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The influence of chemical form on the effects of supplementary malate on serum metabolites and enzymes in finishing bull calves
Livestock Science 137 (2011) 260–263
Contents lists available at ScienceDirect
Livestock Science j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / l i v s c i
Short communication
The influence of chemical form on the effects of supplementary malate on serum metabolites and enzymes in finishing bull calves J. Hernández a, C. Castillo a,⁎, J. Méndez b, V. Pereira a, P. Vázquez a,c, M. López Alonso a, O. Vilariño a, J.L. Benedito a a b c
Departamento de Patología Animal, Facultad de Veterinaria, Universidad de Santiago de Compostela, Campus Universitario s/n, 27002 Lugo, Spain Departamento Técnico, COREN SCL, Ourense, Spain Departamento I+D+I, CESFAC, Madrid, Spain
a r t i c l e
i n f o
Article history: Received 27 April 2010 Received in revised form 3 September 2010 Accepted 3 October 2010 Keywords: Malate salt Malic acid Metabolites Feedlot
a b s t r a c t This study investigated the effects of free malic acid and a commercial malate salt on serum metabolic parameters in finishing-stage Belgian Blue bull calves maintained in a commercial feedlot. Serum levels of glucose, non-esterified fatty acids (NEFA), β-Hydroxibutirate (BHBT) urea nitrogen (SUN), creatinine, total protein (TSP), L-lactate, aspartate aminotransferase (AST) and gamma glutamyl transferase (GGT) were monitored over 86 days in 38 animals randomly allotted to three groups: MA (supplementation of feed with 4-g DL-malic acid per kg on a dry mass (DM) basis; 14 animals), MS (supplementation of feed with 4 g of a commercial disodium/calcium DL-malic acid salt per kg DM; 14 animals), and C (controls with no malate supplement; 10 animals). All the parameters considered except SUN lay within the physiological ranges for intensively reared beef, possibly due to the high crude protein (CP) content of the diet and the forage fiber source (barley straw). However, animals fed either form of malate had lower serum L-lactate and creatinine levels than those that did not receive this supplement, and their NEFA levels fell over time instead of rising. The only parameters differing between the free acid and salt groups were SUN and BHBT, which for unknown reasons, were higher in group MA than in either controls or group MS. That SUN was higher in group MA than in group C is attributed to its favoring CP-degrading ruminal flora. That BHBT was higher in group MA than in groups MS and C could be due to that the acid form can promote butyrate synthesis in rumen. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Currently, the beneficial in vitro effects of the organic acid on a ruminal environment (Martin and Streeter, 1995; Carro et al., 1999; Carro and Ranilla, 2003) is well known. However, the results of in vivo studies have been contradictory (Sanson and Stallcup, 1984; Martin et al., 1999; Carro et al., 2006; Castillo et al., 2008), probably due at least in part to differences in diet composition and/or malate dosage (Callaway et al., 1997; Abbreviations: ADF, acid detergent fiber; ADG, average daily gain; AST, aspartate aminotransferase; BW, body weight; CP, crude protein; DM, dry matter; EE, ether extract; GGT, gamma glytamyltransferase; MA, malic acid; MS, malate salt; NDF, neutral detergent fiber; NEFA, non-esterified fatty acids; SUN, serum urea nitrogen; TSP, total serum protein. ⁎ Corresponding author. Tel.: +34 982 28 59 00; fax: +34 982 28 59 40. E-mail address: [email protected] (C. Castillo). 1871-1413/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.livsci.2010.10.001
Castillo et al., 2004). Another factor that may influence the response to malate is the chemical form in which it is administered. In vitro, free malic acid and its disodium salt have similar effects, except for a lowering of pH by the former (Martin and Streeter, 1995); in a previous research performed by our group (Castillo et al., 2007) we found that the feed:gain ratio of finishing-stage bull calves has been reported to be lower with the free acid than with a commercial disodium/ calcium salt, and their blood acid–base balance was better with the salt than the free acid at a dosage of 4 g per kg of feed. To clarify further the different influences of the free acid and the disodium/calcium salt during the finishing stage, in the study described here we examined their effects on serum levels of glucose, non-esterified fatty acids (NEFA), β-hydroxibutirate (BHBT), urea nitrogen (SUN), creatinine, total protein (TSP), L-lactate, aspartate aminotransferase (AST) and
J. Hernández et al. / Livestock Science 137 (2011) 260–263
gamma glutamyl transferase (GGT) in finishing-stage Belgian Blue bull calves maintained in a commercial feedlot.
261
first day of the growing period. These groups were kept apart and fed as groups (which prevented the recording of individual daily intake).
