J. Anim. Sci. 2006. 84:405-410
© 2006 American Society of Animal Science
Effects of malate on diet digestibility, microbial protein synthesis, plasma metabolites, and performance of growing lambs fed a high-concentrate diet1
M. D. Carro*,
,2,
M. J. Ranilla*,,
F. J. Giráldez
, and
A. R. Mantecón,
* Departamento de Producción Animal I, Universidad de León, 24071;
and
Estación Agrícola Experimental (CSIC). Apdo. 788, 24080; and
and
Unidad Asociada Nutrición-Praticultura CSIC-ULE, León, Spain
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Abstract
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The objective of this study was to evaluate the effects of malate supplementation on growth rate, feed efficiency, and diet digestibility in growing lambs. Twenty-four Merino lambs with a mean BW of 15.3 ± 0.22 kg were divided into 3 homogenous groups. Each group was randomly allocated to 1 of 3 malate (16% disodium malate:84% calcium malate) levels: 0 (control), 4 (MAL-4), or 8 (MAL-8) g/kg of concentrate. Lambs were fed concentrate and barley straw ad libitum for 35 d. After a 20-d period, diet digestibility was determined, and microbial N flow at the duodenum was estimated from the urinary excretion of purine derivatives. Blood samples were taken on d 0, 20, and 35. On d 35, lambs were slaughtered and ruminal fluid samples were collected. There were no effects (P = 0.18 to P = 0.95) of malate on concentrate or straw intake, ADG, carcass yield, and apparent digestibility of OM, CP, NDF, or ADF. Malate supplementation did not influence (P = 0.80) the daily urinary excretion of total purine derivatives, and therefore there were no treatment effects (P = 0.77) on estimated microbial N flow at the duodenum. No differences (P > 0.05) among treatments were observed for plasma concentrations of glucose, cholesterol, triglycerides, urea-N, lactate, or VFA, but malate addition increased (P = 0.003) the molar proportion of butyrate in ruminal fluid (4.29, 6.14, and 5.45% of total VFA for control, MAL-4 and MAL-8, respectively). The use of malate as a feed additive under the conditions of the current study did not influence diet intake or digestion, and consequently did not improve lamb performance.
Key Words: digestibility • intake • lamb • malate • performance
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INTRODUCTION
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Malate could potentially provide an alternative to currently used antimicrobial compounds in ruminant feeding (Martin, 1998
; Castillo et al., 2004). In fact, several papers (Martin and Streeter, 1995; Callaway and Martin, 1996; Carro et al., 1999; Gómez et al., 2005) have shown that adding malate to in vitro ruminal fermentations of different substrates resulted in changes in final pH and production of CH4 and VFA that are analogous to the effects of ionophores. However, in contrast with antimicrobial compounds, malate seems to stimulate rather than inhibit some specific ruminal bacterial populations. Research has shown that malate can stimulate the growth of Selenomonas ruminantium in pure cultures (Nisbet and Martin, 1990, 1993). As S. ruminantium can account for up to 51% of total bacteria in the rumen of animals fed on cereal grains (Caldwell and Bryant, 1966), most of the research conducted to investigate the effects of malate has been carried out using concentrate feeds as the substrate for in vitro experiments or high-concentrate diets for in vivo experiments.
Available information about the effects of malate on in vivo ruminal fermentation and ruminant performance are, however, conflicting. Supplementation of diets with malic acid at rates ranging from 0.6 to 1.1% (DM basis) has been shown to improve ADG and G:F in steers fed high-concentrate diets based on corn (Sanson and Stallcup, 1984; Martin et al., 1999). In contrast, no effects of malic acid supplementation (2.6% of diet DM) on ruminal digestion and rumen microbial efficiency were found by Montaño et al. (1999) in steers fed an 81% steam-flaked barley-based diet, and Kung et al. (1982) reported no effects of malic acid supplementation (0.55 and 1.1% diet DM) on diet digestibility and N retention in steers fed ad libitum a diet based on 49% whole-shelled corn and 49% corn silage. These contrasting results could be due to differences in the composition of the diet and/or to the dose of malic acid.
To our knowledge, there is no information about the influence of malate on digestion and growth performance in lambs; therefore, the aim of this study was to evaluate the effects of 2 doses of malate on feed intake, digestive function, and performance in growing lambs fed a high-concentrate diet.
