a-Tocopherol Concentrations in Serum and Tissues of Sheep Fed Different Sources of Vitamin E1,2,3 L. Ochoa*, L. R. McDowell", S. N. Williams*, N. Wilkinson*, J. Bouchert, and E. L. Lentz*
ABSTRACT: Thirty-five crossbred wethers were used to determine the concentrations of a-tocopherol in serum and tissues after oral supplementation of six different vitamin E product forms. Five wethers were assigned to each of the following treatments: 1) control, no supplemental vitamin E (C), 2) emulsifiable DL-a-tocopheryl acetate-dry (Rovimix E-50% SD), 3) nonemulsifiable DL-atocopheryl acetate-dry (Rovimix E-50% Ads), 4) emulsifiable DL-a-tocopheryl acetate-liquid (Rovimix E-40% Dispersible Liquid Concentrate [DLCI); 5) emulsifiable DL-a-tocopherol-liquid (HoffmannLa Roche, E-40% DLC alcohol), 6) micellized DL-atocopheryl acetate-liquid (Bioglan, Inc., E-20%1; and 7) micellized DL-a-tocopherol-liquid (Bioglan, Inc., E-20%). Animals were supplemented daily with 1,000IU of their respective vitamin E sources for 56 d. Blood samples were collected daily from d 0 to 7 and weekly until d 56. Animals were subsequently killed by exsanguination after stunning and eight different tissues were collected for a-tocopherol analysis. There were effects of day, treatment, and day x treatment interaction on serum a-tocopherol. All supplemented groups were
higher in serum a-tocopherol concentration than were the C wethers. The emulsifiable vitamin E alcohol liquid product form (Treatment 5) yielded higher ( P < .01) serum a-tocopherol concentration than the emulsifiable acetate liquid product (Treatment 4). Sheep on Treatment 5 reached maximum concentration on d 1, sheep on Treatment 6 on d 2, and the sheep on the remaining Treatments by d 3. Blood sera a-tocopherol concentrations stabilized by d 6 in all supplemented groups. Tissue a-tocopherol concentrations for all treatments were higher than those for C, but there were no differences in tissue a-tocopherol concentration among supplemented groups, except that Treatment 2 produced higher ( P e .05)levels than did Treatment 3 in heart. Across all treatments, the highest a-tocopherol concentrations were noted in liver and pancreas and the lowest were found in kidney and gluteus medius. In conclusion, all forms of vitamin E tested resulted in relatively similar serum and tissue concentrations of atocopherol and would be considered suitable supplemental forms of the vitamin.
Key Words: Vitamin E, Diet, Sheep, Biological Activity
J. Anim. Sci. 1992. 70:2568-2573
Introduction Bioavailability of dietary DL-a-tocopherol, DL-atocopheryl acetate, D-a-tocopherol, and D-atocopheryl acetate has been tested in sheep and 'Florida Agric. Exp. Sta. Series No. 00767. 2This research was supported in part by the USDA under CSRC special grant No. 86-CRSR-2-2843 managed by the Caribbean Advisory Group (CBAG). 3Appreciation is extended to R. Merkel, R. Pastrana, E. Espinoza, N. Hidiroglou, A. Prabowo, M. Sandoval, and E. Pott for blood and tissue sampling. Received October 16, 1991. Accepted March 26, 1992.
cattle (Hidiroglou et al., 1987, 1988). These authors concluded that after ingestion of equivalent vitamin E activity (IU) of the seurces tested, D-atocopherol was more potent than D-a-tocopheryl acetate or the L-isomeric forms of vitamin E based on blood a-tocopherol concentrations. However, differences were not observed among forms of vitamin E when assessing overall a-tocopherol tissue concentrations. Additionally, Hidiroglou et al. (19891 reported differences in several blood traits used to access bioavailability in ruminants orally administered DL-a-tocopherol or DL-atocopheryl acetate. However, vitamin E sources were administered on a n equal-weight basis and
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Departments of *Animal Science and +Statistics, University of Florida, Gainesville 3261 1 and 'Hoffmann-La Roche Inc., Nutley, N J 071 10
VITAMIN E IN SERUM AND TISSUES OF SHEEP
Table 1. Basal dieta administered to sheep Ingredient
Percentage 58.75
21.00 12.00 3.00 3.00
1.oo 1.oo 25
*Diet contained 28.8 IU of vitamin E/kg of feed. bProvided, per kilogram of diet: 2.1 g of NaCl, .22 mg of I, ,088 mg of Co, 9 mg of Fe, .725mg of Cu, 2.625 mg of Mn, and 8.75 mg of Zn. CProvidedper kilogram of diet: 5,000 IU of vitamin A and 500 IU of vitamin D,.
