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The Effects of Dietary Soybean Versus Skim Milk Protein on Plasma and Hepatic Concentrations of Zinc in Veal Calves
The Effects of Dietary Soybean Versus Skim Milk Protein on Plasma and Hepatic Concentrations of Zinc in Veal Calves C. XU,1 T. WENSING, and A. C. BEYNEN Department of Large Animal Medicine and Nutrition, Faculty of Veterinary Medicine, Utrecht University, 3508 TD Utrecht, The Netherlands
ABSTRACT We assessed the zinc status of veal calves that were fed milk replacers containing either skim milk protein as the sole source of protein or a mixture of skim milk protein and soybean protein. After the milk replacers had been fed for 26 wk, mean body weight gain was 3 kg lower for calves fed the skim milk plus soybean proteins; this decrease was not significant. Inclusion of dietary protein from soybeans versus milk protein alone reduced plasma concentrations of zinc by 43% and reduced hepatic concentrations of zinc by 81%. The impairment of zinc status that was induced by the inclusion of soybean protein was probably caused by its phytate component. The effect of soybean protein on zinc status was rather specific because plasma and hepatic concentrations of copper were unaffected. Despite the high concentration of zinc (142 mg/kg of dry matter) in the milk replacer that contained milk plus soybean proteins, calves displayed a shortage of zinc because their plasma and hepatic concentrations of zinc were significantly reduced. ( Key words: soy protein, phytic acid, zinc, veal calves) Abbreviation key: SBP = soybean protein, SMP = skim milk protein. INTRODUCTION The consumption of products that contain soybeans reduces the bioavailability of zinc in nonruminants, including humans (4, 6, 15), and the component that is responsible for this reduction is phytic acid (11, 14, 20). Increasingly, soybean protein ( SBP) preparations are being used as an alternative to milk proteins for incorporation into milk replacers in the diets of veal calves ( 1 ) . The influence of dietary SBP on the
Received May 16, 1996. Accepted February 28, 1997. 1Corresponding author. 1997 J Dairy Sci 80:2156–2161
zinc status of veal calves was unknown. Therefore, we measured plasma and hepatic concentrations of zinc in veal calves fed milk replacers that contained either skim milk protein ( SMP) as the sole source of protein or a mixture of SMP and SBP. To assess whether inclusion of SBP specifically influenced zinc status, we also determined plasma and hepatic concentrations of iron and copper. MATERIALS AND METHODS Calves and Diets Sixteen male Dutch Friesian-Holstein calves, about 1 wk of age, were purchased at a local market. Mean BW (and SD) was 45.8 ± 3.0 kg. The calves were housed individually in wooden stalls (70 cm × 170 cm) with slatted floors. The stalls were placed in a ventilated room. Calves were fed twice daily at 0800 and 1900 h; reconstituted milk replacer was presented in plastic buckets. On arrival (wk 0), 8 calves were allocated either to the group receiving the milk replacer that contained SMP or to the group receiving the milk replacer that contained SMP and SBP; BW distributions of the two dietary groups were similar. Calves were fed starter diets for 7 wk followed by finisher diets for another 19 wk. The composition of the milk replacers is documented in Tables 1 and 2. The control milk replacers contained SMP as the sole source of protein. The starter and finisher test diets contained 14 and 50%, respectively, of total protein in the form of SBP. The milk replacers were reconstituted in hot water (70°C ) just before feeding. Upon arrival, calves received 1.0 L of reconstituted milk containing 125 g of starter milk replacers per meal; the volume was gradually increased to 7.0 L per meal after 7 wk. The starter milk replacers were then gradually replaced by the finisher rations (115 g/L of reconstituted milk) over 4 d. After the replacement was complete, the concentration of the finisher control and test milk replacers was gradually increased to 180 g/L within 17 wk, and the volume was increased from 7 to 8.5 L
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SOY PROTEIN AND ZINC STATUS IN VEAL CALVES TABLE 1. Ingredient composition of the diets. Starter diet
Finisher diet
Ingredient
SMP1
SMP + SBP
Casein Soybean concentrate2 Hydrolyzed meat protein Hydrolyzed gluten L-Lysine·HCl D,L-Methionine Whey powder Defatted milk powder Wheat starch Tallow Lard Coconut oil Lecithin Premix3 Calcium formate Dicalcium phosphate
13.05 ... ... ... 0.03 0.02 63.15 ... 5.0 8.0 4.25 4.0 0.9 1.6 ... ...
5.22 4.44 1.25 5.0 0.3 ... 61.48 ... 3.75 5.0 6.32 4.0 0.9 1.6 0.74 ...
SMP
SMP + SBP
(g/100 g of DM) ... ... ... ... 0.57 0.36 33.99 35.50 5.0 8.5 7.5 5.0 1.0 1.65 0.43 0.5
... 12.50 ... ... ... ... 56.5 ... 5.0 8.35 7.5 5.0 1.0 2.9 1.25 ...