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MATERIALS AND METHODS
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Animals, Treatments, and Experimental Procedures
Twenty-four 6-wk-old male Merino lambs (15.3 ± 0.22 kg of BW) were weighed and housed in individual pens (0.80 x 1.40 m). During the experiment (35 d), lambs were fed barley straw and concentrate (Table 1), and had ad libitum access to fresh water. Concentrate ingredients were ground and pelleted. A mixture of 16% sodium malate and 84% calcium malate was added to concentrate to achieve final malate concentrations of 0 (control), 4 (MAL-4), and 8 (MAL-8) g/kg (DM basis). Malate was mixed directly with the concentrate at the time of feeding. Based on results from previous experiments conducted by our group, a ruminal volume of 2.7 L and a mean intake of 1.1 kg of DM/d can be assumed for 25 kg of BW Merino lambs fed similar diets; thus, malate concentrations at the end of the experimental period corresponded to 9.4 and 18.8 mM for MAL-4 and MAL-8, respectively. These levels of malate were chosen based on results from previous in vivo and in vitro studies (Callaway and Martin, 1996; Martin et al., 1999; Montaño et al., 1999). Each experimental treatment was randomly assigned to 8 lambs.
Lambs were offered concentrate and barley straw ad libitum (15% in excess of the previous day’s intake) twice daily at 0900 and 1800. Concentrate and straw refusals were taken daily (0830), pooled weekly for each animal, and sampled for DM, OM, N, NDF, and ADF determination. Samples of offered concentrate and straw were taken weekly for chemical composition analysis.
The experimental protocol was approved by the León University Institutional Animal Care and Use Committee. Lambs were weighed twice per week during the experimental period. On d 20, animals were moved to metabolism cages (0.6 x 1 m, 0.8 m high) equipped for quantitative collection of feces and urine separately. Concentrate, straw, and water were offered as just described. After 4 d of adaptation, feces and urine voided by each lamb in 24 h were quantitatively collected for 6 d. Fecal collection did not involve the use of bags or harnesses. An aliquot (10%) of total fecal output was collected each day for digestibility determination and dried to constant weight before analysis. Urine was collected in a solution of H2SO4 (10%; vol/vol) to keep the pH below 3. The daily volume of urine was determined, and a subsample (20%) was taken daily for each lamb and frozen. Daily samples of feces and urine were pooled to form composite samples. On d 30, lambs were moved again to floor pens.
On d 0, 20, and 35, blood samples were collected from each lamb via jugular venipuncture into heparinized tubes immediately before the morning feeding. Samples were centrifuged (1,000 x g for 15 min at 4°C), and the plasma was immediately frozen (–20°C) until determination of glucose, urea-N, cholesterol, triglycerides, lactate, and VFA. On d 35, lambs were slaughtered at a commercial slaughter facility and HCW were obtained. Carcasses were chilled for 24 h and weighed. The day before slaughtering, feed and water were withdrawn at 2000, and 200 g of concentrate and 400 mL of water were offered to each lamb at 2400 in order to minimize the effects of intake on ruminal response variables. Total ruminal contents from each lamb were removed within 30 min after slaughter, weighed, mixed, and sampled. About 200 g of contents was strained through 2 layers of cheesecloth; the pH was measured immediately; and samples were taken for NH3-N (20 mL were mixed with 20 mL of 0.5 M HCl), VFA [2 mL was added to 2 mL of deproteinizing solution of metaphosphoric (100 mL/L) acid and crotonic (0.06 mL/L) acid], and L-lactate (2 mL of ruminal fluid) analyses.
Chemical composition analyses were conducted on samples of feeds, refusals, and feces. Dry matter, ash, and N were determined according to AOAC (1999) procedures (AOAC official methods 943.01, 942.05, and 976.06, respectively). Neutral-detergent fiber and ADF analyses were carried out as described by Van Soest et al. (1991) and Goering and Van Soest (1970), respectively. Preparation of feed samples for malate analysis and malate analysis by HPLC followed the procedures described by Callaway et al. (1997). Urinary concentrations of urea-N (on wet urinary samples) and plasma metabolites were determined by automated enzymatic methods adapted to a Cobas Integra 400 plus Analyzer (F. Hoffmann-La Roche Ltd, Basel, Switzerland). Concentrations of L-lactate in ruminal fluid were analyzed by an enzymatic-colorimetric method using a diagnostic kit (Sigma, Madrid, Spain). Colorimetric reactions were measured in a Biokinetics ELISA microplate reader (Cultek TL 340; Cultek SL, Madrid, Spain). Concentrations of VFA in plasma and ruminal fluid were determined by using a gas chromatograph (Shimadzu GC 14B; Shimadzu Corporation, Kyoto, Japan) equipped with an autosampler and a GP 60/80 Carbopack C/0.3% Carbowax 20M/0.1% H3PO4 column (Supelco Inc., Spain). Ammonia-N concentration was determined by a colorimetric method (Weatherburn, 1967). Concentration of purine derivatives (allantoin, uric acid, xanthine, and hypoxanthine) was analyzed by HPLC (Balcells et al., 1992), and microbial N flow at the duodenum was estimated from the daily urinary excretion of purine derivatives (Balcells et al., 1991).