not on an equal-activity basis. Research reports are in disagreement as to the bioavailability of various vitamin E sources for ruminants. Hidiroglou and McDowell (19871 reported that the D-form had a higher bioavailability in sheep than DL-a-tocopherol after i.m. injection. The objective of this study was to compare tissue uptake and retention of six sources of vitamin E activity.
Materials and Methods Animals. Thirtyfive yearling crossbred wethers weighing a n average of 36.4 kg were adapted for a 30-d period to the consumption of a basal diet (Table 1) with water available ad libitum. The diet was calculated to be adequate in protein, energy, vitamins, and minerals for this class of animal. The protocol for animal care had been approved by the University Animal Use Committee. Sheep were placed in individual pens (1.4 m2) for a lo-d adjustment period before beginning the 56-d experiment. The basal nonsupplemented diet, which averaged 28.6 IU/kg of vitamin E, was offered at 1 kg/d during the experiment. Lambs were fed .5 kg of feed twice daily (0800 and 1700). Lambs were weighed on d 0, 28, and 56 of the study. Groups of five sheep each were randomly assigned to one of seven treatments as follows: 1) control, no supplemental vitamin E (0,2) emulsifiable DL-a-tocopheryl acetate-dry (Rovimix E-50% SD), 3) nonemulsifiable DL-a-tocopheryl acetatedry (Rovimix E-50% Ads), 4) emulsifiable DL-atocopheryl acetate-liquid (Rovimix E-40% Dispersible Liquid Concentrate [DLCI), 5) emulsifiable DLa-tocopherol-liquid (Hoffmann-LaRoche, E- 40% DLC alcohol), 6) micellized DL-a-tocopheryl acetate-liquid (Bioglan, Inc., E-20% and 7) micellized DL-a-tocopherol-liquid (Bioglan, Inc., E-20%). All vitamin E sources were supplemented to diets to provide 1,000 IU of vitamin E activity per kilogram.
Blood samples were obtained by jugular venipuncture before the morning feeding daily from d 0 to 7 and weekly thereafter until d 56 of the experimental period and immediately centrifuged at 700 x g for 15 min. Serum was separated, frozen, and stored a t -2OOC. All animals were killed by exsanguination on d 56. Portions of tissues were collected in plastic bags immediately after slaughter, placed in an ice chest, frozen, and stored at -2OOC. Tissues collected were liver, kidney, heart, pancreas, adrenal gland, adipose, longissimus muscle, and gluteus medius. Analytical Methods. Quantification of a-tocopherol in tissues, serum, and feed samples was performed by HPLC using a fluorescent detector (Faustman et al., 19891, after tissue homogenization, saponification, and iso-octane double-extraction. Identification and quantification of a-tocopherol were accomplished by comparison of retention time as well as peak areas with atocopherol standards. A Perkin-Elmer 650-150 fluorescent spectrophotometer (Perkin-Elmer, Norwalk, CT), equipped with microflow cell unit, was used for quantification. Fluorescence was measured at a n excitation wavelength of 298 nm and an emission wavelength of 325 nm. The column was a Chromegasphere SI 60, 5 p,15 cm x 4.6 mm i.d. (E.S. Industries, Marlton, NJ). The mobile phase consisted of 4 % tetrahydrofuran in isooctane, with a flow rate of 1.2 to 1.3 mL/min. Statistical Methods. A repeated-measurements analysis, including treatment, day, animal (treatment) and the treatment x day interaction, was performed on logarithms of serum values. Logarithms were taken to stabilize variances. Animals within treatment term was used as a n error term to test the treatment difference. Seven specific contrasts of interest were made among treatment means of serum a-tocopherol concentration. Box’s (1954) and Geisser and Greenhouse’s (1958) adjustments on the F-tests for day and treatment x day effects were made to account for the correlation among the repeated measures on the same individuals. One-way analysis of variance including treatment was calculated for tissue a-tocopherol concentration. A Ryan-Eniot-GabrielWelsch (REGWQ option, SAS GLM procedure) multiple range test of treatment difference in tissue a-tocopherol concentration was completed GAS, 1988).