1SMP
= Skim milk protein; SBP = soybean protein. SBP concentrate contained 0.2 g/kg of zinc in the form of added ZnSO4·H2O. 3Tentego B.V. (Mijdrecht, The Netherlands); the premix contained 2.75 g of Zn/kg in the form of ZnSO4·H2O. 2The
per meal within 4 wk. The concentration and volume were then kept constant until 26 wk (the end of the trial). Collection of Samples and Chemical Analysis Blood samples were taken from the jugular vein into heparinized vacuum tubes at 1000 h every 4 wk. Plasma was collected by centrifugation at a low speed
and was subsequently stored at –20°C until analysis. At the end of the experiment, calves were slaughtered, and liver samples were taken; the samples were stored at –20°C until analysis. Feed and liver samples were digested in 23 M HNO3 for 16 h at 70°C. For copper and zinc analyses, samples of plasma were deproteinized using trichloroacetic acid. Zinc, copper, and iron in the prepared samples were estimated by flame atomic absorption spectroscopy (Perkin Elmer 3110; Perkin-
TABLE 2. Nutrient composition of the diets. Starter diet Composition
SMP1
Finisher diet
SMP + SBP
SMP
SMP + SBP
(g/100 g of DM) Calculated SMP NonSMP CP Crude fat Ash Moisture, g/100 g of diet Analyzed Zinc Copper Iron Phytic acid
23.5 0 23.5 18.4 6.7 3.3
14.9 8.6 23.5 18.0 7.3 3.3
117.7 17.2 58.2 ND2
143.2 18.5 64.7 210
19.3 0 19.3 23.2 7.3 3.3 (mg/kg of DM) 131.1 16.2 8.9 ND
9.6 9.4 19.0 23.3 7.0 3.4
141.0 13.2 26.4 1530
1SMP 2Not
= Skim milk protein; SBP = soybean protein. detectable. Journal of Dairy Science Vol. 80, No. 9, 1997
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TABLE 3. Growth performance of veal calves fed milk replacers containing either skim milk protein (SMP; n = 6 ) or a mixture of SMP and soybean protein (SBP; n = 8). Feed intake1
BW Gain wk
SMP
0–4 4–8 8–12 12–16 16–20 20–24 24–26 0–26
X 13 29 37 34 38 35 20 205
SMP + SBP
SMP
(kg) SD 4 2 3 2 4 12 4 16
SMP + SBP
Feed conversion ratio SMP
(kg of DM) X 12 29 32* 35 40 35 20 202
SD 3 2 4 4 5 9 4 8
23.8 43.0 57.4 68.4 82.8 79.6 42.8 397.8
23.8 43.0 57.4 68.4 82.8 79.6 42.8 397.8
X 0.54 0.68 0.64 0.49 0.46 0.44 0.46 0.51
SMP + SBP (kg of BW gain/kg of DM) SD X SD 0.17 0.51 0.13 0.04 0.67 0.04 0.06 0.55* 0.07 0.03 0.52 0.06 0.04 0.48 0.06 0.15 0.43 0.12 0.10 0.46 0.09 0.04 0.51 0.02
1Standard
deviations are not given for feed intake because calves were fed restricted amounts of milk replacer. *Different from the group fed SMP alone ( P < 0.05) by Student’s t test.
Elmer Corp., Norwalk, CT). Plasma concentrations of iron were measured spectrophotometrically with a commercial test combination (Instruchemie; Hilversum B.V., Hilversum, The Netherlands). Blood concentrations of hemoglobin and hematocrit were measured with an automatic blood analyzer (Sysmex K1000; Charles Goffin, Ijsselstein, The Netherlands). The phytic acid content of the diets was analyzed by HPLC as described by Slump et al. (17). Statistical Analysis
sion was significantly lower. At 26 wk, liver weights of calves fed SMP and SMP plus SBP were 1.78 ± 0.11 and 1.77 ± 0.17% of BW, respectively. Intakes of Zinc, Copper, and Iron Zinc and copper contents of the four milk replacers were similar (Table 2). The finisher diets contained less iron than did the starter diets, and the finisher diet with SMP contained less iron than did the corresponding SMP plus SBP diet. Figure 2 shows the intakes of zinc, copper, and iron. Over the entire
Data were statistically analyzed by either Student’s t test or repeated measure ANOVA using SPSS for Windows 6.0 (18). Significance was declared at P < 0.05. RESULTS Growth Performance Two calves from the SMP group were excluded from data analysis because their performance was extremely poor. Final BW of the 2 calves were 149 and 185 kg. Figure 1 illustrates BW development throughout the study. The mean BW of calves fed SMP plus SBP consistently was 1 to 6 kg lower than that of calves fed SMP as the sole source of protein. However, BW curves for the two groups did not differ ( P > 0.05). At wk 12 only, the BW of calves fed SMP plus SBP (118 ± 5 kg, X ± SD; n = 8 ) was lower ( P = 0.02) than that of calves fed SMP only (124 ± 4; n = 6). Data for BW gain, feed intake, and feed conversion are shown in Table 3. There was no significant effect of SMP plus SBP for the entire feeding period, but, during wk 8 to 12, BW gain was significantly lower for calves fed SMP plus SBP, and feed converJournal of Dairy Science Vol. 80, No. 9, 1997
Figure 1. Time course for BW of veal calves that were fed milk replacers containing either skim milk protein (SMP; ÿ) or SMP plus soybean protein (SBP) ( o) . Results are expressed as means ( ±SD) for 6 and 8 calves fed SMP and SMP plus SBP, respectively. Student’s t test was performed to compare the two groups at the same age: *P < 0.05. Repeated measure analysis of variance revealed no effect ( P = 0.24) of SBP on the growth performance of veal calves.
