. This study was conducted to determine if combining M with plant oil yielded interactions on trans fatty acid concentrations in cultures of mixed ruminal microorganisms or their effects were additive. Four continuous fermentors were fed 14 g of dry feed per day (divided equally between two feedings), consisting of alfalfa hay pellets (30% of DM) and either a high corn (HC) or a high barley (HB) concentrate (70% of DM) in each of two fermentors. Within each grain type, one fermentor was supplemented with M (25 ppm), and the other fermentor was supplemented with 5% soybean oil (SBO) during d 5 to 8. Monensin and SBO were added together in all fermentors during d 9 to 12. Samples were taken at 2 h after the morning feeding on the last day of each period and analyzed for fatty acids by gas-liquid chromatography. A second run of the fermentors followed the same treatment sequence to give additional replication. Average pH across all treatments was 6.15, which was reduced by M but not affected by SBO. Monensin reduced the ratio of acetate to propionate (A:P), which averaged 2.03 across all treatments; fat decreased A:P in cultures not receiving M but increased it in the presence of M. Monensin and SBO altered the concentration of several trans fatty acids, but the only interaction was a grain × M × SBO interaction for trans-10 C18:1. The increase in trans-10 C18:1 by the M and SBO combination exceeded the sum of increases in trans-10 C18:1 for each individual feed additive, but only for HB. For the HC diet, M increased trans-10 C18:1 more than fat alone and more than the M and SBO combination. The results of this study show that M and SBO effects are additive for all trans FA except for trans-10 C18:1. In the case of trans-10 C18:1, M and SBO interacted to give higher trans-10 C18:1 concentrations in ruminal contents than would be expected simply by adding their individual effects, but only for HB. Because some trans fatty acid isomers have been associated with milk fat depression in dairy cows, these results suggest more severe depressions in milk fat content when cows are fed M along with unsaturated plant oils.
Trans fatty acids (FA) are produced as intermediates of FA biohydrogenation by ruminal microorganisms and are linked to milk fat depression in dairy cattle (
identified three factors that affected the concentration of trans C18:1 isomers produced in the rumen:1) the concentration of unsaturated FA, 2) rumen pH, and 3) ionophores. The concentration of unsaturated FA in the rumen can be manipulated by adjusting the amount and source of added fat. Trans C18:1 concentration in ruminal contents of Holstein cows steadily increased as the percentage of soybean oil (SBO) in the diet increased from 0 to 8% (
). Feeding unsaturated plant oils also increases the ruminal and milk concentrations of conjugated linoleic acid (CLA) that have one or more trans double bonds, including some isomers that are highly effective at causing milk fat depression (
Ionophores disrupt ruminal biohydrogenation similar to unsaturated fat supplements. Higher concentrations of linoleic acid, trans C18:1, and CLA were maintained in continuous cultures of ruminal bacteria following infusion of monensin, nigericin, or tetronasin (
). Feeding monensin had similar effects on enhancing linoleic acid and trans FA in milk of lactating cows, and also caused a reduction in milk fat percentage (
, ionophores and other antimicrobials act primarily to inhibit lipolysis, thus reducing the formation of a free carboxyl group that is a requirement for subsequent hydrogenation of double bonds.
This study was conducted to determine if combining monensin with SBO yielded interactions on trans FA concentrations in cultures of mixed ruminal microorganisms or if their effects were additive. A second objective was to identify how monensin and plant oil affected the double bond position of trans C18:1 isomers produced in continuous cultures of mixed ruminal microorganisms.
Materials and Methods
Fermentor Conditions
Whole ruminal contents were taken from a ruminally-fistulated cow fed a predominantly forage diet, transported in vacuum containers to the lab, and filtered through double-layered cheesecloth prior to incubation in the fermentors. Approximately 700 ml of the strained ruminal fluid were transferred into each of the four fermentors. Culture vessels used in this study were an all glass, closed system type with a continuous independent flow of liquid and particulate matter. Air leak into the fermentor is avoided by 1) rubber seals and 2) a continuous flow of CO2 (20 ml/min) that maintains anaerobic conditions as well as a positive internal pressure. Artificial saliva was delivered using a precision pump set at a flow rate of 0.73 ml/min resulting in a fractional dilution rate of 6.8%/h as described by
. The pH was monitored during the entire duration of the experiment. The temperature of ruminal cultures was maintained at 39°C by a circulating water bath. Culture contents were mixed continually at 10 rpm. Fermentors were allowed to stabilize for 4 d to respective diets prior to the administration of additives.