Results Overall means and SE for ADG and feed conversion were .12 f .003 and 8.86 f .342 kg, respectively. There were no differences ( P > .05) among treatments for feed conversion or weight
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Ground yellow corn Cottonseed hulls Soybean meal (44% CP) Alfalfa meal (14% CP) Corn oil Trace minerals and saltb Ground limestone Vitamins A and DC
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OCHOA ET AL.
Table 2. Vitamin E content of diets Supplemental vitamin E, IU/kg
Treatment*
~
d l
-
26
1,000 1.000 1,000
T4
T5 T6 T7
d 14
d 28
d 42
31 1,045 1,025 1,060 1,110 1,050 1,010
28 1,085 1,080 1,125
33 1,070 1,085 1,055 1,070 1,035 910
d 56
~~~~
1,045 1,045
1,085 1,115 1,060 945
1,000
1,000 1,000
965 1,065 960
25
1,085 1,035 1,100 950
1,070 955
&C = control, no supplemental vitamin E; Tz = emulsifiable DL-atocopheryl acetate-dry; T3 = nonemulsifiable DL-a-tocopheryl acetate-dry; T4 = emulsifiable DL-a-tocopheryl acetate-liquid;T5 = emulsifiable DL-atocopherol-liquid;T6 = micellized DL-atocopheryl acetate-liquid; T7 = mcellized DL-a-tocopherol-liquid. bRepresents the average of duplicate vitamin E analyses.
gains. Because animals were fed restrictively and were near maturity, weight gains were not a pertinent criterion. Vitamin E concentration in feed was relatively stable throughout the experiment. All supplemented animals had 1,000 IU/kg of vitamin E in feed, and the unsupplemented control analyzed every 14 d averaged 28.6 IU/kg. The analyzed vitamin E content of feed samples was similar to the calculated content (Table 21. There were day (P c .OOOl), treatment (P c .00011, and day x treatment ( P < .00011 interaction effects on a-tocopherol serum concentration. Treatment means and specific contrasts of interest were used to compare serum a-tocopherol concentration among treatments (Table 31. Animals from all supplemented treatments produced higher ( P e .011 serum a-tocopherol concentrations than did controls. There was no difference ( P > ,051 in serum a-tocopherol concentration for sheep fed between emulsifiable and nonemulsifiable (TreatTable 3. Treatment means and contrasts in a-tocopherol concentration in serum a-Tocopherol, mg/100 mL
Treatmenta
nb
C T2 T3 T4 T5 T6 T7 SE
75 75 75 75
.10 .36 .32 .32
75 75 75
.46
.35 .40
.006
&C = control, no supplemental vitamin E; T2 = emulsifiable DL-a-tocopheryl acetate-dry; T3 = nonemulsifiable DL-a tocopheryl acetate dry; T4 = emulsifiable DL-a-tocopheryl acetate-liquid; T5 = emulsifiable DL-a-tocopherol-liquid; T6 = micellized DL-a-tocopheryl acetate-liquid; T7 = micellized DL-atocopherol-liquid. bThere were five animals per treatment and 15 observations per animal.