2. Materials and methods 2.1. Animals, feeding management and experimental design
2.2. Measurements and analyses
Thirty-eight double-muscled Belgian Blue bull calves (122.9 ± 2.0 kg) were purchased and transported to the commercial study farm (Coren SCL, Ourense, NW Spain) at an age of 3–5 weeks. Adaptation to high-grain diets was carried out using a milk replacer (1-L/20-kg body weight (BW)) combined with a solid starter containing maize, wheat, barley, soybean meal, and vitamin–mineral premix (see footnote to Table 1); water and straw were available ad libitum. The compositions of the diets provided during the growing and finishing periods (14–22 and 23–35 weeks of age) are listed in Table 1; feed, water and barley straw were freely accessible at all times. Fresh feed was provided once a day at 08:00 h. Throughout the study the animals were cared for and managed in accordance with official Spanish guidelines on animal care and with EC Directive 86/609/EEC for animal experiments. Before the first feeding of growing diet the calves were allotted randomly to one of the three experimental groups: MA (supplementation of feed with DL-malic acid (from A. Pintaluba, SA, Reus, Spain) at 4 g/kg of DM; 14 animals), MS (supplementation of feed with disodium/calcium malate (Rumalato®, from Norel and Nature, Madrid, Spain) at 4 g/ kg of DM; 14 animals), and C (controls with no malate supplement; 10 animals). Supplement was given since the
Samples of concentrate were collected at the beginning of each stage and analyzed. Starch, ether extract and ash values were determined as described by Castillo et al. (2007). Before blood sampling, the calves were examined for clinical signs of metabolic disturbances (Lorenz, 2004). Blood samples were collected by jugular venous puncture (Vacutainer® tubes without EDTA) between 09:00 and 11:00 h on the finishing period: on day 0 (at the beginning of the fattening diet) and on days 3, 7, 23, 53 and 86 (the last day, prior to slaughter). Samples were centrifuged (2000 g for 20 min) and the plasma was immediately frozen (−20 °C) for pending analysis. Metabolic parameters were assayed using standardized kits (RAL Técnica para el Laboratorio, Spain, for glucose, urea, creatinine and AST; Gesellschaft für Diagnostica und Biochemica Mbh, Germany, for TSP; Randox Laboratories Ltd., UK, for NEFA and BHBT; and Spinreact, Spain, for L-lactate and GGT).
Table 1 Ingredients and chemical composition of the diets supplied in the present study. Growing
Finishing
Ingredient (g/kg DM) Barley Rye Wheat Corn Molasses Palm oil (98% bypass) Palm kernel oil Soybean meal, 44% CP DDGS a Corn gluten feed Wheat bran Soybean hulls Vitamin/Mineral premix a
326 50 100 100 25 18 – 151 70 100 – 32 28
305 60.0 100 100 25 20 40 96 80 100 42 11 21
Chemical composition (g/kg DM) CP CF ADF NDF EE b NFC c Starch Ash
166 50 66 190 41 545 350 58
155 50 72 216 47 531 350 51
a Vitamin and mineral premix contained per kg DM premix: 10.000 IU of vitamin A, 2.000 IU of vitamin D, 10 IU of vitamin E, 0.4 mg of Co, 16 mg of Cu, 25 mg of Fe, 2 mg of I, 110 mg of Mn, 0.3 mg of Se, and 120 mg of Zn. b EE: ether extract content. c NFC: non-fiber carbohydrates calculated as 100 (CP + ash + NDF + EE).
2.3. Statistical analyses Data were checked for normal distribution using the Shapiro–Wilks test and were subjected ANOVA, with group (TR) as the fixed main effect, sampling date (T) as a repeatedmeasured effect and a T × TR term included in the model. All statistical analyses were performed using SPSS 12.1. The criterion for statistical significance was P ≤ 0.05; P values between 0.05 and 0.1 were considered near significant. 3. Results Table 2 lists metabolic data for each group during the study. All parameters except AST showed a significant influence of sampling date. Comparing the final with the initial values, TSP rose in all groups; glucose and SUN fell appreciably in groups C and MS but much less in group MA; and creatinine fell slightly in groups MA and MS but not in group C. L-lactate increased during the first 3–7 days and decreased following the third day, and GGT behaved somewhat similarly in groups C and MS (which showed a considerable fall in GGT following day 53) but not in group MA (which had higher final than initial GGT levels). BHBT increased in the first 3 days with fluctuations after this; note that the C and MS groups had similar values while MA had higher concentrations, although without statistical relevance. The only parameter for which the T×TR ANOVA term was statistically significant was NEFA, which at the end of the study was slightly higher than at the beginning in group C and slightly lower in groups MA and MS. The group only had a statistically significant effect on L-lactate (P b 0.05), SUN (P b 0.001) and creatinine (P b 0.001). Lactate and creatinine were higher in untreated than in malate-treated animals, but the levels in groups MA and MS were similar. SUN values were significantly higher in group MA than in group MS (P b 0.001), and near significantly higher than in group C.