SOY PROTEIN AND ZINC STATUS IN VEAL CALVES
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experimental period, calves fed the diet with SMP plus SBP ingested about 5 g of zinc more than did those fed the diet with SMP only. Total copper intake was 1.2 g lower, and iron intake was 6.2 g higher, for calves fed SMP plus SBP. Plasma Zinc, Copper, and Iron Plasma concentrations of zinc, copper, and iron are shown in Figure 3. From wk 8, plasma concentrations of zinc for calves fed SMP plus SBP were consistently lower than those for calves that received SMP only. The mean difference in plasma zinc between the two groups was 52.9%. Plasma copper was lower for calves fed SMP plus SBP for either 4 or 8 wk, but initial values of plasma copper for these calves were also lower. From wk 12, plasma concentrations of copper were similar for both groups of calves. Plasma concentrations of iron decreased during the course of the experiment until they became stable at wk 8. Except for wk 4, the control and test milk replacers did not significantly influence plasma concentrations of iron. Hemoglobin and hematocrit fell from initial values of 4.3 ± 0.8 mM and 32 ± 6% ( X ± SD; n = 14), respectively, to final values of 3.1 ± 0.5 mM and 24 ± 4%; type of milk replacer had no effect. Hepatic Zinc, Copper, and Iron Hepatic concentrations of zinc, copper, and iron are illustrated in Figure 4. Concentrations of zinc found in the liver of calves fed SMP plus SBP were five times lower than those found in the liver of calves fed SMP only ( P = 0.002). No significant differences in hepatic concentrations of copper and iron were detected between the two dietary groups. DISCUSSION
Figure 2. Intakes of zinc, copper, and iron expressed in 2-wk intervals for veal calves that were fed milk replacers containing either skim milk protein (SMP; ÿ) or a mixture of SMP and soybean protein ( o) . Standard deviations are not given because the calves were fed restricted amounts of milk replacer.
The plasma concentration of zinc of ruminating calves is directly correlated with dietary zinc in the range of 8 to 80 mg/kg of ration ( 9 ) . Thus, plasma zinc of breeding calves may be used as an indicator of available zinc in the ration. This correlation may also be the same for nonruminating veal calves. When compared with calves that were fed SMP, calves that were fed SMP plus SBP for 26 wk, had plasma and hepatic concentrations of zinc that were reduced by 43 and 81%, respectively. Calves that were fed SMP plus SBP even ingested 5 g of zinc more than did their counterparts fed SMP. Therefore, SMP plus SBP reduced the zinc status of the calves; this effect was rather specific because copper status was unaffected. For species other than veal calves, such as humans (6, 16), swine (4, 14), and rats (11, 20), the phytate Journal of Dairy Science Vol. 80, No. 9, 1997
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Figure 4. Hepatic concentrations of zinc, copper, and iron at the end of the experimental period in veal calves fed milk replacers that contained either skim milk protein (SMP; solid bar) or a mixture of SMP and soybean protein (SBP) (open bar). Results are expressed as means ( ±SD) for 6 and 8 calves fed SMP and SMP plus SBP, respectively. Student’s t test was performed to compare the two groups: **P < 0.01.
Figure 3. Time course of plasma concentrations of zinc, copper, and iron in veal calves that were fed milk replacers containing either skim milk protein (SMP; ÿ) or a mixture of SMP and soybean protein (SBP) ( o) . Results are expressed as means ( ±SD) for 6 and 8 calves fed SMP and SMP plus SBP, respectively. Student’s t test was performed to compare the two groups at the same age: *P < 0.05; **P < 0.01. Repeated measures analysis of variance revealed effects of SBP on plasma concentrations of zinc ( P < 0.001) and copper ( P < 0.01) but not on plasma concentrations of iron ( P = 0.70). Journal of Dairy Science Vol. 80, No. 9, 1997
component of SBP preparations impaired zinc absorption. This inhibition likely occurred in calves fed SMP plus SBP. As would be expected, milk replacers containing SBP were found to contain phytate (Table 2). Dietary phytate may impair zinc absorption through formation in the digesta of phytate and zinc complexes that are unavailable for absorption. Phytate in the diet may also reduce iron absorption by formation of complexes ( 5 ) . The milk replacers used in this study were restricted in iron (Table 2), which is a common practice in rearing veal calves (13). The low iron content of the milk replacers could have allowed for the formation of more complexes consisting of phytate and zinc and thus could have enhanced the influence of phytate on zinc absorption. In preruminant calves fed milk replacers based on SMP and that contained zinc at either 40 or 200 mg/kg of DM, mean hepatic concentrations of zinc were 10.4 and 25.0 mmol/g of DM ( 8 ) . The range of values for hepatic concentrations of zinc that were reported by Jenkins and Hidiroglou ( 8 ) compares well with the range found in this study for the calves fed SMP or SMP plus SBP. The two finisher milk replacers contained about 135 mg of zinc/kg of DM. Thus, a comparison of the results of Jenkins and Hidiroglou ( 8 ) with ours supports the suggestion that SBP in the diet lowered zinc availability. In the present study, the lower BW gain ( 3 kg) for calves fed SMP plus SBP was similar to that observed in previous studies (2, 3). However, the decrease in
SOY PROTEIN AND ZINC STATUS IN VEAL CALVES
BW gain of calves fed SMP plus SBP did not reach statistical significance. The objective of this study was not to demonstrate a significant difference in BW gain between groups, and the number of calves used in this study provided insufficient statistical power to determine differences. Although the antigenic activity of SBP ( 1 0 ) or the reduced fat digestibility in calves fed SBP ( 1 9 ) might have been responsible for the slightly depressed growth performance of the calves fed SMP plus SBP, the question arises as to whether the lower zinc status of calves fed SMP plus SBP was responsible for the impaired growth performance of those calves when compared with that of the calves fed SMP alone. Zinc deficiency reduces growth in young animals ( 7 ) , but the minimum zinc requirement of veal calves is not known. The data reported by Kirchgessner and Heindl ( 9 ) indicated that, for breeding calves ( 4 to 18 wk of age), the minimum requirement to achieve optimal feed conversion and maximal BW gain is 19 mg of zinc/kg of DM. According to Miller et al. (12), the minimum requirement for breeding calves that are 30 to 37 wk old is ≤9 mg of zinc/kg of DM. The concentration of zinc in the milk replacers that contained SMP plus SBP was about 140 mg/kg of DM. Analogous to the soy formulas used in infant nutrition (11), phytate in the finishing milk replacer could have reduced zinc absorption from about 50 to 25%, and the dietary concentration of available zinc still could have been ≥30 mg/kg of DM. A similar conclusion is reached when study results of Jenkins and Hidiroglou ( 8 ) are compared with this one. In that study, a milk replacer that was based on SMP with supplemental ZnO that contained 40 mg of zinc/kg of DM produced a hepatic concentration of zinc of 10.4 mmol/g of DM. According to the studies of Kirchgessner and Heindl ( 9 ) and Miller et al. (12), for calves fed SMP plus SBP, the amount of available zinc in the ration was unlikely to have been below the requirement. In conclusion, this study shows that the partial substitution of SBP for SMP in a milk replacer for veal calves reduced the availability of zinc as was reflected in lower plasma and hepatic concentrations of zinc. In practice, when veal calves are fed a milk replacer that contains SBP in combination with a relatively low concentration of zinc, reduced growth performance, if exhibited, might be related to impaired zinc status. ACKNOWLEDGMENTS This study was supported by The Netherlands Foundation for Nutrition and Health Research, Loders Croklaan B.V., Wormerveer and Tentego B.V., Mijdrecht, The Netherlands.