ment 2 vs Treatment 31 DL-a-tocopheryl acetate dry product forms, and no difference ( P > .05) was found between dry and liquid emulsifiable (Treatment 2 vs Treatment 41 DL-a-tocopheryl acetate product forms. There was no difference ( P > .051 in serum a-tocopherol concentration between emulsifiable and micellized (Treatment 4 vs Treatment 61 DL-a-tocopheryl acetate products, nor was there a difference (P > ,051 between emulsifiable and micellized (Treatment 5 vs Treatment 7) DL-atocopherol products. Micellization of DL-atocoperol or DL-a-tocopheryl acetate in this study did not increase a-tocopherol in serum concentration over that of other product forms tested. Sheep fed the vitamin E alcohol product had a higher ( P c .011 serum a-tocopherol concentration than did sheep fed the acetate when it was supplied in the emulsifiable form (Treatment 4 vs Treatment 51. In micellized form, there was no difference (P > .051 in serum a-tocopherol between alcohol and acetate products (Treatment 6 vs Treatment 71. Overall, serum a-tocopherol concentration across all treatments reached peak concentration on d 3 of the experiment (Figure 11. Animals from Treatment 5 reached peak a-tocopherol concentration on d 1, those from Treatment 6 on d 2, and those from the remaining treatments by d 3 (Figure 11. Serum a-tocopherol concentration had the tendency to stabilize by d 6 of the trial, whereas the control group did not vary ( P > ,051 throughout the trial. Tissue a-tocopherol concentrations in animals for all treatments were higher ( P e .051 than those of C except for the pancreas and heart. For sheep heart tissue, a-tocopherol concentration of the C group was not different from that of Treatment 3; all other treatments were higher than C (P c .051. None of the treatment groups was different from C for a-tocopherol in pancreas tissue. The tissue with the highest a-tocopherol concentration was liver, followed by pancreas (Figure 2).
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C T2 T3
Vitamin E content, IU/kgb
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VITAMIN E IN SERUM AND TISSUES OF SHEEP
Discussion
L
-
0 0
.6
r-
L Q
0 0
.I
0
bts
o 0
1
2
3
4
5
6
7
Days on trial Figure 1. Effect of time and supplemental vitamin E product form on sheep blood serum a-tocopherol concentration over 7 d. Treatment designation: C (A) = control, no supplemental vitamin E; T2 ( 0 ) = emulsifiable DL-a-tocopherylacetate-dry; T3 ( + ) = nonemulsifiable DL-a-tocopheryl acetate-dry; T4 ( * ) = emulsifiable DL-atocopheryl acetate-liquid; T5 (0) = emulsifiable DL-a-tocopherol-liquid;T6 (x) = micellized DL-a-tocopheryl acetate-liquid; T7 (0) = micellized DL-a-tocopherol-liquid.The SE for the 7 and 56 d were .010 and .006, respectively.
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with the highest a-tocopherol concentration in serum (Figure 1). The time required for serum a-tocopherol to It may be suggested that after blood was reach its peak level depends on vitamin source saturated with the vitamin at d 3, a-tocopherol and method of administration. Orally adminisstarted to be deposited in the tissues, which might tered, emulsifiable DL-a-tocopherol liquid (Treat- explain the decrease in concentration of a-tocophment 5) and micellized DL-a-tocopherol(Treatment erol in serum after d 3 (Figure 1). It was observed 7)product forms reached peak concentration on d that the tissue with the highest a-tocopherol 1 and 2, respectively. These data are in agreement concentration was liver, followed by pancreas. with those of Hidiroglou et al. (1989) obtained with Previous results are inconsistent in regard to cattle but are in disagreement with those of which tissue has the highest a-tocopherol concenHidiroglou et al. (19871 obtained with sheep, in tration. Hidiroglou et al., (19871 reported that in which serum a-tocopherol reached maximum peak sheep the tissues with the highest a-tocopherol on d 11. After a single i.m. injection of DL-a- concentration after oral administration followed tocopherol, time of serum maximum peak a- the order pancreas, liver, and spleen. However, tocopherol concentration was 6 d after administraHidiroglou and McDowell (1987) reported that tion (Hidiroglou and McDowell, 1987). When adadrenal tissue was highest, followed by adipose, ministered intraperitoneally, vitamin E peak level pancreas, and liver after a n intramuscular single was observed at d 1 (Hidiroglou et al., 1990). The dose of the vitamin. Hidiroglou et al., (19901 biological activity of a-tocopherol administered to observed that 28 d after i.p. administration of DLsheep was dependent on the method of administraa-tocopherol, a-tocopherol concentration was tion, following the order intravenous, muscular, greatest in adrenal gland, followed by pancreas, and oral (Hidiroglou and Karpinski, 19871, liver, and lung. In this experiment, the treated The data from the current study indicate a sheep exhibited a large variation in a-tocopherol treatment x day interaction for serum a-tocopherol concentration in kidney, adrenal gland, heart, and pancreas. This variation is most likely the reason concentration. However, emulsifiable a-tocopherol why treatment differences could not be detected in liquid (Treatment 5 ) was the highest at d 1 of the pancreatic a-tocopherol Concentration. experiment, and remained in the treatment groups
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OCHOA ET AL.