Statistical Analyses
Data were analyzed as a 1-way ANOVA with 3 concentrations of malate (0, 4, and 8 g/kg of concentrate). When a significant F value (P < 0.05) was detected, means were compared by the least significance difference test. Statistical analyses were performed using the GLM procedures of SAS (SAS Inst. Inc., Cary, NC).
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RESULTS AND DISCUSSION
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One lamb receiving the control treatment was removed during the experiment because of health complications. Therefore, mean values for control treatment are the mean of 7 lambs, whereas data for the other treatments are the mean of 8 lambs. As shown in Table 2, there were no differences in concentrate (P = 0.23) and barley straw (P = 0.52) intake, which agrees with previously reported results (Kung et al., 1982; Martin et al., 1999; Montaño et al., 1999) and indicates that no negative effects of malate on feed intake should be expected when it is included at levels up to 2.6% of dietary DM. Although lambs were fed barley straw ad libitum in the current study, the intake of barley straw was very low and ranged from 2.8 to 9.1% of total DM intake. This low intake of straw has also been found in previous studies conducted by our group in lambs under similar feeding conditions (Manso et al., 1998). The ADG was not affected by malate supplementation either during the whole experiment (35 d; P = 0.18; Table 2) or through the first 20 d (P = 0.21; 265, 278, and 261 g/d for control, MAL-4 and MAL-8 treatments, respectively). Malate supplementation did not affect (P = 0.45 to P = 0.47) final weight of lambs, HCW, or carcass yield in our study (Table 2).
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Table 2. Average initial BW, ADG, DMI, G:F, final BW, carcass weight, and carcass yield in lambs fed ad libitum barley straw and a concentrate supplemented with 0 (Control), 4 (MAL-4), or 8 (MAL-8) g of malate/kg of DM for 35 d
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Consistent with earlier studies (Kung et al., 1982; Montaño et al., 1999), malate did not influence (P = 0.88 to 0.95) apparent digestibility of OM, CP, NDF, or ADF (Table 3). In contrast, some in vitro studies with batch cultures of mixed ruminal microorganisms (Carro and Ranilla, 2003) or Rusitec fermenters (Carro et al., 1999; Gómez et al., 2005) showed that malate increased ruminal degradability of diets of diverse composition. It is possible that malate stimulated diet ruminal degradation in vivo, but digestion in the postruminal tract counteracted this effect. However, Montaño et al. (1999) reported that malic acid supplementation (2.6% diet DM) did not affect ruminal digestion of OM or ADF in steers fed an 81% steam-flaked barley-based diet. Differences in microbial concentrations in the rumen compared with the in vitro systems, which are much more dilute, should be taken into account when considering these results.
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Table 3. Apparent digestibility coefficients, daily urinary excretion of N compounds and purine derivatives, estimated microbial N flow at the duodenum (MNFD), and N retained in lambs fed ad libitum barley straw and a concentrate supplemented with 0 (Control), 4 (MAL-4) or 8 (MAL-8) g of malate/kg of DM for 35 d
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Daily urinary excretion of total N and urea-N varied widely between lambs within treatment, and there were no effects (P = 0.52 and P = 0.89, respectively) of malate supplementation (Table 3
). Daily urinary excretion of allantoin, uric acid, xanthine, and hypoxantine (values not shown) was not affected (P = 0.56 to P = 0.88) by malate supplementation and, therefore, the estimated microbial N flow at the duodenum did not differ (P = 0.77) between treatments. Few studies have investigated the effects of malate on rumen microbial growth. Montaño et al. (1999) used purine bases as a microbial marker to estimate microbial N duodenal flow in steers supplemented daily with 80 g of malate (2.6% of diet), and reported no differences from the control group. Nisbet and Martin (1990, 1993) showed that malate (4 to 12 mM) stimulated the growth of S. ruminantium in pure cultures, but in vivo results seemed to indicate that malate does not stimulate growth of mixed ruminal microorganisms under the conditions of the cited experiments.
There were no differences (P = 0.24 to 1.0) between groups of lambs in plasma concentrations of metabolites either before feeding the experimental diets (d 0; values not shown) or on d 20 and 35 (Table 4). These results agree with those reported by Sanson and Stallcup (1984) and Martin et al. (1999) in steers and by Kung et al. (1982) in cows, indicating that under the conditions of these studies (concentrations of malate up to 1.1% diet DM basis), malate had no effects on plasma metabolites. In the present experiment, total VFA concentrations and molar proportions of acetate, propionate, and butyrate in plasma were not influenced (P = 0.18 to P = 0.96) by malate treatment.
