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Effects of monensin on the chemical composition of the liquid associated microbial fraction in an in vitro rumen fermentation system
Livestock Science 150 (2012) 414–418
Contents lists available at SciVerse ScienceDirect
Livestock Science journal homepage: www.elsevier.com/locate/livsci
Short communication
Effects of monensin on the chemical composition of the liquid associated microbial fraction in an in vitro rumen fermentation system Joaquı´n Miguel Castro-Montoya n, Harinder P.S. Makkar, Klaus Becker Institute for Animal Production in the Tropics and Subtropics (480b), University of Hohenheim, Fruwirthstrasse 12, 70593, Stuttgart, Germany
a r t i c l e in f o
abstract
Article history: Received 17 February 2012 Received in revised form 28 September 2012 Accepted 29 September 2012
The hypothesis of this study was that monensin alters chemical composition of liquid associated rumen microbes. Furthermore, this study generates information on the chemical composition of rumen bacteria as affected by a ionophore by analyzing different parameters within the same experiment. Monensin at 5 and 8 mM was incubated with 380 mg of substrate (hay:concentrate 70:30 w/w) for 24 h in an in vitro gas production system. Monensin elicited no effects on nitrogen and true protein contents of the liquid associated microbial fraction, but sugar content and carbon did change on addition on monensin. Ratio of crude protein to purine bases decreased on adding monensin. True dry matter digestibility and short chain fatty acids production decreased on addition of monensin. Acetate, butyrate and valerate proportions decreased, whereas propionate and branched-chain fatty acid proportions increased. The change in acetate to propionate ratio concurs with the increase in the efficiency of microbial protein synthesis and the decrease in methane production. Results suggest that: monensin influenced the chemical composition of the liquid associated microbial fraction in vitro, and the changed ratio of crude protein to purine bases may lead to under- or overestimations of microbial protein synthesis when purine derivatives are used as markers in the presence of monensin. & 2012 Elsevier B.V. All rights reserved.
Keywords: Microbial nitrogen Microbial sugar Purine bases Monensin
1. Introduction Monensin is a ionophore compound with reported capability to change the rumen fermentation. Increased milk production, decreased methanogenesis and attenuation of
Abbreviations: BCFA, branched-chain fatty acids; CP, crude protein; EMPS, efficiency of microbial protein synthesis; NDS, neutral detergent solution; PB, purine bases; PF, partitioning factor; SCFA, short chain fatty acids; TDMD, true dry matter digestibility; TP, true protein n Corresponding author. Current address: Laboratory for Animal Nutrition and Animal Product Quality, Department of Animal Production, Ghent University, Proefhoevestraat 10, 9090 Melle, Belgium. Tel.: þ 32 9264 9000; fax: þ 32 9264 9099. E-mail address: [email protected] (J.M. Castro-Montoya). 1871-1413/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.livsci.2012.09.026
certain cattle digestive disorders are some of the benefits attributed to monensin (McGuffey et al., 2001; Russell, 1996). These changes are associated with the property of ionophores to alter the microbial populations in the rumen: Gram-positive, proteolytic and obligate amino acid fermenting bacteria are sensitive to ionophores; while, gramnegative bacteria are not sensitive to them (Russell, 1996). Rumen microbes contribute substantially to the nutrients reaching the small intestine. Therefore, information on the chemical composition of the rumen microbes is essential for better understanding the nutrition of the animals. Previous studies, especially during the 1970s and early 1980s, have reported contents of N, amino acids, nucleic acids, carbohydrates, lipids and ash (e.g. Smith and McAllan, 1974; Storm and Ørskov, 1983) and more recently also on purines
J.M. Castro-Montoya et al. / Livestock Science 150 (2012) 414–418
(Rodrı´guez et al., 2000) in rumen microbial fraction. It is clear that some of these components vary with factors such as diet and time after feeding (Smith and McAllan, 1974) but less clear is how the chemical composition of the microbial fraction varies with the inclusion of additives in the diet. Therefore the objective of this study was to add information on the changes of the chemical composition of the liquid associated microbial fraction (LAM) as affected by monensin. The hypothesis of this study was that the addition of monensin changes the chemical composition of LAM in an in vitro fermentation system. 2. Materials and methods 2.1. Methods
415
for the treatment. From these eight syringes, six were used to collect the microbial fraction (see Section 2.1.2) and two were used to perform the protozoa count. Each experiment was repeated on three different days (n ¼3). The experimental design used was completely randomized with three replications for each treatment. After incubation for 24 h gas, methane, protozoa number, true dry matter digestibility (TDMD) and SCFA were determined as described in Castro-Montoya et al. (2011). Partitioning factor (PF) and microbial mass production ¨ were calculated as described by Blummel et al. (1997). The true dry matter digestibility (TDMD) was calculated as the difference between original sample weight (on dry matter basis) and residue after neutral detergent solution treatment and then divided by the original sample weight (on a dry matter basis).