8
0 0 7
1
E) c-
2
+
c
a, 0
c
0
0 0 L
a,
c
a. 0 0
F 8
Serum
Liver
Kidney
Adrenal
Adipose
Heart
"L. Dors."
"G.Medi." Pancreas
TREATMENTS Figure 2. Effect of supplemental vitamin E product form on sheep tissue a-tocopherol concentration. Within a tissue, treatments are shown from left to right in this order: control, no supplemental vitamin E; Treatment 2, emulsifiable DL-a-tocopheryl acetate-dry; Treatment 3, nonemulsifiable DL-a-tocopherylacetate-dry; Treatment 4, emulsifiable DL-a-tocopheryl acetate-liquid; Treatment 5, emulsifiable DL-a-tocopherol-liquid; Treatment 6, micellized DL-a-tocopheryl acetate-liquid; and Treatment 7, micellized DL-a-tocopherol-liquid.The SE for each tissue was as follows: serum, .006; liver, .442; kidney, .51; adrenal, .196; adipose, .184; heart, .121; longissimus muscle, .064; Gluteus medius, .048; and pancreas, 248.
Hidiroglou et al. (1987) reported that sheep supplemented with 400 IU of vitamin E activity. animal-l.d-l for 28 d as DL-a-tocopherol had higher (P e .05) serum a-tocopherol concentrations than those supplemented with DL-a-tocopheryl acetate, but no difference (P c .05)was found in tissue a-tocopherol concentration. Furthermore, the same results were obtained by Hidiroglou et al. (1988) in cattle after a daily supplementation of 1,000 IU of vitamin E activity per animal for a 28-d period. After a single oral dose of 50 mg of vitamin E/kg of BW was given to cattle and 100 mg to sheep, DL-a-tocopherol had greater (P c .05) potency than its ester form (Hidiroglou et al., 19891. However, sources of vitamin E were administered on a n equal-weight basis rather than a n equalactivity basis (i.e., 10% more vitamin E activity administered with the alcohol source). Results of the current investigation would indicate that there was no difference in vitamin E availability from various vitamin E products tested based on tissue a-tocopherol accretion when administered on a n equal-activity basis; however, there was a tendency of the vitamin E alcohol product forms to maintain higher blood serum a-tocopherol concentration than vitamin E acetate product forms. No
difference in serum a-tocopherol concentration was seen among the supplemented groups by the last 2 wk of the trial.
Implications All vitamin E sources tested resulted in relatively similar tissue a-tocopherol concentrations, but there was a tendency for vitamin E alcohol forms to promote greater serum a-tocopherol concentrations than did acetate products. In general, all forms would serve as suitable supplemental sources of vitamin E.
Literature Cited Box, G.E.P. 1954. Some theorems on quadratic forms applied in the study of analysis of variance problems. 11. Effects of inequality of variance and correlation between errors in the two-way classification. Ann. Math. Stat. 25:484. Faustman, C., R. G. Cassens, D. M. Schaefer, D. R. Buege, S. N. Williams, and K. K. Scheller. 1989. Improvement of pigment and lipid stability in Holstein steer beef by dietary supplementation with vitamin E. J. Food Sci. 54:4. Geisser, S., and S. W. Greenhouse. 1958. An extension of Box's results on the use of the F distribution in multivariate
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0 .c.
VITAMIN E IN SERUM AND TISSUES OF SHEEP
Hidiroglou, N., and L. R. McDowell. 1987. Plasma and tissue level of vitamin E in sheep following intramuscular administration in a n oil carrier. Int. J. Vitam. Nutr. Res. 57:261. Hidiroglou, N., L. R. McDowell, and 0. Balbuena. 1989. Plasma tocopherol in sheep and cattle after ingesting free or acetylated tocopherol. J. Dairy Sci. 72:1793. Hidiroglou, N., L. R. McDowell, and R. Pastrana. 1987. Bioavailability of various vitamin E compounds in sheep. Int. J. Vitam. Nutr. Res. 58:189. SAS. 1988. SAS User’s Guide: Statistics. SAS Inst. Inc., Cary, NC.
