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Technical Note: Forms and Route of Vitamin C Supplementation for Cows
Technical Note: Forms and Route of Vitamin C Supplementation for Cows M. HIDIROGLOU Centre for Food and Animal Research, Agriculture and Agri-Food Canada, Ottawa, ON, Canada K1A 0C6
ABSTRACT The purpose of this study was to compare the effect of multiple oral supplementation of two forms of vitamin C on plasma ascorbic acid concentrations in dairy cows. Vitamin C was administrated at the rate of 40 g/d during a 5-d period. The supplements were vitamin C in the form of a fine powder and vitamin C coated with ethyl cellulose. In addition, another group of cows provided with vitamin C in the form of fine powder through an abomasal fistula was enrolled in the study. The effect of vitamin C supplementation was assessed by the change in plasma ascorbic acid concentrations during a 5-d period of vitamin C supplementation. A tendency existed for cows that were dosed with vitamin C in the abomasum to have higher ascorbic acid than cows supplemented orally. In the abomasum-dosed cows, plasma ascorbic acid value varied between 3.6 and 4.4 mg/ml; the maximum ascorbic acid content was 4.4 mg/ml at 54 h. Cows dosed orally with vitamin C coated with ethyl cellulose had higher plasma ascorbic acid concentrations than did cows that received vitamin C in the form of fine powder. In the former group, the maximum ascorbic acid concentration occurred at 30 h after dosing ( 5 mg/ml). ( Key words: cows, vitamin C, plasma) Abbreviation key: VCEC = vitamin C coated with ethyl cellulose, VCFP = vitamin C fine powder. INTRODUCTION L-Ascorbic acid is produced in almost all mammals except humans and guinea pigs ( 8 ) . Vitamin C is a known antioxidant that protects the structural integrity of the cells of the immune system ( 3 ) . Young ruminants may need dietary ascorbic acid, because skin lesions, similar to those that are common with scurvy, have been reported in calves fed a diet with a low vitamin C content (9, 14). The observations of
Received November 4, 1998. Accepted April 5, 1999. 1999 J Dairy Sci 82:1831–1833
Hemingway ( 6 ) indicate that vitamin C may be important in the prevention of scours in calves. Cappa ( 4 ) reported that adult cattle are prone to ascorbic acid deficiency when ascorbic acid synthesis is impaired because exogenous supplies of this vitamin are rapidly destroyed by ruminal microflora. Therefore, a coated form of vitamin C with known stability was tested as a source of vitamin C supplementation for ruminants. The objective of this study was to compare plasma concentration of ascorbic acid after two forms of vitamin C were orally dosed to dairy cows. In addition, the effect of oral or intraabomasal supplementation of vitamin C on plasma ascorbic acid concentrations was compared. MATERIALS AND METHODS Six Holstein cows were used in the experiment. Four cows had ruminal fistulas, and two had abomasal fistulas ( 5 ) . Cows received a mixed diet consisting of 88% forage (70% corn silage, 20% haylage, and 10% hay) and 12% concentrate (DM basis). Cows that produced more than 7 kg of milk/d received additional concentrate in the form of pellets at the rate of 1 kg for every 3 kg of milk over 7 kg ( 1 ) . Cows in this study were cared for under guidelines comparable to those developed by the Canadian Council of Animal Care. Multiple oral supplements of two forms of vitamin C were administered at the rate of 40 g of vitamin C equivalent/d during a 5-d supplementation period. Blood samples were taken twice daily on d 0, 1, 2, and 3 during the morning and afternoon and once daily on d 4, 5, and 6. The vitamin formulations were provided by Hoffmann-La Roche, Inc. (Mississauga, ON, Canada) and consisted of 1 ) vitamin C fine powder ( VCFP) and 2 ) vitamin C coated with ethyl cellulose ( VCEC) . Two cows were assigned to each group. The VCFP is a fine white or slightly yellow odorless powder with a tart taste. The product contains a minimum of 99% ascorbic acid and is stable in dry form. The VCEC is a free-flowing powder that contains a minimum of 97.5% ascorbic acid. The VCFP or VCEC was dissolved in 200 ml of water and was ad-