2.1.1. In vitro gas production method 2.1.1.1. Rumen fluid. The fistulation of the cows and the regular collection of rumen fluid were approved on the 30 June 2009 by the regional council of Stuttgart, Germany under the experiment number A 355/09 TE. The rumen fluid was collected before the morning feeding from two rumen-fistulated Holstein-Friesian cattle, one fed ad libitum on grass silage, and the other on a concentrate containing diet (concentrate 2 kg/day and grass silage ad libitum). The concentrate (in g/kg) comprised of barley (350), wheat (340), maize (100), soybean meal (170), soybean oil (10) and mineral mixture (30). The cows had free access to drinking water. The collected rumen fluid was brought to the laboratory in warm (39 1C) insulated flasks, both fluids were mixed together, homogenized and filtered through 100 mm nylon filter. The glassware used was continuously flushed with CO2, stirred and kept in a water bath at 39 1C. 2.1.1.2. Method. The in vitro Hohenheim gas test (HFT) was used according to the protocol of Makkar et al. (1995) based on the method of Menke et al. (1979), except that double strength buffer and 380 mg of substrate were used. A mixture of hay and concentrate (70:30 w/w) was used as the substrate incubated in 100-ml-capacity calibrated glass syringes having 30 ml of a buffered incubation medium containing rumen microbes. Just before dispensing the buffered rumen fluid, a freshly prepared monensin (sodium salt 90–95% TLC; Sigma-Aldrich, Stenheim, Germany) aqueous solution was injected into the syringes through the syringe nozzle to reach the desired concentration of 5 and 8 mM in 30 ml of the medium. These concentrations were decided from previous experiments in our laboratory were monensin applied at a concentration of 5 mM decreased gas production without decreasing short chain fatty acids (SCFA) production (Selje-Assmann et al., 2008). The set without monensin containing 380 mg of the substrate was used as a control. A corresponding blank consisted of the buffered medium without the substrate but containing monensin at the experimental concentrations. The buffered medium was dispensed into the syringes and the incubation was performed in a water bath at 39 1C for 24 h. On a single day, each set of treatments comprised of ten syringes: two syringes for the blank and eight syringes
2.1.2. Collection of microbial fraction from the liquid associated microbes After 24 h of incubation, the contents of six syringes were filtered through a previously weighed nylon bag (pores size of 25 mm, F57, ANKOM Technology, NY, USA). The filtrate containing microbes was received in a tube, which afterwards was centrifuged at 17,000g at 4 1C for 20 min. The supernatant was removed and the microbial fraction was washed by adding 30 ml distilled water. Centrifugation was repeated and the microbial fraction was lyophilized, ground using a ball mill (Retsch, MM200, Haan, Germany) for 2 min at a frequency of 30 /s and stored at room temperature.
2.1.3. Chemical composition of the liquid associated microbial fraction Reducing sugar content was determined using the method of Hodge and Hofreiter (1962) and expressed as glucose equivalent; purine bases (adenine and guanine) by the method of Makkar and Becker (1999); and microbial protein content by Lowry’s method (Peterson, 1983). For N and C analyses 50 mg of microbial fraction were weighed into a crucible in duplicate and placed on the C/N analyzer system (Vario Max CN Elementar Analysensysteme GmbH. Frankfurt Main, Germany).
2.2. Statistical analysis The main effects of monensin were tested by the GLM procedure of SAS software, according to Y i ¼ m þ b1i þ xi where m is the overall mean, b1i the effect of the ith treatment and xI the error term. Significanct effects (Po0.1) of level of monensin were characterized using orthogonal contrasts testing the probability of linear or quadratic responses. Coefficients for polynomial contrasts were calculated for the unequally spaced treatments using the ILM procedure of SAS software. The values reported are means and standard errors.