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The authors thank Inez Lemmens, Janny T. Broeke, and Marjory Pollak for analytical assistance and Arie C. Westeneng and Jan van Dasselaar for taking care of the calves. REFERENCES 1 Best, P. 1995. User-friendly milk replacers. Feed Int. 16:6. 2 Beynen, A. C., and L.G.M. van Gils. 1983. Postprandial changes in the levels of lipids, glucose, urea and nonprotein nitrogen in the serum of veal calves fed milk replacers containing either skim milk powder or soybean protein concentrate. Z. Tierphysiol. Tierernaehr. Futtermittelkd. 49:49. 3 Beynen, A. C., L.G.M. van Gils, K. E. Scholz, and C. E. West. 1983. Serum cholesterol levels of calves and rabbits fed milk replacers containing skim milk powder or soybean protein concentrate. Nutr. Rep. Int. 27:757. 4 Bobilya, D. J., M. R. Ellersieck, D. T. Gordon, and T. L. Veum. 1991. Bioavailabilities of zinc from nonfat dry milk, lowfat plain yogurt, and soy flour in diets fed to neonatal pigs. J. Agric. Food Chem. 39:1246. 5 Davies, N. T., and R. Nightingale. 1975. The effects of phytate on intestinal absorption and secretion of zinc, and whole-body retention of zinc, copper, iron and manganese in rats. Br. J. Nutr. 34:243. 6 Erdman, J. W., Jr., and E. J. Fordyce. 1989. Soy products and human diet. Am. J. Clin. Nutr. 49:725. 7 Hambidge, K. M., C. E. Casey, and N. F. Krebs. 1986. Zinc. Page 1 in Trace Elements in Human and Animal Nutrition. W. Mertz, ed. Acad. Press, Orlando, FL. 8 Jenkins, K. J., and M. Hidiroglou. 1991. Tolerance of the preruminant calf for excess manganese or zinc in milk replacer. J. Dairy Sci. 74:1047. 9 Kirchgessner, M., and U. Heindl. 1993. Investigation about the determination of zinc requirement of calves. J. Anim. Physiol. Anim. Nutr. 70:38. 10 Lalle`s, J. P. 1993. Nutritional and antinutritional aspects of soyabean and field pea proteins used in veal calf production: a review. Livest. Prod. Sci. 34:181. 11 Lo¨nnerdal, B., J. G. Bell, A. G. Hendrickx, R. A. Burns, and C. L. Keen. 1988. Effect of phytate removal on zinc absorption from soy formula. Am. J. Clin. Nutr. 48:1301. 12 Miller, W. J., C. M. Clifton, and N. W. Cameron. 1963. Zinc requirement of Holstein bull calves to nine months of age. J. Dairy Sci. 46:715. 13 Miltenburg, G.A.J., T. Wensing, F.J.M. Smulders, and H. J. Breukink. 1992. Relationship between blood hemoglobin, plasma and tissue iron, muscle heme pigment, and carcass color of veal. J. Anim. Sci. 70:2766. 14 O’Dell, B. L. 1969. Effect of dietary components upon zinc availability. Am. J. Clin. Nutr. 22:1315. 15 O’Dell, B. L., C. E. Burpo, and J. E. Savage. 1972. Evaluation of zinc availability in foodstuffs of plant and animal origin. J. Nutr. 102:653. 16 Reinhold, J. G., K. Nasr, A. Lahimgarzadeh, and H. Hedayati. 1973. Effects of purified phytate and phytate-rich bread upon metabolism of zinc, calcium, phosphorus, and nitrogen in man. Lancet i:283. 17 Slump, P., K. D. Bos, and M.G.E. Wolters. 1987. Improved determination of phytate by HPLC. Page 151 in Euro Food Chem. IV. Rapid Analysis in Food Processing and Food Control. Vol. I. Norwegian Food Res. Inst., Leon, Norway. 18 SPSS for Windows, Release 6.0. 1993. SPSS Inc., Chicago, IL. 19 Van Kempen, G.J.M., and J. Huisman. 1991. Introductory remarks: some aspects of skim-milk replacement by other protein sources in veal-calf diets. Page 201 in New Trends in Veal Calf Production. J.H.M. Metz and C. M. Groenestein, ed. Pudoc, Wageningen, The Netherlands. 20 Zhou, J. R., E. J. Fordyce, V. Raboy, D. B. Dickinson, M. S. Wong, R. A. Burns, and J. W. Erdman, Jr. 1992. Reduction of phytic acid in soybean products improves zinc bioavailability in rats. J. Nutr. 122:2466. Journal of Dairy Science Vol. 80, No. 9, 1997