Diets and Additives
A total of 14 g of feed (DM basis) was placed in each fermentor daily in two equal portions at 0800 and 1500 h. Diets consisted of a pelleted concentrate mix and alfalfa hay (70:30, DM basis). Two concentrate mixes were formulated using either corn or barley as the grain source. Soybean meal (49% CP) was included in both mixes to keep the crude protein levels similar. Both mixes included a vitamin and mineral premix (Table 1). The FA content of alfalfa hay was 2.3% and that of the concentrate mix 1 and 2 was 1.0 and 0.8%, respectively. Total FA content of soybean oil was 88.1% and consisted predominantly of C18:2 (55%) and cis C18:1 (22.5%).
Consisted of 0.8% limestone; 0.4% salt; 0.09% magnesium oxide; 0.68% sodium bicarbonate; 0.06% potassium chloride and 0.12% vitamin trace mineral mix which provided per kilogram of diet: 66mg of S, 46mg of Zn, 46mg of Mn, 14mg of Cu, 11.6mg of Fe, 0.8mg of I, 0.7mg of Co, 0.3mg of Se, 7075 IU of vitamin A, 1769 IU of vitamin D, and 21 IU of vitamin E.
3.0
3.0
1 Consisted of 0.8% limestone; 0.4% salt; 0.09% magnesium oxide; 0.68% sodium bicarbonate; 0.06% potassium chloride and 0.12% vitamin trace mineral mix which provided per kilogram of diet: 66 mg of S, 46 mg of Zn, 46 mg of Mn, 14 mg of Cu, 11.6 mg of Fe, 0.8 mg of I, 0.7 mg of Co, 0.3 mg of Se, 7075 IU of vitamin A, 1769 IU of vitamin D, and 21 IU of vitamin E.
Two fermentors received the high corn diet and the other two fermentors received the high barley diet. After 4 d of adaptation to the respective diets, one fermentor within each grain source received monensin (25 ppm of diet DM) and the other received supplemental SBO (5% of diet DM) during d 5 to d 8 (Table 2). Both monensin and SBO were included in all fermentors during d 9 to 12. The same treatment protocol was repeated a second time to give additional replication. Therefore, the experiment consisted of four continuous fermentors operated for two 12-d runs. Each 12-d run was divided into three periods that included:
Period 2: 25 ppm monensin for 4 d or 5% SBO for 4 d.
Period 3: 25 ppm monensin for 4 d and 5% SBO for 4 d.
Monensin was purchased from Sigma Chemical Co. (St. Louis, MO), and SBO (Wesson brand) was purchased at a local grocery store. Samples for VFA, ammonia, pH, and methane were taken each day of the experiment at 2 h after the morning feeding. Analysis of VFA was done by GLC as described by
A separate sample (5-ml) of the mixed cultures was taken on the last day of each period at 2 h after the morning feeding and frozen for analysis of long-chain fatty acids. The frozen samples were freeze-dried, methylated (
), and then analyzed for FA composition by GLC. Fatty acid methyl esters were separated on a 100-m × 0.25-mm × 0.2-μm film thickness CP-Sil 88 capillary column (Chrompack, Middleburg, The Netherlands). Each sample was run twice as described by
, first using a temperature gradient (70 to 240°C) to determine total fatty acid profile and then isothermally (160°C) to separate cis and trans mononenes. Chromatograms of trans FA were similar to the chromatographs reported by
, which provided a basis for peak identification. Further verification of peak identity was established by comparison of peak retention times to known standards.