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3. Results The addition of monensin at 5 and 8 mM to the incubation medium showed no effects on the true protein and N contents of LAM (Table 1). Carbon decreased linearly by adding monensin (Po0.05). Sugar and PB content decreased with increasing levels of monensin (Po0.05). Furthermore, the ratio of CP to PB and TP to PB increased when monensin was used (Po0.05). Protozoa number decreased with increasing levels of monensin (Po0.05) (Table 2). Adding monensin increased the efficiency of microbial protein synthesis (EMPS) (expressed both as mg N/mmol SCFA and g N/kg TDMD) and the partitioning of nutrients toward microbial mass (PF) (Po0.05). Monensin decreased CH4 production (Po0.05). The microbial mass was not
affected, while the true dry matter digestibility and gas production decreased (Po0.05) (Table 2). Acetate, C4, C5 proportions, total SCFA and the C2:C3 ratio (Po0.05) decreased with increasing levels of monensin. Proportions of C3 and branched chain fatty acid (BCFA) increased (Po0.05) (Table 3). 4. Discussion This study was performed to test the hypothesis that monensin can change the chemical composition of the rumen liquid associated microbial fraction in an in vitro system. Decreases in sugar content of the microbial fraction are in conformity with decreases in protozoa number upon addition of monensin. Protozoa are known
Table 1 Effects of monensin on the chemical composition of the liquid associated microbial fraction. Variable
Chemical composition Sugar (g/100 g)c True protein (g/100 g) Nitrogen (g/100 g) Carbon (g/100 g)
Monensin (mM)
SEM
0
5
8
23.4 42.7 7.5 44.9
20.5 43.4 7.3 46.3
18.2 43.8 7.6 42.3
2.62 (496) 2.95 (399) 5.57 (795) 57.7 54.6
2.62 (496) 2.93 (396) 5.56 (792) 60.1 55.3
Purine bases (mmol/100 mg microbial fraction) Guaninee 3.11 (470) Adeninee 3.95 (534) d Purine bases (PB) 7.06 (1004) Crude protein/PBe 47.1 True protein/PBe 42.5
Constrasta,
b
L
Q
0.801 0.379 0.531 0.676
0.02 – – 0.04
0.72 – – 0.01
0.085 0.074 0.154 0.003 0.132
0.002 0.002 o 0.001 0.002 0.04
0.05 o 0.001 0.02 0.22 0.94
a Contrasts were evaluated only if the main effect of treatment was significant (P o0.10). The lack of a main effect of treatment is designated by –. b Probability of a linear (L) or quadratic (Q) effect of monensin concentration. c Expressed as glucose equivalents. d Values in parentheses are mg purine bases 100 mg 1 microbial fraction; purine bases: guanineþ adenine. e Milligram crude protein (or true protein) to mg purine bases (on 100 mg microbial fraction basis).
Table 2 Effects of monensin on fermentation parameters and efficiency of microbial protein synthesis. Variable
Substrate fermentation TDMD (%)c Partitioning factor (mg/ml) Gas production (ml/syringe) CH4 100/mg TDMD Microbial mass (mg)d Protozoa number ( 106 ml 1) Efficiency of microbial protein synthesis g N/kg TDMD mg N/mmol SCFA mmol PB/g TDMD mmol PB/mmol SCFA
Monensin (mM)
SEM
0
5
8
75.1 3.6 79.9 5.1 113.0 98.6
62.2 4.1 57.6 3.8 108.4 71.25
60.7 4.1 54.0 3.8 100.7 74.5
33.7 8.3 25.6 6.3
33.2 8.1 24.3 5.9
29.7 6.04 27.9 5.7
Constrasta,
b
L
Q
1.205 0.038 0.787 0.232 2.697 7.853
0.01 o0.001 o0.001 0.003 – 0.02
0.26 0.02 0.02 0.07 – 0.13
0.831 0.428 0.804 0.173
0.02 0.01 0.04 –
0.13 0.07 0.94 –
a Contrasts were evaluated only if the main effect of treatment was significant (P o0.10). The lack of a main effect of treatment is designated by –. b Probability of a linear (L) or quadratic (Q) effect of monensin concentration. c True dry matter digestibility. d In 30 ml incubation medium.
J.M. Castro-Montoya et al. / Livestock Science 150 (2012) 414–418
417
Table 3 Individual short chain fatty acid proportions and total short chain fatty acids production on addition of monensin. Variable
Acetate (C2) Propionate (C3) Butyrate (C4) Valerate (C5) BCFA Total SCFA (mmol) C2:C3
Monensin (mM)
SEM
0
5
8
64.0 22 12.5 1.2 0.21 46.9 2.91
56.4 31.5 10.2 0.9 0.73 31.9 1.80
55.3 32.9 10.2 0.8 0.83 31.5 1.69
1.403 1.860 0.554 0.069 0.044 2.647 0.059
Contrasta,
b
L
Q
o 0.001 o 0.001 0.03 0.004 0.005 o 0.001 o 0.001
0.01 0.07 0.4 0.47 0.12 o 0.001 o 0.001
a Contrasts were evaluated only if the main effect of treatment was significant (P o 0.10). The lack of a main effect of treatment is designated by –. b Probability of a linear (L) or quadratic (Q) effect of monensin concentration.