Downloaded from https://academic.oup.com/jas/article-abstract/70/8/2568/4632065 by Washington University in St. Louis user on 20 September 2018
analysis. Ann. Math. Stat. 28:885. Hidiroglou, M., and K. Karpinski. 1987. Vitamin E kinetics in sheep. Br. J. Nutr. 58:113. Hidiroglou, N., G. Butler, and L. R. McDowell. 1990. Plasma and tissue vitamin E concentrations in sheep after administration of a single intraperitoneal dose of dl-a-tocopherol. J. Anim. Sci. 88:782. Hidiroglou, N., L. F. Laflamme, and L. R. McDowell. 1988. Blood plasma and tissue concentrations of vitamin E in beef cattle as influenced by supplementation of various tocopherol compounds. J. Anim. Sci. 86:3227.
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ABSTRACT: Thirty-five crossbred wethers were used to determine the concentrations of a-tocopherol in serum and tissues after oral supplementation of six different vitamin E product forms. Five wethers were assigned to each of the following treatments: 1) control, no supplemental vitamin E (C), 2) emulsifiable DL-a-tocopheryl acetate-dry (Rovimix E-50% SD), 3) nonemulsifiable DL-atocopheryl acetate-dry (Rovimix E-50% Ads), 4) emulsifiable DL-a-tocopheryl acetate-liquid (Rovimix E-40% Dispersible Liquid Concentrate [DLCI); 5) emulsifiable DL-a-tocopherol-liquid (HoffmannLa Roche, E-40% DLC alcohol), 6) micellized DL-atocopheryl acetate-liquid (Bioglan, Inc., E-20%1; and 7) micellized DL-a-tocopherol-liquid (Bioglan, Inc., E-20%). Animals were supplemented daily with 1,000IU of their respective vitamin E sources for 56 d. Blood samples were collected daily from d 0 to 7 and weekly until d 56. Animals were subsequently killed by exsanguination after stunning and eight different tissues were collected for a-tocopherol analysis. There were effects of day, treatment, and day x treatment interaction on serum a-tocopherol. All supplemented groups were
higher in serum a-tocopherol concentration than were the C wethers. The emulsifiable vitamin E alcohol liquid product form (Treatment 5) yielded higher ( P < .01) serum a-tocopherol concentration than the emulsifiable acetate liquid product (Treatment 4). Sheep on Treatment 5 reached maximum concentration on d 1, sheep on Treatment 6 on d 2, and the sheep on the remaining Treatments by d 3. Blood sera a-tocopherol concentrations stabilized by d 6 in all supplemented groups. Tissue a-tocopherol concentrations for all treatments were higher than those for C, but there were no differences in tissue a-tocopherol concentration among supplemented groups, except that Treatment 2 produced higher ( P e .05)levels than did Treatment 3 in heart. Across all treatments, the highest a-tocopherol concentrations were noted in liver and pancreas and the lowest were found in kidney and gluteus medius. In conclusion, all forms of vitamin E tested resulted in relatively similar serum and tissue concentrations of atocopherol and would be considered suitable supplemental forms of the vitamin.
Key Words: Vitamin E, Diet, Sheep, Biological Activity
J. Anim. Sci. 1992. 70:2568-2573
Introduction Bioavailability of dietary DL-a-tocopherol, DL-atocopheryl acetate, D-a-tocopherol, and D-atocopheryl acetate has been tested in sheep and 'Florida Agric. Exp. Sta. Series No. 00767. 2This research was supported in part by the USDA under CSRC special grant No. 86-CRSR-2-2843 managed by the Caribbean Advisory Group (CBAG). 3Appreciation is extended to R. Merkel, R. Pastrana, E. Espinoza, N. Hidiroglou, A. Prabowo, M. Sandoval, and E. Pott for blood and tissue sampling. Received October 16, 1991. Accepted March 26, 1992.