Statistical Analyses and FA Terminology
Fermentation data from the last 2 d of each treatment period and FA data from d 4 of each period were analyzed according to a randomized complete block design using the Proc Mixed procedure of
. Sources of variation in the model included grain source, monensin, SBO, and all possible interactions tested against residual error.
Fatty acids are designated by number of carbons:number of double bonds. Unsaturated FA identified as C18:1, C18:2, and C18:3 refer to oleic, linoleic, and linolenic acids, respectively. Trans FA are identified according to their positional (location of double bonds) and geometrical (cis or trans) isomers.
Results and Discussion
Total VFA concentrations ranged between 54 to 56 mM, and A:P ranged from 1.7 to 2.4 in the fermentors across all treatments (Table 3). These numbers were lower than usually reported in continuous cultures. For instance, total VFA concentrations and A:P were 104.3 and 2.71 (
) in continuous cultures with no additives. In these previous studies, feeding rates and fiber concentration of the diets were generally higher (50% forage compared to 30% forage in the present study). The lower forage content in this study did not appear to cause an acidity problem in the cultures, as pH values remained within normal limits, i.e., 6.0 to 6.3.
Table 3Main effects of grain source, monensin, and soybean oil on VFA, methane, ammonia, and digestibility in continuous cultures of mixed ruminal microbes.
Compared to corn, fermentation of the barley diet increased (P < 0.05) total VFA, isobutyrate, isovalerate, and ammonia concentrations, but grain source had no effect on pH (Table 3). Monensin addition did not affect total VFA concentration, but it increased (P < 0.05) valerate concentration and reduced (P < 0.05) isobutyrate, pH, and ammonia. Adding SBO had no effect on total VFA, ammonia, or pH, but it increased (P < 0.05) valerate concentration from 2.0 to 2.6 mol/100 mol. Digestibility, calculated as the g feed used for VFA plus methane per 100 g dry feed added to the fermentors each day, was not affected by grain source, monensin, or SBO.
There were several treatment interactions affecting acetate, propionate, and butyrate concentrations. Monensin increased (P < 0.05) acetate concentration in the culture contents (from 55.2 to 58.9 mol/100 mol) when the grain source was corn, but decreased (P < 0.05) acetate concentration (from 55.4 to 50.3 mol/100 mol) when the grain source was barley. Monensin decreased (P < 0.05) butyrate concentrations for both corn (from 14.4 to 10.9 mol/100 mol) and barley (from 16.0 to 8.3 mol/100 mol), but the drop was greater for the barley diet. Monensin by SBO interactions were observed for propionate and butyrate. Propionate concentration increased (P < 0.05) when SBO was added without monensin (from 22.4 to 26.9 mol/100 mol), but its concentration decreased (P < 0.05) when SBO was added with monensin (from 35.3 to 30.5 mol/100 mol). Butyrate concentration decreased (P < 0.05) when SBO was added without monensin (from 16.5 to 13.9 mol/100 mol), but it was not affected by SBO when added with monensin (9.0 and 10.2 mol/100 mol).
Overall, monensin had a greater effect on reducing A:P when the diet contained barley instead of corn, as shown by the grain source by monensin interaction in Figure 1. Also, adding SBO reduced (P < 0.05) A:P and methane production but only in the absence of monensin. When monensin was present, SBO had no effect on A:P and methane (Figure 1). Unsaturated plant oils, such as SBO, typically interfere with ruminal fermentation, causing a drop in A:P and methane, similar to the effects of ionophores. This interaction suggests that ruminal microorganisms already suppressed by the action of one antimicrobial agent, such as an ionophore, are less susceptible to the suppressive effects of a second antimicrobial agent, such as unsaturated FA.
Figure 1A) The grain source by monensin (M) interaction on acetate to propionate ratio (A:P) in continuous fermentors, and the monensin by soybean oil (SBO) interaction on B) A:P and C) methane production. Error bars are pooled SEM.