to engulf starch, therefore, the decrease in protozoa number seems to be the explanation for the lower sugar content of LAM by adding monensin. Furthermore, protozoa has a lower N content than liquid associated bacteria (Gonza´lez-Ronquillo et al., 2004), but in this study, the N content of LAM was not affected and only a numerical decrease of 7% compared with control was noted (at 5 mM). Crude protein and true protein were not affected by adding monensin. Crude protein can be described as the sum of true protein (peptides 41200 Da), oligopeptides o1200 Da, free amino acids and other organic substances containing nitrogen (Koschuh et al., 2004). The results of this study suggest that the differences between the fractions included in the true protein and crude protein remain unaffected by the addition of monensin. In in vivo studies a reduction in the excretion of urinary allantoin, a product of hepatic oxidation of purine bases, was observed on the addition of monensin (Isichei and Bergen, 1980; Poos et al., 1979). The decrease in PB could be related with the negative effects of monensin on protozoa, because these ciliates have been reported to have a purine content of almost half of that of bacteria (Broderick and Merchen, 1992). Monensin has been found to increase microbial protein synthesis (Jalc et al., 1992), which is in accordance with our findings. On the other hand, previous studies also have shown that monensin decreases in vitro CH4 production (e.g. Callaway et al., 1997; Russell and Strobe, 1988), in agreement with our results. Monensin has little direct effect on methanogens, but it inhibits hydrogen producing bacteria, while promoting propionate production, therefore inhibiting methanogenesis (Callaway et al., 1997). Addition of monensin to the in vitro systems decreases the dry matter digestibility of the substrate (e.g. Jalc and Laukova´, 2002; Wang et al., 2004), as found in this study. Unlike in vivo, in vitro systems may allow an excessive accumulation of end products of the fermentation, which likely inhibit some microbial activity, leading to the depression in TDMD, as stated by Nisa et al. (1999). Accordingly, total SCFA production also decreases. The lower C2 and C4 proportions by adding monensin could be related to monensin’s capacity to inhibit gram-positive bacteria, which influences the processes related to acetate and butyrate production (Russell, 1996). Conversely, gram-negative bacteria, linked to propionate production
are not affected by ionophores (Russell, 1996). In agreement with the results of this study, Gehman et al. (2008) reported an increase of the BCFA proportion. The increase in BCFA proportions might reflect an increase in deamination of the branched-chain amino acid valine and leucine and in general a higher degradation of dietary proteins in the rumen. 5. Conclusions Little information exists about the effects of monensin on the chemical composition of the microbial fraction. This study shows that monensin does not change N and true protein content of the liquid associated microbial matter in an in vitro system after 24 h; however, sugar, C and purine bases content did change. Since both the crude protein to purine bases ratio and the true protein to purine bases ratio varied, the use of purine bases or purine derivatives to estimate microbial protein synthesis should be done with caution upon addition of monensin. Further studies, particularly effects on solid associated microbes and in vivo validation of the effects observed are required. Conflict of interest statement The authors confirm that there is no conflict of interest.
Acknowledgments Joaquı´n Miguel Castro Montoya is grateful to DAAD (Deutscher Akademischer Austauchdienst) for financial support during the course of this work. References ¨ Blummel, M., Makkar, H.P.S., Becker, K., 1997. In vitro gas production: a technique revisited. J. Anim. Physiol. Anim. Nutr. 77, 24–34. Broderick, A., Merchen, N., 1992. Markers for quantifying microbial protein synthesis in the rumen. J. Dairy Sci. 75, 2618–2632. Callaway, R., Carneiro De Melo, A., Russell, J., 1997. The effect of nisin and monensin on ruminal fermentations in vitro. Curr. Microbiol. 35, 90–96. Castro-Montoya, J.M., Makkar, H.P.S., Becker, K., 2011. Chemical composition of rumen microbial fraction and fermentation parameters as
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affected by tannins and saponins using an in vitro rumen fermentation system. Can. J. Anim. Sci. 91, 433–448. Gehman, A.M., Kononoff, P.J., Mullins, C.R., Janicek, B.N., 2008. Evaluation of nitrogen utilization and the effects of monensin in dairy cows fed brown midrib corn silage. J. Dairy Sci. 91, 288–300. Gonza´lez-Ronquillo, M., Balcells, J., Belenguer, A., Castrillo, C., Mota, M., 2004. A comparison of purine derivatives excretion with conventional methods as indices of microbial yield in dairy cows. J. Dairy Sci. 87, 2211–2221. Hodge, J., Hofreiter, B., 1962. Determination of reducing sugars and carbohydrates. In: Whistler, R.L., Wolfram, M.L. (Eds.), Methods in Carbohydrate Chemistry, vol. 1. , Academic Press, New York, London, pp. 380–394. Isichei, C.O., Bergen, W.G., 1980. The effect of monensin on the composition of abomasal nitrogen flow in steers fed grain and silage rations. J. Anim. Sci. 51 (suppl.), 371A–372A. Jalc, D., Laukova´, A., 2002. Effect of nisin and monensin on rumen fermentation in the artificial rumen. Berl. Munch. Tierarztl. Wochenschr 115 (1–2), 6–10. Jalc, D., Baran, M., Vendra´k, T., Siroka, P., 1992. Effect of monensin on fermentation of hay and wheat bran investigated by the Rumen Simulation Technique (Rusitec). 2. End-products of fermentation and protein synthesis. Arch. Tierernahr. 42, 153–158. Koschuh, W., Povoden, G., Hong Thang, V., Kromus, S., Kulbe, K.D., Novalin, S., Krotscheck., C., 2004. Production of leaf protein concentrate from ryegrass (Lolium perenne x multiflorum) and alfalfa (Medicago sativa subsp. sativa). Comparison between heat coagulation/centrifugation and ultrafiltration. Desalination 163, 253–259. Makkar, H.P.S., Becker, K., 1999. Purine quantification in digesta from ruminants by spectrophotometic and HPLC methods. Br. J. Nutr. 81, 107–112. ¨ Makkar, H.P.S., Blummel, M., Becker, K., 1995. In vitro effects and interactions of tannins and saponins and fate of tannins in rumen. J. Sci. Food Agric. 69, 481–493. McGuffey, R.K., Richardson, L.F., Wilkinson, J.D., 2001. Ionophores for dairy cattle: current status and future outlook. J. Dairy Sci. 84 (E suppl.), E194–E203.