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Table 4. Plasma concentrations of glucose, urea-N, cholesterol, triglycerides, lactate, and VFA in lambs fed ad libitum barley straw and a concentrate supplemented with 0 (Control), 4 (MAL-4), or 8 (MAL-8) g of malate/kg of DM for 35 d
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No differences (P = 0.65 to P = 0.86) between treatments in ruminal pH or concentrations of ammonia-N, L-lactate, or total VFA were detected (Table 5). In the studies of Martin et al. (1999) and Montaño et al. (1999), malate did not affect total VFA concentration in the rumen or molar proportions of acetate, propionate, and butyrate. In contrast, increases of VFA production caused by malate supplementation have been consistently found when different substrates were incubated in batch cultures of mixed ruminal microorganisms (Callaway and Martin, 1996; Carro and Ranilla, 2003; Tejido et al., 2005) or in Rusitec fermenters (Carro et al., 1999; Gómez et al., 2005).
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Table 5. Ruminal pH and concentrations of NH3-N, L-lactate, and VFA in lambs fed ad libitum barley straw and a concentrate supplemented with 0 (Control), 4 (MAL-4), or 8 (MAL-8) g of malate/kg of DM for 35 d
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These inconsistent responses could be related to the different experimental conditions found in vivo and in vitro; thus, ruminal DM content can vary with several factors, ranging from 10 to 25%, whereas in the in vitro systems, DM content of digesta represented about 2 to 4% of total volume. Although in all these studies malate was added at final concentrations of 4 to 12 mM, when concentrations are expressed as g of malate per 100 g of diet, marked differences between experiments were observed. In the in vitro experiments with batch cultures (Callaway and Martin, 1996; Carro and Ranilla, 2003; Tejido et al., 2005), 4, 8, and 12 mM malate represented, respectively, 6.8, 12.7, and 17.1% of incubated substrate (DM basis); in Rusitec fermenters (Carro et al., 1999; Gómez et al., 2005), 6.55 and 9.37 mM malate represented, respectively, 3.4 and 7.1% of incubated substrate (DM basis). Malate concentrations (DM basis) in vivo were, however, much lower, and ranged from 0.28 (Martin et al., 1999) to 2.6% of total diet DM (Montaño et al., 1999). Malate concentrations in our study (0.38 and 0.75% of total diet [DM basis] for MAL-4 and MAL-8, respectively) were intermediate. It is possible that greater levels of malate would be necessary to detect significant effects on in vivo VFA production and diet degradability.
In the current study, malate supplementation produced an increase (P = 0.003) in the molar proportion of butyrate (4.29, 6.14, and 5.45 mol/100 mol for control, MAL-4, and MAL-8, respectively), which is in agreement with previously results reported for dairy cows receiving malic acid at 0.38, 0.55, or 0.77% of total diet (Kung et al., 1982) and in several in vitro experiments (Callaway and Martin, 1996; Carro and Ranilla, 2003; Gómez et al., 2005). The reasons for the increased molar proportions of butyrate is unclear, since malate is fermented by ruminal microorganisms to acetate and propionate but not to butyrate. In addition, it should be noted that ruminal VFA concentrations in our experiment were determined 8 h after feeding and therefore do not reflect actual ruminal VFA concentrations in the postfeeding period.
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IMPLICATIONS
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Supplementing the diet of growing lambs with malate at 3.8 or 7.5 g/kg of total dietary dry matter did not affect feed intake, total tract digestibility, or lamb growth performance. Malate treatment produced an increase in molar proportion of butyrate in ruminal fluid but did not change any of the other ruminal variables measured. Comparison of the present results with those obtained in vitro might indicate that greater levels of malate than those used in this experiment would be necessary to stimulate ruminal fermentation in vivo and to improve ruminant animal performance. However, the high cost of malate ($2.90/kg) could make its use impractical as a feed additive in lamb production systems. More studies with diets of variable composition and different doses of malate are required to assess the conditions that influence the effectiveness of malate in ruminant feeding.
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Footnotes
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1 Funding was provided by the Spanish M.C.Y.T. (Project AGL2001-0130) and the J.C.Y.L. (Project LE62/03). M. J. Ranilla gratefully acknowledges receipt of a research contract from the M.C.Y.T. (Programa Ramón y Cajal). The authors are grateful to J. Balcells for conducting analyses of urinary purine derivatives. 
2 Corresponding author: dp1mct{at}unileon.es
Received for publication April 4, 2005.
Accepted for publication September 12, 2005.
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Abstract
INTRODUCTION