cattle (Hidiroglou et al., 1987, 1988). These authors concluded that after ingestion of equivalent vitamin E activity (IU) of the seurces tested, D-atocopherol was more potent than D-a-tocopheryl acetate or the L-isomeric forms of vitamin E based on blood a-tocopherol concentrations. However, differences were not observed among forms of vitamin E when assessing overall a-tocopherol tissue concentrations. Additionally, Hidiroglou et al. (19891 reported differences in several blood traits used to access bioavailability in ruminants orally administered DL-a-tocopherol or DL-atocopheryl acetate. However, vitamin E sources were administered on a n equal-weight basis and
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Departments of *Animal Science and +Statistics, University of Florida, Gainesville 3261 1 and 'Hoffmann-La Roche Inc., Nutley, N J 071 10
VITAMIN E IN SERUM AND TISSUES OF SHEEP
Table 1. Basal dieta administered to sheep Ingredient
Percentage 58.75
21.00 12.00 3.00 3.00
1.oo 1.oo 25
*Diet contained 28.8 IU of vitamin E/kg of feed. bProvided, per kilogram of diet: 2.1 g of NaCl, .22 mg of I, ,088 mg of Co, 9 mg of Fe, .725mg of Cu, 2.625 mg of Mn, and 8.75 mg of Zn. CProvidedper kilogram of diet: 5,000 IU of vitamin A and 500 IU of vitamin D,.
not on an equal-activity basis. Research reports are in disagreement as to the bioavailability of various vitamin E sources for ruminants. Hidiroglou and McDowell (19871 reported that the D-form had a higher bioavailability in sheep than DL-a-tocopherol after i.m. injection. The objective of this study was to compare tissue uptake and retention of six sources of vitamin E activity.
Materials and Methods Animals. Thirtyfive yearling crossbred wethers weighing a n average of 36.4 kg were adapted for a 30-d period to the consumption of a basal diet (Table 1) with water available ad libitum. The diet was calculated to be adequate in protein, energy, vitamins, and minerals for this class of animal. The protocol for animal care had been approved by the University Animal Use Committee. Sheep were placed in individual pens (1.4 m2) for a lo-d adjustment period before beginning the 56-d experiment. The basal nonsupplemented diet, which averaged 28.6 IU/kg of vitamin E, was offered at 1 kg/d during the experiment. Lambs were fed .5 kg of feed twice daily (0800 and 1700). Lambs were weighed on d 0, 28, and 56 of the study. Groups of five sheep each were randomly assigned to one of seven treatments as follows: 1) control, no supplemental vitamin E (0,2) emulsifiable DL-a-tocopheryl acetate-dry (Rovimix E-50% SD), 3) nonemulsifiable DL-a-tocopheryl acetatedry (Rovimix E-50% Ads), 4) emulsifiable DL-atocopheryl acetate-liquid (Rovimix E-40% Dispersible Liquid Concentrate [DLCI), 5) emulsifiable DLa-tocopherol-liquid (Hoffmann-LaRoche, E- 40% DLC alcohol), 6) micellized DL-a-tocopheryl acetate-liquid (Bioglan, Inc., E-20% and 7) micellized DL-a-tocopherol-liquid (Bioglan, Inc., E-20%). All vitamin E sources were supplemented to diets to provide 1,000 IU of vitamin E activity per kilogram.
Blood samples were obtained by jugular venipuncture before the morning feeding daily from d 0 to 7 and weekly thereafter until d 56 of the experimental period and immediately centrifuged at 700 x g for 15 min. Serum was separated, frozen, and stored a t -2OOC. All animals were killed by exsanguination on d 56. Portions of tissues were collected in plastic bags immediately after slaughter, placed in an ice chest, frozen, and stored at -2OOC. Tissues collected were liver, kidney, heart, pancreas, adrenal gland, adipose, longissimus muscle, and gluteus medius. Analytical Methods. Quantification of a-tocopherol in tissues, serum, and feed samples was performed by HPLC using a fluorescent detector (Faustman et al., 19891, after tissue homogenization, saponification, and iso-octane double-extraction. Identification and quantification of a-tocopherol were accomplished by comparison of retention time as well as peak areas with atocopherol standards. A Perkin-Elmer 650-150 fluorescent spectrophotometer (Perkin-Elmer, Norwalk, CT), equipped with microflow cell unit, was used for quantification. Fluorescence was measured at a n excitation wavelength of 298 nm and an emission wavelength of 325 nm. The column was a Chromegasphere SI 60, 5 p,15 cm x 4.6 mm i.d. (E.S. Industries, Marlton, NJ). The mobile phase consisted of 4 % tetrahydrofuran in isooctane, with a flow rate of 1.2 to 1.3 mL/min. Statistical Methods. A repeated-measurements analysis, including treatment, day, animal (treatment) and the treatment x day interaction, was performed on logarithms of serum values. Logarithms were taken to stabilize variances. Animals within treatment term was used as a n error term to test the treatment difference. Seven specific contrasts of interest were made among treatment means of serum a-tocopherol concentration. Box’s (1954) and Geisser and Greenhouse’s (1958) adjustments on the F-tests for day and treatment x day effects were made to account for the correlation among the repeated measures on the same individuals. One-way analysis of variance including treatment was calculated for tissue a-tocopherol concentration. A Ryan-Eniot-GabrielWelsch (REGWQ option, SAS GLM procedure) multiple range test of treatment difference in tissue a-tocopherol concentration was completed GAS, 1988).