Menke, K.H., Raab, L., Salewski, A., Steingass, H., Fritz, D., Schneider, W., 1979. The estimation of the digestibility and metabolizable energy content of ruminant feedstuffs from the gas production when they are incubated with rumen liquor in vitro. J. Agric. Sci. 92, 217–222. Nisa, M.U., Sarwar, M., Bilal, Q., 1999. Review: effect of ionophores on metabolic energetics in cattle. Int. J. Agri. Biol. 1, 66–69. Peterson, G.L., 1983. Determination of total protein. Methods Enzymol. 91, 95–121. Poos, M.I., Hanson, T.L., Klopfenstein, T.J., 1979. Monensin effects on diet digestibility, ruminal protein bypass and microbial protein synthesis. J. Anim. Sci. 48, 1516–1524. Rodrı´guez, C.A., Gonza´lez, J., Alvir, M.R., Repetto, J.L., Centeno, C., Lamrani, F., 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. Russell, J.B., Strobel, H.J., 1988. Effects of additives on in vitro ruminal fermentation: a comparison of monensin and bacitracin, another gram-positive antibiotic. J. Anim. Sci. 66, 552–558. Russell, J.B., 1996. Mechanisms of ionophore action in ruminal bacteria. Pages E1–E18 in Scientific Update on Rumensin_/Tylan_/Micotil_ for the Professional Feedlot consultant. Elanco Anim Health, Indianapolis, IN. Selje-Assmann, N., Hoffmann, E.M., Becker, K., 2008. A batch incubation assay to screen plant samples and extracts for their ability to inhibit rumen protein degradation. Anim. Feed Sci. Technol. 145, 302–318. Smith, R.H., McAllan, A.B., 1974. Some factors influencing the chemical composition of mixed rumen bacteria. Br. J. Nutr. 31, 27–34. Storm, E., Ørskov, E.R., 1983. The nutritive value of rumen microorganisms in ruminants. 1. Large-scale isolation and chemical composition of rumen micro-organisms. Br. J. Nutr. 50 (2), 463–470. Wang, Y., Alexander, T.W., McAllister, T.A., 2004. In vitro effects of Monensin and Tween 80 on ruminal fermentation of barley grain: barley silage-based diets for beef cattle. Anim. Feed Sci. Technol. 16, 197–209.
Contents lists available at SciVerse ScienceDirect
Livestock Science journal homepage: www.elsevier.com/locate/livsci
Short communication
Effects of monensin on the chemical composition of the liquid associated microbial fraction in an in vitro rumen fermentation system Joaquı´n Miguel Castro-Montoya n, Harinder P.S. Makkar, Klaus Becker Institute for Animal Production in the Tropics and Subtropics (480b), University of Hohenheim, Fruwirthstrasse 12, 70593, Stuttgart, Germany
a r t i c l e in f o
abstract
Article history: Received 17 February 2012 Received in revised form 28 September 2012 Accepted 29 September 2012
The hypothesis of this study was that monensin alters chemical composition of liquid associated rumen microbes. Furthermore, this study generates information on the chemical composition of rumen bacteria as affected by a ionophore by analyzing different parameters within the same experiment. Monensin at 5 and 8 mM was incubated with 380 mg of substrate (hay:concentrate 70:30 w/w) for 24 h in an in vitro gas production system. Monensin elicited no effects on nitrogen and true protein contents of the liquid associated microbial fraction, but sugar content and carbon did change on addition on monensin. Ratio of crude protein to purine bases decreased on adding monensin. True dry matter digestibility and short chain fatty acids production decreased on addition of monensin. Acetate, butyrate and valerate proportions decreased, whereas propionate and branched-chain fatty acid proportions increased. The change in acetate to propionate ratio concurs with the increase in the efficiency of microbial protein synthesis and the decrease in methane production. Results suggest that: monensin influenced the chemical composition of the liquid associated microbial fraction in vitro, and the changed ratio of crude protein to purine bases may lead to under- or overestimations of microbial protein synthesis when purine derivatives are used as markers in the presence of monensin. & 2012 Elsevier B.V. All rights reserved.