Fatty acid composition of the fermentor contents 2 h after feeding on d 4 of each period is shown in Table 4. Interactions occurred for C16:0, C18:0, C18:1, and C18:2. The proportion of C18:3 was not affected by grain source or monensin, but it was reduced (P < 0.05) by the addition of SBO. Adding SBO to the fermentors also reduced (P < 0.05) the proportion of C16:0 in the culture contents, especially when the grain source was barley (Figure 2A). The proportion of C18:0 was reduced (P < 0.05) by monensin but only when the fermentors were not supplemented with SBO. Based on the lower C18:0 (Figure 2B) and higher total trans-C18:1 (Table 4) concentrations in the fermenters, it appears that monensin interfered with the terminal step of biohydrogenation where trans mononenes are converted to stearic acid.
Table 4Main effects of grain source, monensin, and soybean oil on major FA in continuous cultures of mixed ruminal microbes.
Figure 2A) The grain source by soybean oil (SBO) interaction on C16:0 and B) the monensin (M) by SBO interaction on C18:0 concentrations in cultures of mixed ruminal microbes grown in continuous fermentors. Error bars are pooled SEM.
Grain source × monensin × SBO interactions for C18:1 and C18:2 are shown in Figure 3. When the fermentors were fed the corn diet with no additives, the proportions of C18:1 and C18:2 were higher than when they were fed the barley diet. In the case of C18:1, this reflects differences in the FA composition of the two grain sources. The corn used in this study contained higher C18:1 than the barley source (30.7 and 14.2% of the total FA, respectively). However, the C18:2 content of the two grain sources were similar (51.9 and 54.9% of total FA for corn and barley, respectively), suggesting that the lower concentration of C18:2 for the barley diet could be attributed to enhanced biohydrogenation. Higher rates of starch fermentation for barley compared to corn could account for this if it contributed to lower pH in the rumen. Low ruminal pH inhibits lipolysis and biohydrogenation (
). However, in this study, fermentor pH did not differ for the corn and barley diets (Table 3).
Figure 3The grain source by monensin (M) by soybean oil (SBO) interactions for A) C18:1 and B) C18:2 concentrations in cultures of mixed ruminal microbes grown in continuous fermentors. Error bars are pooled SEM.
The effects of monensin and SBO on C18:1 and C18:2 differed depending on grain source. When the grain source was corn, monensin and SBO only had an effect on C18:1 when the two additives were combined (Figure 3A). The opposite occurred for the barley diet. Monensin or SBO alone increased (P < 0.05) C18:1, but the combination of monensin and SBO had no effect on C18:1 concentration. In the case of C18:2, monensin and SBO alone or in combination all reduced (P < 0.05) C18:2 when the grain source was corn. But when the grain source was barley, only the combination of monensin and SBO reduced (P < 0.05) C18:2 in the cultures (Figure 3B). These results suggest that monensin and fat additives are likely to reduce or have little effect on unsaturated FA concentration in ruminal contents when corn is the primary starch source. However, monensin and SBO could increase unsaturated FA concentration in the rumen if grains with higher rates of digestion are present in the diet, such as barley.
Among the trans FA, total trans C18:1 concentration in the fermentor contents was higher (P < 0.05) for corn compared to barley (Table 4). Total trans C18:1 concentration increased (P < 0.05) when either monensin or SBO was added to the fermentors. The addition of SBO increased total trans C18:1 isomers nearly 100%, compared to a 53% increase caused by the addition of monensin. The concentration of total CLA isomers was not affected by monensin or SBO, but was lower (P < 0.05) for the barley diet compared to the corn diet. The three treatments each affected a different CLA isomer. The barley diet had a lower (P < 0.05) cis-9, trans-11 C18:2 concentration than the corn diet, monensin decreased (P < 0.05) trans-9, trans-11 C18:2; and SBO increased (P < 0.05) trans-10, cis-12 C18:2.