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J. Hernández et al. / Livestock Science 137 (2011) 260–263
Table 2 Mean values of the studied serum parameters during the study period. The effects of sampling date and treatment are summarized in the rightmost columns. P-value c
Days 0 Glucose (mg/dL) Ca MA MS NEFA (mmol/L) C MA MS L-lactate (mmol/L) C MA MS BHBT (mmol/L) C MA MS TSP (g/dL) C MA MS SUN (mg/dL) C MA MS Creatinine (mg/dL) C MA MS AST (IU/L) C MA MS GGT (IU/L) C MA MS a b c
3
102.2 91.3 106.1
7
96.8 91.1 97.4
23
94.6 97.3 101.3
53
94.0 94.1 100.1
90.4 92.1 93.8
86 88.0 88.8 92.3
Mean ± Pooled SEM
b
T
TR
T × TR
b0.05
0.52
0.54
b0.001
0.20
b0.05
b0.001
b 0.05
0.36
b0.05
0.23
0.09
b0.001
0.51
0.34
b0.001
b 0.001
0.06
b0.001
b 0.001
0.09
0.6
0.27
0.43
b0.001
0.94
0.63
94.3 ± 4.4 92.5 ± 3.7 98.5 ± 3.7
0.46 0.47 0.47
0.43 0.47 0.45
0.45 0.42 0.44
0.51 0.47 0.50
0.49 0.46 0.47
0.50 0.45 0.45
0.47 ± 0.01 0.46 ± 0.00 0.46 ± 0.00
0.47 0.40 0.44
0.58 0.53 0.53
0.72 0.54 0.49
0.70 0.58 0.51
0.57 0.45 0.44
0.56 0.39 0.43
0.60 ± 0.02 0.48 ± 0.02 0.48 ± 0.02
0.18 0.19 0.18
0.23 0.3 0.21
0.21 0.21 0.21
0.23 0.33 0.22
0.15 0.38 0.20
0.16 0.26 0.21
0.19 0.28 0.17
6.8 6.9 7.1
7.0 7.1 7.5
6.7 6.6 6.8
7.3 7.6 7.8
7.9 7.5 7.7
7.6 7.5 7.5
7.2 ± 0.1 7.2 ± 0.1 7.4 ± 0.1
26.6 28.2 23.1
23.6 20.4 14.0
21.2 23.0 19.4
18.8 25.0 15.1
18.6 21.1 15.0
21.6 28.0 19.4
21.7 ± 1.0 24.3 ± 0.9 17.7 ± 0.9
1.3 1.2 1.2
1.2 1.1 1.1
1.4 1.1 1.2
1.1 1.0 1.0
1.3 1.1 1.1
1.3 1.1 1.1
17.8 19.2 17.4
12.4 18.1 15.8
13.9 19.9 16.1
15.9 17.7 17.1
12.0 18.0 21.0
18.3 19.2 16.7
15.1 ± 1.6 17.4 ± 1.4 18.7 ± 1.4
5.8 5.5 6.5
7.4 7.9 8.2
8.0 7.3 6.6
6.1 6.5 6.7
7.4 6.4 7.2
5.3 6.2 5.5
6.7 ± 0.4 6.8 ± 0.3 6.6 ± 0.3
1.3 ± 0.02 1.1 ± 0.02 1.1 ± 0.02
C: control group (no malate supplement); MA: group with malic acid supplementation; MS: group with malate salt mixture (Rumalato®) supplementation. Pooled standard error of the mean. T = time effect; TR = group effect; T × TR = time × group interaction.