Keywords: Microbial nitrogen Microbial sugar Purine bases Monensin
1. Introduction Monensin is a ionophore compound with reported capability to change the rumen fermentation. Increased milk production, decreased methanogenesis and attenuation of
Abbreviations: BCFA, branched-chain fatty acids; CP, crude protein; EMPS, efficiency of microbial protein synthesis; NDS, neutral detergent solution; PB, purine bases; PF, partitioning factor; SCFA, short chain fatty acids; TDMD, true dry matter digestibility; TP, true protein n Corresponding author. Current address: Laboratory for Animal Nutrition and Animal Product Quality, Department of Animal Production, Ghent University, Proefhoevestraat 10, 9090 Melle, Belgium. Tel.: þ 32 9264 9000; fax: þ 32 9264 9099. E-mail address: [email protected] (J.M. Castro-Montoya). 1871-1413/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.livsci.2012.09.026
certain cattle digestive disorders are some of the benefits attributed to monensin (McGuffey et al., 2001; Russell, 1996). These changes are associated with the property of ionophores to alter the microbial populations in the rumen: Gram-positive, proteolytic and obligate amino acid fermenting bacteria are sensitive to ionophores; while, gramnegative bacteria are not sensitive to them (Russell, 1996). Rumen microbes contribute substantially to the nutrients reaching the small intestine. Therefore, information on the chemical composition of the rumen microbes is essential for better understanding the nutrition of the animals. Previous studies, especially during the 1970s and early 1980s, have reported contents of N, amino acids, nucleic acids, carbohydrates, lipids and ash (e.g. Smith and McAllan, 1974; Storm and Ørskov, 1983) and more recently also on purines
J.M. Castro-Montoya et al. / Livestock Science 150 (2012) 414–418
(Rodrı´guez et al., 2000) in rumen microbial fraction. It is clear that some of these components vary with factors such as diet and time after feeding (Smith and McAllan, 1974) but less clear is how the chemical composition of the microbial fraction varies with the inclusion of additives in the diet. Therefore the objective of this study was to add information on the changes of the chemical composition of the liquid associated microbial fraction (LAM) as affected by monensin. The hypothesis of this study was that the addition of monensin changes the chemical composition of LAM in an in vitro fermentation system. 2. Materials and methods 2.1. Methods
415
for the treatment. From these eight syringes, six were used to collect the microbial fraction (see Section 2.1.2) and two were used to perform the protozoa count. Each experiment was repeated on three different days (n ¼3). The experimental design used was completely randomized with three replications for each treatment. After incubation for 24 h gas, methane, protozoa number, true dry matter digestibility (TDMD) and SCFA were determined as described in Castro-Montoya et al. (2011). Partitioning factor (PF) and microbial mass production ¨ were calculated as described by Blummel et al. (1997). The true dry matter digestibility (TDMD) was calculated as the difference between original sample weight (on dry matter basis) and residue after neutral detergent solution treatment and then divided by the original sample weight (on a dry matter basis).
2.1.1. In vitro gas production method 2.1.1.1. Rumen fluid. The fistulation of the cows and the regular collection of rumen fluid were approved on the 30 June 2009 by the regional council of Stuttgart, Germany under the experiment number A 355/09 TE. The rumen fluid was collected before the morning feeding from two rumen-fistulated Holstein-Friesian cattle, one fed ad libitum on grass silage, and the other on a concentrate containing diet (concentrate 2 kg/day and grass silage ad libitum). The concentrate (in g/kg) comprised of barley (350), wheat (340), maize (100), soybean meal (170), soybean oil (10) and mineral mixture (30). The cows had free access to drinking water. The collected rumen fluid was brought to the laboratory in warm (39 1C) insulated flasks, both fluids were mixed together, homogenized and filtered through 100 mm nylon filter. The glassware used was continuously flushed with CO2, stirred and kept in a water bath at 39 1C. 2.1.1.2. Method. The in vitro Hohenheim gas test (HFT) was used according to the protocol of Makkar et al. (1995) based on the method of Menke et al. (1979), except that double strength buffer and 380 mg of substrate were used. A mixture of hay and concentrate (70:30 w/w) was used as the substrate incubated in 100-ml-capacity calibrated glass syringes having 30 ml of a buffered incubation medium containing rumen microbes. Just before dispensing the buffered rumen fluid, a freshly prepared monensin (sodium salt 90–95% TLC; Sigma-Aldrich, Stenheim, Germany) aqueous solution was injected into the syringes through the syringe nozzle to reach the desired concentration of 5 and 8 mM in 30 ml of the medium. These concentrations were decided from previous experiments in our laboratory were monensin applied at a concentration of 5 mM decreased gas production without decreasing short chain fatty acids (SCFA) production (Selje-Assmann et al., 2008). The set without monensin containing 380 mg of the substrate was used as a control. A corresponding blank consisted of the buffered medium without the substrate but containing monensin at the experimental concentrations. The buffered medium was dispensed into the syringes and the incubation was performed in a water bath at 39 1C for 24 h. On a single day, each set of treatments comprised of ten syringes: two syringes for the blank and eight syringes
2.1.2. Collection of microbial fraction from the liquid associated microbes After 24 h of incubation, the contents of six syringes were filtered through a previously weighed nylon bag (pores size of 25 mm, F57, ANKOM Technology, NY, USA). The filtrate containing microbes was received in a tube, which afterwards was centrifuged at 17,000g at 4 1C for 20 min. The supernatant was removed and the microbial fraction was washed by adding 30 ml distilled water. Centrifugation was repeated and the microbial fraction was lyophilized, ground using a ball mill (Retsch, MM200, Haan, Germany) for 2 min at a frequency of 30 /s and stored at room temperature.