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HIDIROGLOU TABLE 1. Means and standard errors of plasma ascorbic acid concentration ( mg/ml) following administration of VCEC (vitamin C coated with ethyl cellulose) into the rumen or VCFP (vitamin C fine powder) into the abomasum or into the rumen. All treatments consisted of 40 g/d of vitamin C for 5 d. VCEC Rumen
Hours after dosing
Mean
0 6 24 30 48 54 72 78 96 120 1441
3.45x 3.65a,x 4.57a,y 5.00a,y 4.45a,y 4.67a,y 4.50a,y 4.79a,y 4.68a,y 4.89a,y 4.83a,y
a,bMeans x,yMeans
VCFP Abomasum SE
Mean
0.12 0.29 0.27 0.46 0.41 0.46 0.35 0.21 0.10 0.19 0.04
3.38x 3.56a,x 3.75b,y 4.35b,y 4.41a,y 4.33ab,y 4.40a,y 4.34ab,y 4.12b,y 4.21b,y 4.12b,y
VCFP Rumen SE
Mean
SE
0.14 0.20 0.07 0.14 0.30 0.64 0.05 0.16 0.05 0.01 0.03
3.11x 2.98b,x 3.08b,x 3.73b,y 3.84b,y 4.10b,y 3.87b,y 4.00b,y 3.90b,y 3.73b,y 3.60b,y
0.56 0.56 0.62 0.43 0.42 0.66 0.43 0.62 0.67 0.41 0.39
in the same row with different superscripts are different ( P < 0.05). in the same column with different superscripts are different from initial values at 0 h ( P <
0.05). 124 h after the last dosing.
ministered daily for 5 d either orally or abomasally every morning. Blood samples were taken from the jugular vein immediately before vitamin C dosing. The samples were centrifuged, and plasma was separated. Each sample was immediately mixed with 2.55% metaphosphoric acid in the ratio of 1:2 (vol/ vol). The sample mixtures were frozen at –70°C until analyzed fewer than 3 d later. The ascorbic acid concentrations in samples were measured by HPLC using an electrochemical detector ( 2 ) . The statistical analysis was conducted using repeated measures of option of the general linear model procedure of SAS (13). The model included the effects of treatment, time, and the interaction between treatment and time. Duncan multiple range test was used to test differences between means, once a significant effect of treatments was indicated by ANOVA. Significance was determined at P < 0.05. RESULTS AND DISCUSSION Plasma ascorbic acid concentrations before supplementation varied between 3.2 and 3.5 mg/ml. The effects of treatment and time were significant, while the interaction between treatment and time was not significant. The plasma ascorbic acid concentrations in cows that received VCFP orally or abomasally and the concentrations in cows that received VCEC orally are shown in Table 1. Supplementation of vitamin C increased plasma ascorbic acid concentrations in all three groups. Cows Journal of Dairy Science Vol. 82, No. 8, 1999
that received VCEC had higher plasma ascorbic acid concentrations than did cows that received VCFP either orally (all time points) or abomasally (four of nine comparisons) ( P < 0.05). According to Liu et al. (10), ascorbic acid is metabolized very rapidly in cattle. Those researchers reported that following intravenous infusion of 300 g of sodium ascorbate to cattle, the plasma ascorbic acid concentration was highest 10 min after completion of the jugular infusion and returned to the basal level by 5 h postinfusion. Vitamin C administration directly into the abomasum resulted in slightly