4. Discussion No indications of acidosis-related metabolic disorder (Lorenz, 2004) were observed in any animal at any time, and the mean values of all parameters except SUN were within the established normal ranges for cattle under intensive conditions (Latimer et al., 2003; Kaneko et al., 2008). The health of the animals may have been favored by the amount of crude protein in these diets, which was greater than the recommended 12–13% (Bailey and Duff, 2005), and the forage fiber source, barley straw, that contains more longfiber NDF than traditional silage forage, promoting chewing activity and saliva secretion (Krause et al., 1998). Animal performance was reported elsewhere (Castillo et al., 2007). Briefly, calves performance was not affected by treatment during the finishing period, although a numerically better feed conversion rate (feed:gain ratio) was obtained in the MA supplemented group, with similar values for the MS and C groups. The lower L-lactate levels in malate-treated animals should lead to less absorption of lactate into the bloodstream (Martin et al., 1999) but contradict the results of a similar
study of bull calves fed a maize-based diet, in which lactate levels were higher in malate-treated animals than in controls (Castillo et al., 2008). The effectiveness of malate may therefore depend on the nature of the grain that predominates in the diet. In the present study, the effect of malate on serum lactate levels was independent of whether it was administered as free acid or as its disodium and calcium salts. The finding that creatinine was higher in the control group than in the others contradicts those of Sanson and Stallcup (1984) and suggests that malate increased the efficiency of the diet provided in the present study. However, if creatinine is taken as an index of muscular mass and leanness (Latimer et al., 2003), our result suggests that the greater productive performance reached by the MA group (see Castillo et al., 2007) may have been due to greater fat deposition rather than to greater muscle formation. In view of this, it would be of interest to investigate whether it might be possible to lower diet cost by reducing CP content without adversely affecting the performance of malate-treated animals. SUN is known to increase with the intake and ruminal degradability of CP (Khan et al., 2007), to the extent that Russell and Roussel (2007) consider high SUN levels to be
J. Hernández et al. / Livestock Science 137 (2011) 260–263
indicative of excess degradable protein in the diet. Since in this study group MA had the lowest average daily intake, its having the highest SUN levels suggests that this chemical form of the organic would favor the ruminal flora responsible for CP degradation, in keeping with reports that malic acid improves ruminal fermentation, feed digestion, and production performance (Martin et al., 1999; Liu et al., 2008), and it may explain why group MA had the smallest feed:gain ratio (Castillo et al., 2007). The higher BHBT concentrations in the MA group than in MS or C suggest that the acid form of the supplement could create an acid environment in rumen, thus, favoring butyrate production. In addition, BHBT values in MS are in accordance with glucose concentrations and the in vitro results described by Carro et al. (2006), who found that the malate salt favors propionate production (a glycogenic precursor). In general, the observed effects of sampling date are in keeping with the accepted improvement of ruminal function with age (Khan et al., 2007) and with the above-noted effects of malate treatment. The finding that NEFA rose in the control group but fell in the others suggests that malate treatment may have prevented lipolysis (Kaneko et al., 2008), which would be in keeping with the notion that the higher ADG in group MA was due fundamentally to fat deposition. In conclusion, under the conditions of this study, supplementation with malate significantly reduces the serum L-lactate and creatinine levels of Belgian Blue bull calves and is associated with a decrease rather than an increase in NEFA levels over time. These effects did not depend on whether malate is administered as the free acid or as a mixture of its disodium and calcium salts. However, in this study animals fed the free acid had higher SUN and BHBT levels than both salt-treated and untreated animals. References Bailey, C.R., Duff, G.C., 2005. Protein requirements for finishing beef cattle. Retrieved March 1, 2006, from: http://animal.cals.arizona.edu/swnmc/ papers/2005/. Callaway, T.R., Martin, S.A., Wampler, J.L., Hill, N.S., Hill, G.M., 1997. Malate content of forage varieties commonly fed to cattle. J. Dairy Sci. 80, 1651–1655.