2.1.3. Chemical composition of the liquid associated microbial fraction Reducing sugar content was determined using the method of Hodge and Hofreiter (1962) and expressed as glucose equivalent; purine bases (adenine and guanine) by the method of Makkar and Becker (1999); and microbial protein content by Lowry’s method (Peterson, 1983). For N and C analyses 50 mg of microbial fraction were weighed into a crucible in duplicate and placed on the C/N analyzer system (Vario Max CN Elementar Analysensysteme GmbH. Frankfurt Main, Germany).
2.2. Statistical analysis The main effects of monensin were tested by the GLM procedure of SAS software, according to Y i ¼ m þ b1i þ xi where m is the overall mean, b1i the effect of the ith treatment and xI the error term. Significanct effects (Po0.1) of level of monensin were characterized using orthogonal contrasts testing the probability of linear or quadratic responses. Coefficients for polynomial contrasts were calculated for the unequally spaced treatments using the ILM procedure of SAS software. The values reported are means and standard errors.
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3. Results The addition of monensin at 5 and 8 mM to the incubation medium showed no effects on the true protein and N contents of LAM (Table 1). Carbon decreased linearly by adding monensin (Po0.05). Sugar and PB content decreased with increasing levels of monensin (Po0.05). Furthermore, the ratio of CP to PB and TP to PB increased when monensin was used (Po0.05). Protozoa number decreased with increasing levels of monensin (Po0.05) (Table 2). Adding monensin increased the efficiency of microbial protein synthesis (EMPS) (expressed both as mg N/mmol SCFA and g N/kg TDMD) and the partitioning of nutrients toward microbial mass (PF) (Po0.05). Monensin decreased CH4 production (Po0.05). The microbial mass was not
affected, while the true dry matter digestibility and gas production decreased (Po0.05) (Table 2). Acetate, C4, C5 proportions, total SCFA and the C2:C3 ratio (Po0.05) decreased with increasing levels of monensin. Proportions of C3 and branched chain fatty acid (BCFA) increased (Po0.05) (Table 3). 4. Discussion This study was performed to test the hypothesis that monensin can change the chemical composition of the rumen liquid associated microbial fraction in an in vitro system. Decreases in sugar content of the microbial fraction are in conformity with decreases in protozoa number upon addition of monensin. Protozoa are known
Table 1 Effects of monensin on the chemical composition of the liquid associated microbial fraction. Variable
Chemical composition Sugar (g/100 g)c True protein (g/100 g) Nitrogen (g/100 g) Carbon (g/100 g)
Monensin (mM)
SEM
0
5
8
23.4 42.7 7.5 44.9
20.5 43.4 7.3 46.3
18.2 43.8 7.6 42.3
2.62 (496) 2.95 (399) 5.57 (795) 57.7 54.6
2.62 (496) 2.93 (396) 5.56 (792) 60.1 55.3
Purine bases (mmol/100 mg microbial fraction) Guaninee 3.11 (470) Adeninee 3.95 (534) d Purine bases (PB) 7.06 (1004) Crude protein/PBe 47.1 True protein/PBe 42.5
Constrasta,
b
L
Q
0.801 0.379 0.531 0.676
0.02 – – 0.04
0.72 – – 0.01
0.085 0.074 0.154 0.003 0.132
0.002 0.002 o 0.001 0.002 0.04
0.05 o 0.001 0.02 0.22 0.94
a Contrasts were evaluated only if the main effect of treatment was significant (P o0.10). The lack of a main effect of treatment is designated by –. b Probability of a linear (L) or quadratic (Q) effect of monensin concentration. c Expressed as glucose equivalents. d Values in parentheses are mg purine bases 100 mg 1 microbial fraction; purine bases: guanineþ adenine. e Milligram crude protein (or true protein) to mg purine bases (on 100 mg microbial fraction basis).