ABSTRACT We assessed the zinc status of veal calves that were fed milk replacers containing either skim milk protein as the sole source of protein or a mixture of skim milk protein and soybean protein. After the milk replacers had been fed for 26 wk, mean body weight gain was 3 kg lower for calves fed the skim milk plus soybean proteins; this decrease was not significant. Inclusion of dietary protein from soybeans versus milk protein alone reduced plasma concentrations of zinc by 43% and reduced hepatic concentrations of zinc by 81%. The impairment of zinc status that was induced by the inclusion of soybean protein was probably caused by its phytate component. The effect of soybean protein on zinc status was rather specific because plasma and hepatic concentrations of copper were unaffected. Despite the high concentration of zinc (142 mg/kg of dry matter) in the milk replacer that contained milk plus soybean proteins, calves displayed a shortage of zinc because their plasma and hepatic concentrations of zinc were significantly reduced. ( Key words: soy protein, phytic acid, zinc, veal calves) Abbreviation key: SBP = soybean protein, SMP = skim milk protein. INTRODUCTION The consumption of products that contain soybeans reduces the bioavailability of zinc in nonruminants, including humans (4, 6, 15), and the component that is responsible for this reduction is phytic acid (11, 14, 20). Increasingly, soybean protein ( SBP) preparations are being used as an alternative to milk proteins for incorporation into milk replacers in the diets of veal calves ( 1 ) . The influence of dietary SBP on the
Received May 16, 1996. Accepted February 28, 1997. 1Corresponding author. 1997 J Dairy Sci 80:2156–2161
zinc status of veal calves was unknown. Therefore, we measured plasma and hepatic concentrations of zinc in veal calves fed milk replacers that contained either skim milk protein ( SMP) as the sole source of protein or a mixture of SMP and SBP. To assess whether inclusion of SBP specifically influenced zinc status, we also determined plasma and hepatic concentrations of iron and copper. MATERIALS AND METHODS Calves and Diets Sixteen male Dutch Friesian-Holstein calves, about 1 wk of age, were purchased at a local market. Mean BW (and SD) was 45.8 ± 3.0 kg. The calves were housed individually in wooden stalls (70 cm × 170 cm) with slatted floors. The stalls were placed in a ventilated room. Calves were fed twice daily at 0800 and 1900 h; reconstituted milk replacer was presented in plastic buckets. On arrival (wk 0), 8 calves were allocated either to the group receiving the milk replacer that contained SMP or to the group receiving the milk replacer that contained SMP and SBP; BW distributions of the two dietary groups were similar. Calves were fed starter diets for 7 wk followed by finisher diets for another 19 wk. The composition of the milk replacers is documented in Tables 1 and 2. The control milk replacers contained SMP as the sole source of protein. The starter and finisher test diets contained 14 and 50%, respectively, of total protein in the form of SBP. The milk replacers were reconstituted in hot water (70°C ) just before feeding. Upon arrival, calves received 1.0 L of reconstituted milk containing 125 g of starter milk replacers per meal; the volume was gradually increased to 7.0 L per meal after 7 wk. The starter milk replacers were then gradually replaced by the finisher rations (115 g/L of reconstituted milk) over 4 d. After the replacement was complete, the concentration of the finisher control and test milk replacers was gradually increased to 180 g/L within 17 wk, and the volume was increased from 7 to 8.5 L
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SOY PROTEIN AND ZINC STATUS IN VEAL CALVES TABLE 1. Ingredient composition of the diets. Starter diet
Finisher diet
Ingredient
SMP1
SMP + SBP
Casein Soybean concentrate2 Hydrolyzed meat protein Hydrolyzed gluten L-Lysine·HCl D,L-Methionine Whey powder Defatted milk powder Wheat starch Tallow Lard Coconut oil Lecithin Premix3 Calcium formate Dicalcium phosphate
13.05 ... ... ... 0.03 0.02 63.15 ... 5.0 8.0 4.25 4.0 0.9 1.6 ... ...
5.22 4.44 1.25 5.0 0.3 ... 61.48 ... 3.75 5.0 6.32 4.0 0.9 1.6 0.74 ...
SMP
SMP + SBP
(g/100 g of DM) ... ... ... ... 0.57 0.36 33.99 35.50 5.0 8.5 7.5 5.0 1.0 1.65 0.43 0.5
... 12.50 ... ... ... ... 56.5 ... 5.0 8.35 7.5 5.0 1.0 2.9 1.25 ...