higher plasma ascorbic acid concentrations than did oral supplementation of the vitamin (three of nine comparisons, P < 0.05). MacLeod et al. (11, 12) reported that oral supplementation of 80 g of ascorbyl-2-polyphosphate or 20 g of vitamin C equivalent for 31 d to dairy cattle significantly increased plasma ascorbic acid concentrations over those of control cows. Results from the present study suggested that the supplementation of dairy cows with 40 g of vitamin C equivalent for 5 d appeared to increase plasma ascorbic acid concentrations, and a peak concentration was reached by 30 h. Comparison between oral and abomasal supplementation supports the notion that unprotected vitamin C is partially destroyed in the rumen. Cows that received VCEC appeared to have higher plasma ascorbic acid concentrations than did cows that received VCFP. The mean plasma ascorbic acid concentration was higher in the group that received VCEC ( P < 0.05) than in the other two groups at 24 h after ascorbic
TECHNICAL NOTE: DISTRIBUTION OF VITAMIN C IN COWS
acid supplementation ceased. Hill et al. ( 7 ) reported an increase in plasma ascorbic acid after daily oral administration of 20 g of VCEC to feedlot heifers, but not when growing steers were dosed with 20 g of VCEC. They proposed additional research to define ruminal degradation and absorption of VCEC. The findings presented here, together with results from the literature, suggest that more complete knowledge is needed about the physiological factors ( 1 5 ) which control the metabolism of VCEC in cattle. REFERENCES 1 Batra, T. R., M. Hidiroglou, and M. W. Smith. 1992. Effect of vitamin E on incidence of mastitis in dairy cattle. Can. J. Anim. Sci. 72:287–297. 2 Behrens, W. A., and R. Madere. 1987. A highly sensitive high performance liquid chromatography method for the estimation of ascorbic acid and dehydro-ascorbic acid in tissues, biological fluids and food. Anal. Biochem. 165:102–107. 3 Bendich, A. 1993. Physiological role of antioxidants in the immune system. J. Dairy Sci. 76:2789–2794. 4 Cappa, C. 1958. Le metabolisme de la vitamin C chez les ruminants. Riv. Zootech. 31:299–308.
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5 Dougherty, R. W. 1981. Experimental Surgery in Farm Animals. Iowa State Univ. Press, Ames. 6 Hemingway, D. C. 1991. Vitamin C in the prevention of neonatal calf diarrhea. Can. Vet. J. 32:184. 7 Hill, G. W., S. N. Williams, M. Frigg, S. E. Williams and B. G. Mullinix. 1993. Plasma ascorbic acid response in ruminants to an orally administrated coated ascorbic acid product. J. Anim. Sci. 71(Suppl. 1):233.(Abstr.). 8 Horning, D. 1975. Metabolism of ascorbic acid. World Rev. Nutr. Diet. 23:225–258. 9 Itze, L. 1984. Ascorbic acid metabolism in ruminants. Pages 120–130 in Ascorbic Acid in Domestic Animals. I. Wegger, ed. Royal Danish Agric. Soc., Copenhagen, Denmark. 10 Liu, L., K. K. Scheffer, and D. M. Shuefea. 1994. Jugular infusion of vitamin C and color stability of beef. J. Anim. Sci. 72(Suppl. 1):372. (Abstr.). 11 MacLeod, D. D., X. Zhang, J. J. Kennelly, and L. Ozimek. 1996. Ascorbyl-2-polyphosphate as a source of ascorbic acid for dairy cattle. J. Dairy Sci. 79(Suppl. 1):233.(Abstr.) 12 Macleod, D. D., L. Ozimed, and J. J. Kennelly. 1996. Supplemental vitamin C may enhance immune function in dairy cows. Adv. Dairy Technol. 8:227–235. 13 SAS User’s Guide: Statistics, Version 5. 1985. SAS Inst., Inc., Cary, NC. 14 Scott, D. W. 1981. Vitamin C-responsive dermatoses in calves. Bovine Pract. 2:22–27. 15 Toutain, P. L., D. Bechu, and M. Hidiroglou. 1997. Ascorbic acid disposition kinetics in the plasma and tissues of calves. Am. J. Physiol. 273:R1585–1597.