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Carro, M.D., Ranilla, M.J., Giráldez, F.J., Mantecón, A.R., 2006. Effects of malate on diet digestibility, microbial protein synthesis, plasma metabolites and performance on growing lambs fed a high-concentrate diet. J. Anim. Sci. 84, 405–410. Carro, M.D., López, S., Valdés, C., Ovejero, F.J., 1999. Effect of DL-malate on mixed ruminal microorganism fermentation using the rumen simulation technique (RUSITEC). Anim. Feed Sci. Technol. 79, 279–288. Carro, M.D., Ranilla, M.J., 2003. Effect of the addition of malate on in vitro rumen fermentation of cereal grains. Br. J. Nutr. 89, 181–187. Castillo, C., Benedito, J.L., Méndez, J., Pereira, V., López-Alonso, M., Miranda, M., Hernádez, J., 2004. Organic acids as a substitute for monensin in diets for beef cattle. Anim. Feed Sci. Technol. 115, 101–116. Castillo, C., Benedito, J.L., Pereira, V., Vázquez, P., López Alonso, M., Méndez, J., Hernández, J., 2007. Malic acid supplementation in growing/finishing feedlot bull calves: influence of chemical form on blood acid–base balance and productive performance. Anim. Feed Sci. Technol. 135, 222–235. Castillo, C., Benedito, J.L., Pereira, V., Méndez, J., Vázquez, P., López Alonso, M., Hernández, J., 2008. Effects of malate supplementation on acid–base balance and productive performance in growing/finishing bull calves fed a high-grain diet. Arch. Anim. Nutr. 62, 70–81. Kaneko, J.J., Harvey, J.W. Bruss, M.L., 2008. Clinical biochemistry of domestic animals, 6th ed., Ed. Academic Press, Orlando, USA. Khan, M.A., Lee, H.J., Lee, W.S., Kim, H.S., Ki, K.S., Park, S.J., Ha, J.K., Choi, Y.J., 2007. Starch source evaluation in calf starter: I—Feed consumption, body weight gain, structural growth and blood metabolites in Holstein calves. J. Dairy Sci 90, 5259–5268. Krause, M., Beauchemin, K.A., Rode, L.M., Farr, B.I., Norgaard, P., 1998. Fibrolytic enzyme treatment of barley grain and source of forage in highgrain diets fed to growing cattle. J. Anim. Sci. 76, 2912–2920. Latimer, K.S., Mahaffey, E.A., Prasse, K.W., 2003. Duncan and Prasse's Veterinary Laboratory Medicine: Clinical Pathology, 4ª ed. Iowa State University Press, USA. Liu, Q., Wang, C., Yang, W.Z., Dong, Q., Dong, K.H., Huang, Y.X., Yang, X.M., He, D.C., 2008. Effects of malic acid on rumen fermentation, urinary excretion of purine derivatives and feed digestibility in steers. Animal 3, 32–39. Lorenz, I., 2004. Investigations on the influence of serum D-lactate levels on clinical signs in calves with metabolic acidosis. Vet. J. 168, 323–327. Martin, S.A., Streeter, M.N., 1995. Effect of malate on in vivo mixed ruminal microorganism fermentation. J. Anim. Sci. 73, 2141–2145. Martin, S.A., Streeter, M.N., Nisbet, D.J., Hill, G.M., Williams, S.E., 1999. Effects of DL-malate on ruminal metabolism and performance of cattle fed highconcentrate diet. J. Anim. Sci. 77, 1008–1015. Russell, K.E., Roussel, A.J., 2007. Evaluation of the ruminant serum chemistry profile. Vet. Clin. Food. Anim. 23, 403–426. Sanson, D.W., Stallcup, O.T., 1984. Growth response and serum constituents of Holstein bulls fed malic acid. Nutr. Rep. Int. 30, 1261–1267 (abst).
Contents lists available at ScienceDirect
Livestock Science j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / l i v s c i
Short communication
The influence of chemical form on the effects of supplementary malate on serum metabolites and enzymes in finishing bull calves J. Hernández a, C. Castillo a,⁎, J. Méndez b, V. Pereira a, P. Vázquez a,c, M. López Alonso a, O. Vilariño a, J.L. Benedito a a b c
Departamento de Patología Animal, Facultad de Veterinaria, Universidad de Santiago de Compostela, Campus Universitario s/n, 27002 Lugo, Spain Departamento Técnico, COREN SCL, Ourense, Spain Departamento I+D+I, CESFAC, Madrid, Spain
a r t i c l e
i n f o
Article history: Received 27 April 2010 Received in revised form 3 September 2010 Accepted 3 October 2010 Keywords: Malate salt Malic acid Metabolites Feedlot
a b s t r a c t This study investigated the effects of free malic acid and a commercial malate salt on serum metabolic parameters in finishing-stage Belgian Blue bull calves maintained in a commercial feedlot. Serum levels of glucose, non-esterified fatty acids (NEFA), β-Hydroxibutirate (BHBT) urea nitrogen (SUN), creatinine, total protein (TSP), L-lactate, aspartate aminotransferase (AST) and gamma glutamyl transferase (GGT) were monitored over 86 days in 38 animals randomly allotted to three groups: MA (supplementation of feed with 4-g DL-malic acid per kg on a dry mass (DM) basis; 14 animals), MS (supplementation of feed with 4 g of a commercial disodium/calcium DL-malic acid salt per kg DM; 14 animals), and C (controls with no malate supplement; 10 animals). All the parameters considered except SUN lay within the physiological ranges for intensively reared beef, possibly due to the high crude protein (CP) content of the diet and the forage fiber source (barley straw). However, animals fed either form of malate had lower serum L-lactate and creatinine levels than those that did not receive this supplement, and their NEFA levels fell over time instead of rising. The only parameters differing between the free acid and salt groups were SUN and BHBT, which for unknown reasons, were higher in group MA than in either controls or group MS. That SUN was higher in group MA than in group C is attributed to its favoring CP-degrading ruminal flora. That BHBT was higher in group MA than in groups MS and C could be due to that the acid form can promote butyrate synthesis in rumen. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Currently, the beneficial in vitro effects of the organic acid on a ruminal environment (Martin and Streeter, 1995; Carro et al., 1999; Carro and Ranilla, 2003) is well known. However, the results of in vivo studies have been contradictory (Sanson and Stallcup, 1984; Martin et al., 1999; Carro et al., 2006; Castillo et al., 2008), probably due at least in part to differences in diet composition and/or malate dosage (Callaway et al., 1997; Abbreviations: ADF, acid detergent fiber; ADG, average daily gain; AST, aspartate aminotransferase; BW, body weight; CP, crude protein; DM, dry matter; EE, ether extract; GGT, gamma glytamyltransferase; MA, malic acid; MS, malate salt; NDF, neutral detergent fiber; NEFA, non-esterified fatty acids; SUN, serum urea nitrogen; TSP, total serum protein. ⁎ Corresponding author. Tel.: +34 982 28 59 00; fax: +34 982 28 59 40. E-mail address: [email protected] (C. Castillo). 1871-1413/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.livsci.2010.10.001