1SMP
= Skim milk protein; SBP = soybean protein. SBP concentrate contained 0.2 g/kg of zinc in the form of added ZnSO4·H2O. 3Tentego B.V. (Mijdrecht, The Netherlands); the premix contained 2.75 g of Zn/kg in the form of ZnSO4·H2O. 2The
per meal within 4 wk. The concentration and volume were then kept constant until 26 wk (the end of the trial). Collection of Samples and Chemical Analysis Blood samples were taken from the jugular vein into heparinized vacuum tubes at 1000 h every 4 wk. Plasma was collected by centrifugation at a low speed
and was subsequently stored at –20°C until analysis. At the end of the experiment, calves were slaughtered, and liver samples were taken; the samples were stored at –20°C until analysis. Feed and liver samples were digested in 23 M HNO3 for 16 h at 70°C. For copper and zinc analyses, samples of plasma were deproteinized using trichloroacetic acid. Zinc, copper, and iron in the prepared samples were estimated by flame atomic absorption spectroscopy (Perkin Elmer 3110; Perkin-
TABLE 2. Nutrient composition of the diets. Starter diet Composition
SMP1
Finisher diet
SMP + SBP
SMP
SMP + SBP
(g/100 g of DM) Calculated SMP NonSMP CP Crude fat Ash Moisture, g/100 g of diet Analyzed Zinc Copper Iron Phytic acid
23.5 0 23.5 18.4 6.7 3.3
14.9 8.6 23.5 18.0 7.3 3.3
117.7 17.2 58.2 ND2
143.2 18.5 64.7 210
19.3 0 19.3 23.2 7.3 3.3 (mg/kg of DM) 131.1 16.2 8.9 ND
9.6 9.4 19.0 23.3 7.0 3.4
141.0 13.2 26.4 1530
1SMP 2Not
= Skim milk protein; SBP = soybean protein. detectable. Journal of Dairy Science Vol. 80, No. 9, 1997
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TABLE 3. Growth performance of veal calves fed milk replacers containing either skim milk protein (SMP; n = 6 ) or a mixture of SMP and soybean protein (SBP; n = 8). Feed intake1
BW Gain wk
SMP
0–4 4–8 8–12 12–16 16–20 20–24 24–26 0–26
X 13 29 37 34 38 35 20 205
SMP + SBP
SMP
(kg) SD 4 2 3 2 4 12 4 16
SMP + SBP
Feed conversion ratio SMP
(kg of DM) X 12 29 32* 35 40 35 20 202
SD 3 2 4 4 5 9 4 8
23.8 43.0 57.4 68.4 82.8 79.6 42.8 397.8
23.8 43.0 57.4 68.4 82.8 79.6 42.8 397.8
X 0.54 0.68 0.64 0.49 0.46 0.44 0.46 0.51
SMP + SBP (kg of BW gain/kg of DM) SD X SD 0.17 0.51 0.13 0.04 0.67 0.04 0.06 0.55* 0.07 0.03 0.52 0.06 0.04 0.48 0.06 0.15 0.43 0.12 0.10 0.46 0.09 0.04 0.51 0.02
1Standard
deviations are not given for feed intake because calves were fed restricted amounts of milk replacer. *Different from the group fed SMP alone ( P < 0.05) by Student’s t test.
Elmer Corp., Norwalk, CT). Plasma concentrations of iron were measured spectrophotometrically with a commercial test combination (Instruchemie; Hilversum B.V., Hilversum, The Netherlands). Blood concentrations of hemoglobin and hematocrit were measured with an automatic blood analyzer (Sysmex K1000; Charles Goffin, Ijsselstein, The Netherlands). The phytic acid content of the diets was analyzed by HPLC as described by Slump et al. (17). Statistical Analysis
sion was significantly lower. At 26 wk, liver weights of calves fed SMP and SMP plus SBP were 1.78 ± 0.11 and 1.77 ± 0.17% of BW, respectively. Intakes of Zinc, Copper, and Iron Zinc and copper contents of the four milk replacers were similar (Table 2). The finisher diets contained less iron than did the starter diets, and the finisher diet with SMP contained less iron than did the corresponding SMP plus SBP diet. Figure 2 shows the intakes of zinc, copper, and iron. Over the entire
Data were statistically analyzed by either Student’s t test or repeated measure ANOVA using SPSS for Windows 6.0 (18). Significance was declared at P < 0.05. RESULTS Growth Performance Two calves from the SMP group were excluded from data analysis because their performance was extremely poor. Final BW of the 2 calves were 149 and 185 kg. Figure 1 illustrates BW development throughout the study. The mean BW of calves fed SMP plus SBP consistently was 1 to 6 kg lower than that of calves fed SMP as the sole source of protein. However, BW curves for the two groups did not differ ( P > 0.05). At wk 12 only, the BW of calves fed SMP plus SBP (118 ± 5 kg, X ± SD; n = 8 ) was lower ( P = 0.02) than that of calves fed SMP only (124 ± 4; n = 6). Data for BW gain, feed intake, and feed conversion are shown in Table 3. There was no significant effect of SMP plus SBP for the entire feeding period, but, during wk 8 to 12, BW gain was significantly lower for calves fed SMP plus SBP, and feed converJournal of Dairy Science Vol. 80, No. 9, 1997
Figure 1. Time course for BW of veal calves that were fed milk replacers containing either skim milk protein (SMP; ÿ) or SMP plus soybean protein (SBP) ( o) . Results are expressed as means ( ±SD) for 6 and 8 calves fed SMP and SMP plus SBP, respectively. Student’s t test was performed to compare the two groups at the same age: *P < 0.05. Repeated measure analysis of variance revealed no effect ( P = 0.24) of SBP on the growth performance of veal calves.