Journal of Dairy Science Vol. 82, No. 8, 1999
ABSTRACT The purpose of this study was to compare the effect of multiple oral supplementation of two forms of vitamin C on plasma ascorbic acid concentrations in dairy cows. Vitamin C was administrated at the rate of 40 g/d during a 5-d period. The supplements were vitamin C in the form of a fine powder and vitamin C coated with ethyl cellulose. In addition, another group of cows provided with vitamin C in the form of fine powder through an abomasal fistula was enrolled in the study. The effect of vitamin C supplementation was assessed by the change in plasma ascorbic acid concentrations during a 5-d period of vitamin C supplementation. A tendency existed for cows that were dosed with vitamin C in the abomasum to have higher ascorbic acid than cows supplemented orally. In the abomasum-dosed cows, plasma ascorbic acid value varied between 3.6 and 4.4 mg/ml; the maximum ascorbic acid content was 4.4 mg/ml at 54 h. Cows dosed orally with vitamin C coated with ethyl cellulose had higher plasma ascorbic acid concentrations than did cows that received vitamin C in the form of fine powder. In the former group, the maximum ascorbic acid concentration occurred at 30 h after dosing ( 5 mg/ml). ( Key words: cows, vitamin C, plasma) Abbreviation key: VCEC = vitamin C coated with ethyl cellulose, VCFP = vitamin C fine powder. INTRODUCTION L-Ascorbic acid is produced in almost all mammals except humans and guinea pigs ( 8 ) . Vitamin C is a known antioxidant that protects the structural integrity of the cells of the immune system ( 3 ) . Young ruminants may need dietary ascorbic acid, because skin lesions, similar to those that are common with scurvy, have been reported in calves fed a diet with a low vitamin C content (9, 14). The observations of
Received November 4, 1998. Accepted April 5, 1999. 1999 J Dairy Sci 82:1831–1833
Hemingway ( 6 ) indicate that vitamin C may be important in the prevention of scours in calves. Cappa ( 4 ) reported that adult cattle are prone to ascorbic acid deficiency when ascorbic acid synthesis is impaired because exogenous supplies of this vitamin are rapidly destroyed by ruminal microflora. Therefore, a coated form of vitamin C with known stability was tested as a source of vitamin C supplementation for ruminants. The objective of this study was to compare plasma concentration of ascorbic acid after two forms of vitamin C were orally dosed to dairy cows. In addition, the effect of oral or intraabomasal supplementation of vitamin C on plasma ascorbic acid concentrations was compared. MATERIALS AND METHODS Six Holstein cows were used in the experiment. Four cows had ruminal fistulas, and two had abomasal fistulas ( 5 ) . Cows received a mixed diet consisting of 88% forage (70% corn silage, 20% haylage, and 10% hay) and 12% concentrate (DM basis). Cows that produced more than 7 kg of milk/d received additional concentrate in the form of pellets at the rate of 1 kg for every 3 kg of milk over 7 kg ( 1 ) . Cows in this study were cared for under guidelines comparable to those developed by the Canadian Council of Animal Care. Multiple oral supplements of two forms of vitamin C were administered at the rate of 40 g of vitamin C equivalent/d during a 5-d supplementation period. Blood samples were taken twice daily on d 0, 1, 2, and 3 during the morning and afternoon and once daily on d 4, 5, and 6. The vitamin formulations were provided by Hoffmann-La Roche, Inc. (Mississauga, ON, Canada) and consisted of 1 ) vitamin C fine powder ( VCFP) and 2 ) vitamin C coated with ethyl cellulose ( VCEC) . Two cows were assigned to each group. The VCFP is a fine white or slightly yellow odorless powder with a tart taste. The product contains a minimum of 99% ascorbic acid and is stable in dry form. The VCEC is a free-flowing powder that contains a minimum of 97.5% ascorbic acid. The VCFP or VCEC was dissolved in 200 ml of water and was ad-