Table 2 Effects of monensin on fermentation parameters and efficiency of microbial protein synthesis. Variable
Substrate fermentation TDMD (%)c Partitioning factor (mg/ml) Gas production (ml/syringe) CH4 100/mg TDMD Microbial mass (mg)d Protozoa number ( 106 ml 1) Efficiency of microbial protein synthesis g N/kg TDMD mg N/mmol SCFA mmol PB/g TDMD mmol PB/mmol SCFA
Monensin (mM)
SEM
0
5
8
75.1 3.6 79.9 5.1 113.0 98.6
62.2 4.1 57.6 3.8 108.4 71.25
60.7 4.1 54.0 3.8 100.7 74.5
33.7 8.3 25.6 6.3
33.2 8.1 24.3 5.9
29.7 6.04 27.9 5.7
Constrasta,
b
L
Q
1.205 0.038 0.787 0.232 2.697 7.853
0.01 o0.001 o0.001 0.003 – 0.02
0.26 0.02 0.02 0.07 – 0.13
0.831 0.428 0.804 0.173
0.02 0.01 0.04 –
0.13 0.07 0.94 –
a Contrasts were evaluated only if the main effect of treatment was significant (P o0.10). The lack of a main effect of treatment is designated by –. b Probability of a linear (L) or quadratic (Q) effect of monensin concentration. c True dry matter digestibility. d In 30 ml incubation medium.
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Table 3 Individual short chain fatty acid proportions and total short chain fatty acids production on addition of monensin. Variable
Acetate (C2) Propionate (C3) Butyrate (C4) Valerate (C5) BCFA Total SCFA (mmol) C2:C3
Monensin (mM)
SEM
0
5
8
64.0 22 12.5 1.2 0.21 46.9 2.91
56.4 31.5 10.2 0.9 0.73 31.9 1.80
55.3 32.9 10.2 0.8 0.83 31.5 1.69
1.403 1.860 0.554 0.069 0.044 2.647 0.059
Contrasta,
b
L
Q
o 0.001 o 0.001 0.03 0.004 0.005 o 0.001 o 0.001
0.01 0.07 0.4 0.47 0.12 o 0.001 o 0.001
a Contrasts were evaluated only if the main effect of treatment was significant (P o 0.10). The lack of a main effect of treatment is designated by –. b Probability of a linear (L) or quadratic (Q) effect of monensin concentration.
to engulf starch, therefore, the decrease in protozoa number seems to be the explanation for the lower sugar content of LAM by adding monensin. Furthermore, protozoa has a lower N content than liquid associated bacteria (Gonza´lez-Ronquillo et al., 2004), but in this study, the N content of LAM was not affected and only a numerical decrease of 7% compared with control was noted (at 5 mM). Crude protein and true protein were not affected by adding monensin. Crude protein can be described as the sum of true protein (peptides 41200 Da), oligopeptides o1200 Da, free amino acids and other organic substances containing nitrogen (Koschuh et al., 2004). The results of this study suggest that the differences between the fractions included in the true protein and crude protein remain unaffected by the addition of monensin. In in vivo studies a reduction in the excretion of urinary allantoin, a product of hepatic oxidation of purine bases, was observed on the addition of monensin (Isichei and Bergen, 1980; Poos et al., 1979). The decrease in PB could be related with the negative effects of monensin on protozoa, because these ciliates have been reported to have a purine content of almost half of that of bacteria (Broderick and Merchen, 1992). Monensin has been found to increase microbial protein synthesis (Jalc et al., 1992), which is in accordance with our findings. On the other hand, previous studies also have shown that monensin decreases in vitro CH4 production (e.g. Callaway et al., 1997; Russell and Strobe, 1988), in agreement with our results. Monensin has little direct effect on methanogens, but it inhibits hydrogen producing bacteria, while promoting propionate production, therefore inhibiting methanogenesis (Callaway et al., 1997). Addition of monensin to the in vitro systems decreases the dry matter digestibility of the substrate (e.g. Jalc and Laukova´, 2002; Wang et al., 2004), as found in this study. Unlike in vivo, in vitro systems may allow an excessive accumulation of end products of the fermentation, which likely inhibit some microbial activity, leading to the depression in TDMD, as stated by Nisa et al. (1999). Accordingly, total SCFA production also decreases. The lower C2 and C4 proportions by adding monensin could be related to monensin’s capacity to inhibit gram-positive bacteria, which influences the processes related to acetate and butyrate production (Russell, 1996). Conversely, gram-negative bacteria, linked to propionate production
are not affected by ionophores (Russell, 1996). In agreement with the results of this study, Gehman et al. (2008) reported an increase of the BCFA proportion. The increase in BCFA proportions might reflect an increase in deamination of the branched-chain amino acid valine and leucine and in general a higher degradation of dietary proteins in the rumen. 5. Conclusions Little information exists about the effects of monensin on the chemical composition of the microbial fraction. This study shows that monensin does not change N and true protein content of the liquid associated microbial matter in an in vitro system after 24 h; however, sugar, C and purine bases content did change. Since both the crude protein to purine bases ratio and the true protein to purine bases ratio varied, the use of purine bases or purine derivatives to estimate microbial protein synthesis should be done with caution upon addition of monensin. Further studies, particularly effects on solid associated microbes and in vivo validation of the effects observed are required. Conflict of interest statement The authors confirm that there is no conflict of interest.
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