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experimental period, calves fed the diet with SMP plus SBP ingested about 5 g of zinc more than did those fed the diet with SMP only. Total copper intake was 1.2 g lower, and iron intake was 6.2 g higher, for calves fed SMP plus SBP. Plasma Zinc, Copper, and Iron Plasma concentrations of zinc, copper, and iron are shown in Figure 3. From wk 8, plasma concentrations of zinc for calves fed SMP plus SBP were consistently lower than those for calves that received SMP only. The mean difference in plasma zinc between the two groups was 52.9%. Plasma copper was lower for calves fed SMP plus SBP for either 4 or 8 wk, but initial values of plasma copper for these calves were also lower. From wk 12, plasma concentrations of copper were similar for both groups of calves. Plasma concentrations of iron decreased during the course of the experiment until they became stable at wk 8. Except for wk 4, the control and test milk replacers did not significantly influence plasma concentrations of iron. Hemoglobin and hematocrit fell from initial values of 4.3 ± 0.8 mM and 32 ± 6% ( X ± SD; n = 14), respectively, to final values of 3.1 ± 0.5 mM and 24 ± 4%; type of milk replacer had no effect. Hepatic Zinc, Copper, and Iron Hepatic concentrations of zinc, copper, and iron are illustrated in Figure 4. Concentrations of zinc found in the liver of calves fed SMP plus SBP were five times lower than those found in the liver of calves fed SMP only ( P = 0.002). No significant differences in hepatic concentrations of copper and iron were detected between the two dietary groups. DISCUSSION
Figure 2. Intakes of zinc, copper, and iron expressed in 2-wk intervals for veal calves that were fed milk replacers containing either skim milk protein (SMP; ÿ) or a mixture of SMP and soybean protein ( o) . Standard deviations are not given because the calves were fed restricted amounts of milk replacer.
The plasma concentration of zinc of ruminating calves is directly correlated with dietary zinc in the range of 8 to 80 mg/kg of ration ( 9 ) . Thus, plasma zinc of breeding calves may be used as an indicator of available zinc in the ration. This correlation may also be the same for nonruminating veal calves. When compared with calves that were fed SMP, calves that were fed SMP plus SBP for 26 wk, had plasma and hepatic concentrations of zinc that were reduced by 43 and 81%, respectively. Calves that were fed SMP plus SBP even ingested 5 g of zinc more than did their counterparts fed SMP. Therefore, SMP plus SBP reduced the zinc status of the calves; this effect was rather specific because copper status was unaffected. For species other than veal calves, such as humans (6, 16), swine (4, 14), and rats (11, 20), the phytate Journal of Dairy Science Vol. 80, No. 9, 1997
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Figure 4. Hepatic concentrations of zinc, copper, and iron at the end of the experimental period in veal calves fed milk replacers that contained either skim milk protein (SMP; solid bar) or a mixture of SMP and soybean protein (SBP) (open bar). Results are expressed as means ( ±SD) for 6 and 8 calves fed SMP and SMP plus SBP, respectively. Student’s t test was performed to compare the two groups: **P < 0.01.
Figure 3. Time course of plasma concentrations of zinc, copper, and iron in veal calves that were fed milk replacers containing either skim milk protein (SMP; ÿ) or a mixture of SMP and soybean protein (SBP) ( o) . Results are expressed as means ( ±SD) for 6 and 8 calves fed SMP and SMP plus SBP, respectively. Student’s t test was performed to compare the two groups at the same age: *P < 0.05; **P < 0.01. Repeated measures analysis of variance revealed effects of SBP on plasma concentrations of zinc ( P < 0.001) and copper ( P < 0.01) but not on plasma concentrations of iron ( P = 0.70). Journal of Dairy Science Vol. 80, No. 9, 1997
component of SBP preparations impaired zinc absorption. This inhibition likely occurred in calves fed SMP plus SBP. As would be expected, milk replacers containing SBP were found to contain phytate (Table 2). Dietary phytate may impair zinc absorption through formation in the digesta of phytate and zinc complexes that are unavailable for absorption. Phytate in the diet may also reduce iron absorption by formation of complexes ( 5 ) . The milk replacers used in this study were restricted in iron (Table 2), which is a common practice in rearing veal calves (13). The low iron content of the milk replacers could have allowed for the formation of more complexes consisting of phytate and zinc and thus could have enhanced the influence of phytate on zinc absorption. In preruminant calves fed milk replacers based on SMP and that contained zinc at either 40 or 200 mg/kg of DM, mean hepatic concentrations of zinc were 10.4 and 25.0 mmol/g of DM ( 8 ) . The range of values for hepatic concentrations of zinc that were reported by Jenkins and Hidiroglou ( 8 ) compares well with the range found in this study for the calves fed SMP or SMP plus SBP. The two finisher milk replacers contained about 135 mg of zinc/kg of DM. Thus, a comparison of the results of Jenkins and Hidiroglou ( 8 ) with ours supports the suggestion that SBP in the diet lowered zinc availability. In the present study, the lower BW gain ( 3 kg) for calves fed SMP plus SBP was similar to that observed in previous studies (2, 3). However, the decrease in
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BW gain of calves fed SMP plus SBP did not reach statistical significance. The objective of this study was not to demonstrate a significant difference in BW gain between groups, and the number of calves used in this study provided insufficient statistical power to determine differences. Although the antigenic activity of SBP ( 1 0 ) or the reduced fat digestibility in calves fed SBP ( 1 9 ) might have been responsible for the slightly depressed growth performance of the calves fed SMP plus SBP, the question arises as to whether the lower zinc status of calves fed SMP plus SBP was responsible for the impaired growth performance of those calves when compared with that of the calves fed SMP alone. Zinc deficiency reduces growth in young animals ( 7 ) , but the minimum zinc requirement of veal calves is not known. The data reported by Kirchgessner and Heindl ( 9 ) indicated that, for breeding calves ( 4 to 18 wk of age), the minimum requirement to achieve optimal feed conversion and maximal BW gain is 19 mg of zinc/kg of DM. According to Miller et al. (12), the minimum requirement for breeding calves that are 30 to 37 wk old is ≤9 mg of zinc/kg of DM. The concentration of zinc in the milk replacers that contained SMP plus SBP was about 140 mg/kg of DM. Analogous to the soy formulas used in infant nutrition (11), phytate in the finishing milk replacer could have reduced zinc absorption from about 50 to 25%, and the dietary concentration of available zinc still could have been ≥30 mg/kg of DM. A similar conclusion is reached when study results of Jenkins and Hidiroglou ( 8 ) are compared with this one. In that study, a milk replacer that was based on SMP with supplemental ZnO that contained 40 mg of zinc/kg of DM produced a hepatic concentration of zinc of 10.4 mmol/g of DM. According to the studies of Kirchgessner and Heindl ( 9 ) and Miller et al. (12), for calves fed SMP plus SBP, the amount of available zinc in the ration was unlikely to have been below the requirement. In conclusion, this study shows that the partial substitution of SBP for SMP in a milk replacer for veal calves reduced the availability of zinc as was reflected in lower plasma and hepatic concentrations of zinc. In practice, when veal calves are fed a milk replacer that contains SBP in combination with a relatively low concentration of zinc, reduced growth performance, if exhibited, might be related to impaired zinc status. ACKNOWLEDGMENTS This study was supported by The Netherlands Foundation for Nutrition and Health Research, Loders Croklaan B.V., Wormerveer and Tentego B.V., Mijdrecht, The Netherlands.