Castillo et al., 2004). Another factor that may influence the response to malate is the chemical form in which it is administered. In vitro, free malic acid and its disodium salt have similar effects, except for a lowering of pH by the former (Martin and Streeter, 1995); in a previous research performed by our group (Castillo et al., 2007) we found that the feed:gain ratio of finishing-stage bull calves has been reported to be lower with the free acid than with a commercial disodium/ calcium salt, and their blood acid–base balance was better with the salt than the free acid at a dosage of 4 g per kg of feed. To clarify further the different influences of the free acid and the disodium/calcium salt during the finishing stage, in the study described here we examined their effects on serum levels of glucose, non-esterified fatty acids (NEFA), β-hydroxibutirate (BHBT), urea nitrogen (SUN), creatinine, total protein (TSP), L-lactate, aspartate aminotransferase (AST) and
J. Hernández et al. / Livestock Science 137 (2011) 260–263
gamma glutamyl transferase (GGT) in finishing-stage Belgian Blue bull calves maintained in a commercial feedlot.
261
first day of the growing period. These groups were kept apart and fed as groups (which prevented the recording of individual daily intake).
2. Materials and methods 2.1. Animals, feeding management and experimental design
2.2. Measurements and analyses
Thirty-eight double-muscled Belgian Blue bull calves (122.9 ± 2.0 kg) were purchased and transported to the commercial study farm (Coren SCL, Ourense, NW Spain) at an age of 3–5 weeks. Adaptation to high-grain diets was carried out using a milk replacer (1-L/20-kg body weight (BW)) combined with a solid starter containing maize, wheat, barley, soybean meal, and vitamin–mineral premix (see footnote to Table 1); water and straw were available ad libitum. The compositions of the diets provided during the growing and finishing periods (14–22 and 23–35 weeks of age) are listed in Table 1; feed, water and barley straw were freely accessible at all times. Fresh feed was provided once a day at 08:00 h. Throughout the study the animals were cared for and managed in accordance with official Spanish guidelines on animal care and with EC Directive 86/609/EEC for animal experiments. Before the first feeding of growing diet the calves were allotted randomly to one of the three experimental groups: MA (supplementation of feed with DL-malic acid (from A. Pintaluba, SA, Reus, Spain) at 4 g/kg of DM; 14 animals), MS (supplementation of feed with disodium/calcium malate (Rumalato®, from Norel and Nature, Madrid, Spain) at 4 g/ kg of DM; 14 animals), and C (controls with no malate supplement; 10 animals). Supplement was given since the
Samples of concentrate were collected at the beginning of each stage and analyzed. Starch, ether extract and ash values were determined as described by Castillo et al. (2007). Before blood sampling, the calves were examined for clinical signs of metabolic disturbances (Lorenz, 2004). Blood samples were collected by jugular venous puncture (Vacutainer® tubes without EDTA) between 09:00 and 11:00 h on the finishing period: on day 0 (at the beginning of the fattening diet) and on days 3, 7, 23, 53 and 86 (the last day, prior to slaughter). Samples were centrifuged (2000 g for 20 min) and the plasma was immediately frozen (−20 °C) for pending analysis. Metabolic parameters were assayed using standardized kits (RAL Técnica para el Laboratorio, Spain, for glucose, urea, creatinine and AST; Gesellschaft für Diagnostica und Biochemica Mbh, Germany, for TSP; Randox Laboratories Ltd., UK, for NEFA and BHBT; and Spinreact, Spain, for L-lactate and GGT).
Table 1 Ingredients and chemical composition of the diets supplied in the present study. Growing
Finishing
Ingredient (g/kg DM) Barley Rye Wheat Corn Molasses Palm oil (98% bypass) Palm kernel oil Soybean meal, 44% CP DDGS a Corn gluten feed Wheat bran Soybean hulls Vitamin/Mineral premix a
326 50 100 100 25 18 – 151 70 100 – 32 28
305 60.0 100 100 25 20 40 96 80 100 42 11 21
Chemical composition (g/kg DM) CP CF ADF NDF EE b NFC c Starch Ash
166 50 66 190 41 545 350 58
155 50 72 216 47 531 350 51
a Vitamin and mineral premix contained per kg DM premix: 10.000 IU of vitamin A, 2.000 IU of vitamin D, 10 IU of vitamin E, 0.4 mg of Co, 16 mg of Cu, 25 mg of Fe, 2 mg of I, 110 mg of Mn, 0.3 mg of Se, and 120 mg of Zn. b EE: ether extract content. c NFC: non-fiber carbohydrates calculated as 100 (CP + ash + NDF + EE).