The treatments also changed the concentration of individual trans mononene isomers present in the fermentor contents (Table 4). The addition of SBO increased (P < 0.05) trans-12 C18:1 and trans-10 C18:1 concentrations, but reduced (P < 0.05) the concentration of trans-11 C18:1. Monensin increased (P < 0.05) trans-10 C18:1 isomer but had no effect on the concentration of the trans-11 or trans-12 isomers. The only trans C18:1 isomer affected by grain source was trans-11, which was higher (P < 0.05) for corn than for barley. The trans-6 C18:1 isomer was not affected by any treatment. Therefore, the majority of the increases in trans C18:1 by monensin and SBO could be attributed to an increase in a single isomer, i.e., trans-10 C18:1.
The only treatment interaction among the trans FA was for trans-10 C18:1. Adding monensin or SBO individually to the fermentors increased (P < 0.05) trans-10 C18:1 concentration, with a greater increase for SBO (Figure 4). However, when monensin and SBO were combined, trans-10 C18:1 concentration increased, but only when the grain source was barley. For the corn diet, the trans-10 C18:1 concentration in the fermentors was similar for SBO and the combination of monensin and SBO. The three-way interaction for trans-10 C18:1 has implications on milk fat depression in lactating dairy cows. Among the CLA isomers, the presence of a trans-10 double bond appears to be required for depression of milk fat synthesis. Earlier studies showed that infusion of trans-10, cis-12 C18:2 into the abomasum of lactating dairy cows led to a 42 to 44% reduction in milk fat (
), but abomasal infusion of cis-9, trans-11 C18:2 did not affect milk fat percentage. If the presence of a trans-10 double bond in monenes similarly reduces milk fat synthesis, then combining monensin with fat in dairy rations could reduce milk fat percentage considerably more than feeding either monensin or fat alone.
Figure 4The grain source by monensin (M) by soybean oil (SBO) interactions for trans-10 C18:1 concentration in cultures of mixed ruminal microbes grown in continuous fermentors. Error bars are pooled SEM.
The question remains as to why the monensin-fat combination increased trans-10 C18:1 concentration the most only when the grain source was barley. Barley contains 57 to 58% starch compared to 72% starch in corn (
). Although starch content was lower for the barley diet, perhaps the higher degradability of the starch interfered with the completeness of biohydrogenation causing accumulation of the trans intermediates. If this occurred, it cannot be attributed to low pH conditions in the fermentors because fermentor pH was the same for both grain sources (Table 3). Instead, perhaps the higher starch degradability of barley caused a shift in the microbial species associated with the completeness of biohydrogenation, namely the Group B bacteria (
). Ruminal microorganisms capable of fatty acid hydrogenation are often divided into Groups A and B based on their end-products and patterns of isomerization during biohydrogenation (
). Bacterial species in Group A hydrogenate linoleic acid to trans C18:1 but appear incapable of hydrogenating mononenes. Group B bacteria can hydrogenate a wide range of monenes, including trans-11 C18:1, to stearic acid.
Conclusions
Monensin and SBO were added to continuous cultures of mixed ruminal microorganisms in this study to determine how they affected trans fatty acid concentrations when added singly or in combination. Among several isomers of trans mononenes and CLA that were examined, the monensin and SBO increased the concentration of the trans-10 C18:1 isomer the most. Also, combining monensin and SBO increased trans-10 C18:1 more than either additive alone but only if the grain source was barley. The results of this study suggest that the source of starch supplied in high grain diets can accentuate the ability of antimicrobial agents, such as ionophores and unsaturated fatty acids, to disrupt biohydrogenation and increase selected trans FA in the rumen. Because some trans fatty acid isomers have been associated with milk fat depression in dairy cows, these results suggest that monensin and plant oils fed together in dairy rations can depress milk fat percentage more severely than either additive alone, particularly in diets high in rapidly-fermentable starch sources, such as barley.
Acknowledgments
Approved as technical contribution no. 4758 of the South Carolina Agricultural Experiment Station, Clemson University. Additional support was provided by Elanco Animal Health, Indianapolis, IN.
References
Bateman II, H.G.
Jenkins T.C.
Influence of soybean oil in high fiber diets fed to nonlactating cows on ruminal unsaturated FA and nutrient digestibility.
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