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The authors thank Inez Lemmens, Janny T. Broeke, and Marjory Pollak for analytical assistance and Arie C. Westeneng and Jan van Dasselaar for taking care of the calves. REFERENCES 1 Best, P. 1995. User-friendly milk replacers. Feed Int. 16:6. 2 Beynen, A. C., and L.G.M. van Gils. 1983. Postprandial changes in the levels of lipids, glucose, urea and nonprotein nitrogen in the serum of veal calves fed milk replacers containing either skim milk powder or soybean protein concentrate. Z. Tierphysiol. Tierernaehr. Futtermittelkd. 49:49. 3 Beynen, A. C., L.G.M. van Gils, K. E. Scholz, and C. E. West. 1983. Serum cholesterol levels of calves and rabbits fed milk replacers containing skim milk powder or soybean protein concentrate. Nutr. Rep. Int. 27:757. 4 Bobilya, D. J., M. R. Ellersieck, D. T. Gordon, and T. L. Veum. 1991. Bioavailabilities of zinc from nonfat dry milk, lowfat plain yogurt, and soy flour in diets fed to neonatal pigs. J. Agric. Food Chem. 39:1246. 5 Davies, N. T., and R. Nightingale. 1975. The effects of phytate on intestinal absorption and secretion of zinc, and whole-body retention of zinc, copper, iron and manganese in rats. Br. J. Nutr. 34:243. 6 Erdman, J. W., Jr., and E. J. Fordyce. 1989. Soy products and human diet. Am. J. Clin. Nutr. 49:725. 7 Hambidge, K. M., C. E. Casey, and N. F. Krebs. 1986. Zinc. Page 1 in Trace Elements in Human and Animal Nutrition. W. Mertz, ed. Acad. Press, Orlando, FL. 8 Jenkins, K. J., and M. Hidiroglou. 1991. Tolerance of the preruminant calf for excess manganese or zinc in milk replacer. J. Dairy Sci. 74:1047. 9 Kirchgessner, M., and U. Heindl. 1993. Investigation about the determination of zinc requirement of calves. J. Anim. Physiol. Anim. Nutr. 70:38. 10 Lalle`s, J. P. 1993. Nutritional and antinutritional aspects of soyabean and field pea proteins used in veal calf production: a review. Livest. Prod. Sci. 34:181. 11 Lo¨nnerdal, B., J. G. Bell, A. G. Hendrickx, R. A. Burns, and C. L. Keen. 1988. Effect of phytate removal on zinc absorption from soy formula. Am. J. Clin. Nutr. 48:1301. 12 Miller, W. J., C. M. Clifton, and N. W. Cameron. 1963. Zinc requirement of Holstein bull calves to nine months of age. J. Dairy Sci. 46:715. 13 Miltenburg, G.A.J., T. Wensing, F.J.M. Smulders, and H. J. Breukink. 1992. Relationship between blood hemoglobin, plasma and tissue iron, muscle heme pigment, and carcass color of veal. J. Anim. Sci. 70:2766. 14 O’Dell, B. L. 1969. Effect of dietary components upon zinc availability. Am. J. Clin. Nutr. 22:1315. 15 O’Dell, B. L., C. E. Burpo, and J. E. Savage. 1972. Evaluation of zinc availability in foodstuffs of plant and animal origin. J. Nutr. 102:653. 16 Reinhold, J. G., K. Nasr, A. Lahimgarzadeh, and H. Hedayati. 1973. Effects of purified phytate and phytate-rich bread upon metabolism of zinc, calcium, phosphorus, and nitrogen in man. Lancet i:283. 17 Slump, P., K. D. Bos, and M.G.E. Wolters. 1987. Improved determination of phytate by HPLC. Page 151 in Euro Food Chem. IV. Rapid Analysis in Food Processing and Food Control. Vol. I. Norwegian Food Res. Inst., Leon, Norway. 18 SPSS for Windows, Release 6.0. 1993. SPSS Inc., Chicago, IL. 19 Van Kempen, G.J.M., and J. Huisman. 1991. Introductory remarks: some aspects of skim-milk replacement by other protein sources in veal-calf diets. Page 201 in New Trends in Veal Calf Production. J.H.M. Metz and C. M. Groenestein, ed. Pudoc, Wageningen, The Netherlands. 20 Zhou, J. R., E. J. Fordyce, V. Raboy, D. B. Dickinson, M. S. Wong, R. A. Burns, and J. W. Erdman, Jr. 1992. Reduction of phytic acid in soybean products improves zinc bioavailability in rats. J. Nutr. 122:2466. Journal of Dairy Science Vol. 80, No. 9, 1997