2.3. Statistical analyses Data were checked for normal distribution using the Shapiro–Wilks test and were subjected ANOVA, with group (TR) as the fixed main effect, sampling date (T) as a repeatedmeasured effect and a T × TR term included in the model. All statistical analyses were performed using SPSS 12.1. The criterion for statistical significance was P ≤ 0.05; P values between 0.05 and 0.1 were considered near significant. 3. Results Table 2 lists metabolic data for each group during the study. All parameters except AST showed a significant influence of sampling date. Comparing the final with the initial values, TSP rose in all groups; glucose and SUN fell appreciably in groups C and MS but much less in group MA; and creatinine fell slightly in groups MA and MS but not in group C. L-lactate increased during the first 3–7 days and decreased following the third day, and GGT behaved somewhat similarly in groups C and MS (which showed a considerable fall in GGT following day 53) but not in group MA (which had higher final than initial GGT levels). BHBT increased in the first 3 days with fluctuations after this; note that the C and MS groups had similar values while MA had higher concentrations, although without statistical relevance. The only parameter for which the T×TR ANOVA term was statistically significant was NEFA, which at the end of the study was slightly higher than at the beginning in group C and slightly lower in groups MA and MS. The group only had a statistically significant effect on L-lactate (P b 0.05), SUN (P b 0.001) and creatinine (P b 0.001). Lactate and creatinine were higher in untreated than in malate-treated animals, but the levels in groups MA and MS were similar. SUN values were significantly higher in group MA than in group MS (P b 0.001), and near significantly higher than in group C.
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Table 2 Mean values of the studied serum parameters during the study period. The effects of sampling date and treatment are summarized in the rightmost columns. P-value c
Days 0 Glucose (mg/dL) Ca MA MS NEFA (mmol/L) C MA MS L-lactate (mmol/L) C MA MS BHBT (mmol/L) C MA MS TSP (g/dL) C MA MS SUN (mg/dL) C MA MS Creatinine (mg/dL) C MA MS AST (IU/L) C MA MS GGT (IU/L) C MA MS a b c
3
102.2 91.3 106.1
7
96.8 91.1 97.4
23
94.6 97.3 101.3
53
94.0 94.1 100.1
90.4 92.1 93.8
86 88.0 88.8 92.3
Mean ± Pooled SEM
b
T
TR
T × TR
b0.05
0.52
0.54
b0.001
0.20
b0.05
b0.001
b 0.05
0.36
b0.05
0.23
0.09
b0.001
0.51
0.34
b0.001
b 0.001
0.06
b0.001
b 0.001
0.09
0.6
0.27
0.43
b0.001
0.94
0.63
94.3 ± 4.4 92.5 ± 3.7 98.5 ± 3.7
0.46 0.47 0.47
0.43 0.47 0.45
0.45 0.42 0.44
0.51 0.47 0.50
0.49 0.46 0.47
0.50 0.45 0.45
0.47 ± 0.01 0.46 ± 0.00 0.46 ± 0.00
0.47 0.40 0.44
0.58 0.53 0.53
0.72 0.54 0.49
0.70 0.58 0.51
0.57 0.45 0.44
0.56 0.39 0.43
0.60 ± 0.02 0.48 ± 0.02 0.48 ± 0.02
0.18 0.19 0.18
0.23 0.3 0.21
0.21 0.21 0.21
0.23 0.33 0.22
0.15 0.38 0.20
0.16 0.26 0.21
0.19 0.28 0.17
6.8 6.9 7.1
7.0 7.1 7.5
6.7 6.6 6.8
7.3 7.6 7.8
7.9 7.5 7.7
7.6 7.5 7.5
7.2 ± 0.1 7.2 ± 0.1 7.4 ± 0.1
26.6 28.2 23.1
23.6 20.4 14.0
21.2 23.0 19.4
18.8 25.0 15.1
18.6 21.1 15.0
21.6 28.0 19.4
21.7 ± 1.0 24.3 ± 0.9 17.7 ± 0.9
1.3 1.2 1.2
1.2 1.1 1.1
1.4 1.1 1.2
1.1 1.0 1.0
1.3 1.1 1.1
1.3 1.1 1.1
17.8 19.2 17.4
12.4 18.1 15.8
13.9 19.9 16.1
15.9 17.7 17.1
12.0 18.0 21.0
18.3 19.2 16.7
15.1 ± 1.6 17.4 ± 1.4 18.7 ± 1.4
5.8 5.5 6.5
7.4 7.9 8.2
8.0 7.3 6.6
6.1 6.5 6.7
7.4 6.4 7.2
5.3 6.2 5.5
6.7 ± 0.4 6.8 ± 0.3 6.6 ± 0.3
1.3 ± 0.02 1.1 ± 0.02 1.1 ± 0.02
C: control group (no malate supplement); MA: group with malic acid supplementation; MS: group with malate salt mixture (Rumalato®) supplementation. Pooled standard error of the mean. T = time effect; TR = group effect; T × TR = time × group interaction.