1831
1832
HIDIROGLOU TABLE 1. Means and standard errors of plasma ascorbic acid concentration ( mg/ml) following administration of VCEC (vitamin C coated with ethyl cellulose) into the rumen or VCFP (vitamin C fine powder) into the abomasum or into the rumen. All treatments consisted of 40 g/d of vitamin C for 5 d. VCEC Rumen
Hours after dosing
Mean
0 6 24 30 48 54 72 78 96 120 1441
3.45x 3.65a,x 4.57a,y 5.00a,y 4.45a,y 4.67a,y 4.50a,y 4.79a,y 4.68a,y 4.89a,y 4.83a,y
a,bMeans x,yMeans
VCFP Abomasum SE
Mean
0.12 0.29 0.27 0.46 0.41 0.46 0.35 0.21 0.10 0.19 0.04
3.38x 3.56a,x 3.75b,y 4.35b,y 4.41a,y 4.33ab,y 4.40a,y 4.34ab,y 4.12b,y 4.21b,y 4.12b,y
VCFP Rumen SE
Mean
SE
0.14 0.20 0.07 0.14 0.30 0.64 0.05 0.16 0.05 0.01 0.03
3.11x 2.98b,x 3.08b,x 3.73b,y 3.84b,y 4.10b,y 3.87b,y 4.00b,y 3.90b,y 3.73b,y 3.60b,y
0.56 0.56 0.62 0.43 0.42 0.66 0.43 0.62 0.67 0.41 0.39
in the same row with different superscripts are different ( P < 0.05). in the same column with different superscripts are different from initial values at 0 h ( P <
0.05). 124 h after the last dosing.
ministered daily for 5 d either orally or abomasally every morning. Blood samples were taken from the jugular vein immediately before vitamin C dosing. The samples were centrifuged, and plasma was separated. Each sample was immediately mixed with 2.55% metaphosphoric acid in the ratio of 1:2 (vol/ vol). The sample mixtures were frozen at –70°C until analyzed fewer than 3 d later. The ascorbic acid concentrations in samples were measured by HPLC using an electrochemical detector ( 2 ) . The statistical analysis was conducted using repeated measures of option of the general linear model procedure of SAS (13). The model included the effects of treatment, time, and the interaction between treatment and time. Duncan multiple range test was used to test differences between means, once a significant effect of treatments was indicated by ANOVA. Significance was determined at P < 0.05. RESULTS AND DISCUSSION Plasma ascorbic acid concentrations before supplementation varied between 3.2 and 3.5 mg/ml. The effects of treatment and time were significant, while the interaction between treatment and time was not significant. The plasma ascorbic acid concentrations in cows that received VCFP orally or abomasally and the concentrations in cows that received VCEC orally are shown in Table 1. Supplementation of vitamin C increased plasma ascorbic acid concentrations in all three groups. Cows Journal of Dairy Science Vol. 82, No. 8, 1999
that received VCEC had higher plasma ascorbic acid concentrations than did cows that received VCFP either orally (all time points) or abomasally (four of nine comparisons) ( P < 0.05). According to Liu et al. (10), ascorbic acid is metabolized very rapidly in cattle. Those researchers reported that following intravenous infusion of 300 g of sodium ascorbate to cattle, the plasma ascorbic acid concentration was highest 10 min after completion of the jugular infusion and returned to the basal level by 5 h postinfusion. Vitamin C administration directly into the abomasum resulted in slightly higher plasma ascorbic acid concentrations than did oral supplementation of the vitamin (three of nine comparisons, P < 0.05). MacLeod