4. Discussion No indications of acidosis-related metabolic disorder (Lorenz, 2004) were observed in any animal at any time, and the mean values of all parameters except SUN were within the established normal ranges for cattle under intensive conditions (Latimer et al., 2003; Kaneko et al., 2008). The health of the animals may have been favored by the amount of crude protein in these diets, which was greater than the recommended 12–13% (Bailey and Duff, 2005), and the forage fiber source, barley straw, that contains more longfiber NDF than traditional silage forage, promoting chewing activity and saliva secretion (Krause et al., 1998). Animal performance was reported elsewhere (Castillo et al., 2007). Briefly, calves performance was not affected by treatment during the finishing period, although a numerically better feed conversion rate (feed:gain ratio) was obtained in the MA supplemented group, with similar values for the MS and C groups. The lower L-lactate levels in malate-treated animals should lead to less absorption of lactate into the bloodstream (Martin et al., 1999) but contradict the results of a similar
study of bull calves fed a maize-based diet, in which lactate levels were higher in malate-treated animals than in controls (Castillo et al., 2008). The effectiveness of malate may therefore depend on the nature of the grain that predominates in the diet. In the present study, the effect of malate on serum lactate levels was independent of whether it was administered as free acid or as its disodium and calcium salts. The finding that creatinine was higher in the control group than in the others contradicts those of Sanson and Stallcup (1984) and suggests that malate increased the efficiency of the diet provided in the present study. However, if creatinine is taken as an index of muscular mass and leanness (Latimer et al., 2003), our result suggests that the greater productive performance reached by the MA group (see Castillo et al., 2007) may have been due to greater fat deposition rather than to greater muscle formation. In view of this, it would be of interest to investigate whether it might be possible to lower diet cost by reducing CP content without adversely affecting the performance of malate-treated animals. SUN is known to increase with the intake and ruminal degradability of CP (Khan et al., 2007), to the extent that Russell and Roussel (2007) consider high SUN levels to be
J. Hernández et al. / Livestock Science 137 (2011) 260–263
indicative of excess degradable protein in the diet. Since in this study group MA had the lowest average daily intake, its having the highest SUN levels suggests that this chemical form of the organic would favor the ruminal flora responsible for CP degradation, in keeping with reports that malic acid improves ruminal fermentation, feed digestion, and production performance (Martin et al., 1999; Liu et al., 2008), and it may explain why group MA had the smallest feed:gain ratio (Castillo et al., 2007). The higher BHBT concentrations in the MA group than in MS or C suggest that the acid form of the supplement could create an acid environment in rumen, thus, favoring butyrate production. In addition, BHBT values in MS are in accordance with glucose concentrations and the in vitro results described by Carro et al. (2006), who found that the malate salt favors propionate production (a glycogenic precursor). In general, the observed effects of sampling date are in keeping with the accepted improvement of ruminal function with age (Khan et al., 2007) and with the above-noted effects of malate treatment. The finding that NEFA rose in the control group but fell in the others suggests that malate treatment may have prevented lipolysis (Kaneko et al., 2008), which would be in keeping with the notion that the higher ADG in group MA was due fundamentally to fat deposition. In conclusion, under the conditions of this study, supplementation with malate significantly reduces the serum L-lactate and creatinine levels of Belgian Blue bull calves and is associated with a decrease rather than an increase in NEFA levels over time. These effects did not depend on whether malate is administered as the free acid or as a mixture of its disodium and calcium salts. However, in this study animals fed the free acid had higher SUN and BHBT levels than both salt-treated and untreated animals. References Bailey, C.R., Duff, G.C., 2005. Protein requirements for finishing beef cattle. Retrieved March 1, 2006, from: http://animal.cals.arizona.edu/swnmc/ papers/2005/. Callaway, T.R., Martin, S.A., Wampler, J.L., Hill, N.S., Hill, G.M., 1997. Malate content of forage varieties commonly fed to cattle. J. Dairy Sci. 80, 1651–1655.
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