et al. (11, 12) reported that oral supplementation of 80 g of ascorbyl-2-polyphosphate or 20 g of vitamin C equivalent for 31 d to dairy cattle significantly increased plasma ascorbic acid concentrations over those of control cows. Results from the present study suggested that the supplementation of dairy cows with 40 g of vitamin C equivalent for 5 d appeared to increase plasma ascorbic acid concentrations, and a peak concentration was reached by 30 h. Comparison between oral and abomasal supplementation supports the notion that unprotected vitamin C is partially destroyed in the rumen. Cows that received VCEC appeared to have higher plasma ascorbic acid concentrations than did cows that received VCFP. The mean plasma ascorbic acid concentration was higher in the group that received VCEC ( P < 0.05) than in the other two groups at 24 h after ascorbic
TECHNICAL NOTE: DISTRIBUTION OF VITAMIN C IN COWS
acid supplementation ceased. Hill et al. ( 7 ) reported an increase in plasma ascorbic acid after daily oral administration of 20 g of VCEC to feedlot heifers, but not when growing steers were dosed with 20 g of VCEC. They proposed additional research to define ruminal degradation and absorption of VCEC. The findings presented here, together with results from the literature, suggest that more complete knowledge is needed about the physiological factors ( 1 5 ) which control the metabolism of VCEC in cattle. REFERENCES 1 Batra, T. R., M. Hidiroglou, and M. W. Smith. 1992. Effect of vitamin E on incidence of mastitis in dairy cattle. Can. J. Anim. Sci. 72:287–297. 2 Behrens, W. A., and R. Madere. 1987. A highly sensitive high performance liquid chromatography method for the estimation of ascorbic acid and dehydro-ascorbic acid in tissues, biological fluids and food. Anal. Biochem. 165:102–107. 3 Bendich, A. 1993. Physiological role of antioxidants in the immune system. J. Dairy Sci. 76:2789–2794. 4 Cappa, C. 1958. Le metabolisme de la vitamin C chez les ruminants. Riv. Zootech. 31:299–308.
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5 Dougherty, R. W. 1981. Experimental Surgery in Farm Animals. Iowa State Univ. Press, Ames. 6 Hemingway, D. C. 1991. Vitamin C in the prevention of neonatal calf diarrhea. Can. Vet. J. 32:184. 7 Hill, G. W., S. N. Williams, M. Frigg, S. E. Williams and B. G. Mullinix. 1993. Plasma ascorbic acid response in ruminants to an orally administrated coated ascorbic acid product. J. Anim. Sci. 71(Suppl. 1):233.(Abstr.). 8 Horning, D. 1975. Metabolism of ascorbic acid. World Rev. Nutr. Diet. 23:225–258. 9 Itze, L. 1984. Ascorbic acid metabolism in ruminants. Pages 120–130 in Ascorbic Acid in Domestic Animals. I. Wegger, ed. Royal Danish Agric. Soc., Copenhagen, Denmark. 10 Liu, L., K. K. Scheffer, and D. M. Shuefea. 1994. Jugular infusion of vitamin C and color stability of beef. J. Anim. Sci. 72(Suppl. 1):372. (Abstr.). 11 MacLeod, D. D., X. Zhang, J. J. Kennelly, and L. Ozimek. 1996. Ascorbyl-2-polyphosphate as a source of ascorbic acid for dairy cattle. J. Dairy Sci. 79(Suppl. 1):233.(Abstr.) 12 Macleod, D. D., L. Ozimed, and J. J. Kennelly. 1996. Supplemental vitamin C may enhance immune function in dairy cows. Adv. Dairy Technol. 8:227–235. 13 SAS User’s Guide: Statistics, Version 5. 1985. SAS Inst., Inc., Cary, NC. 14 Scott, D. W. 1981. Vitamin C-responsive dermatoses in calves. Bovine Pract. 2:22–27. 15 Toutain, P. L., D. Bechu, and M. Hidiroglou. 1997. Ascorbic acid disposition kinetics in the plasma and tissues of calves. Am. J. Physiol. 273:R1585–1597.
Journal of Dairy Science Vol. 82, No. 8, 1999