Results Overall means and SE for ADG and feed conversion were .12 f .003 and 8.86 f .342 kg, respectively. There were no differences ( P > .05) among treatments for feed conversion or weight
Downloaded from https://academic.oup.com/jas/article-abstract/70/8/2568/4632065 by Washington University in St. Louis user on 20 September 2018
Ground yellow corn Cottonseed hulls Soybean meal (44% CP) Alfalfa meal (14% CP) Corn oil Trace minerals and saltb Ground limestone Vitamins A and DC
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OCHOA ET AL.
Table 2. Vitamin E content of diets Supplemental vitamin E, IU/kg
Treatment*
~
d l
-
26
1,000 1.000 1,000
T4
T5 T6 T7
d 14
d 28
d 42
31 1,045 1,025 1,060 1,110 1,050 1,010
28 1,085 1,080 1,125
33 1,070 1,085 1,055 1,070 1,035 910
d 56
~~~~
1,045 1,045
1,085 1,115 1,060 945
1,000
1,000 1,000
965 1,065 960
25
1,085 1,035 1,100 950
1,070 955
&C = control, no supplemental vitamin E; Tz = emulsifiable DL-atocopheryl acetate-dry; T3 = nonemulsifiable DL-a-tocopheryl acetate-dry; T4 = emulsifiable DL-a-tocopheryl acetate-liquid;T5 = emulsifiable DL-atocopherol-liquid;T6 = micellized DL-atocopheryl acetate-liquid; T7 = mcellized DL-a-tocopherol-liquid. bRepresents the average of duplicate vitamin E analyses.
gains. Because animals were fed restrictively and were near maturity, weight gains were not a pertinent criterion. Vitamin E concentration in feed was relatively stable throughout the experiment. All supplemented animals had 1,000 IU/kg of vitamin E in feed, and the unsupplemented control analyzed every 14 d averaged 28.6 IU/kg. The analyzed vitamin E content of feed samples was similar to the calculated content (Table 21. There were day (P c .OOOl), treatment (P c .00011, and day x treatment ( P < .00011 interaction effects on a-tocopherol serum concentration. Treatment means and specific contrasts of interest were used to compare serum a-tocopherol concentration among treatments (Table 31. Animals from all supplemented treatments produced higher ( P e .011 serum a-tocopherol concentrations than did controls. There was no difference ( P > ,051 in serum a-tocopherol concentration for sheep fed between emulsifiable and nonemulsifiable (TreatTable 3. Treatment means and contrasts in a-tocopherol concentration in serum a-Tocopherol, mg/100 mL
Treatmenta
nb
C T2 T3 T4 T5 T6 T7 SE
75 75 75 75
.10 .36 .32 .32
75 75 75
.46
.35 .40
.006
&C = control, no supplemental vitamin E; T2 = emulsifiable DL-a-tocopheryl acetate-dry; T3 = nonemulsifiable DL-a tocopheryl acetate dry; T4 = emulsifiable DL-a-tocopheryl acetate-liquid; T5 = emulsifiable DL-a-tocopherol-liquid; T6 = micellized DL-a-tocopheryl acetate-liquid; T7 = micellized DL-atocopherol-liquid. bThere were five animals per treatment and 15 observations per animal.
ment 2 vs Treatment 31 DL-a-tocopheryl acetate dry product forms, and no difference ( P > .05) was found between dry and liquid emulsifiable (Treatment 2 vs Treatment 41 DL-a-tocopheryl acetate product forms. There was no difference ( P > .051 in serum a-tocopherol concentration between emulsifiable and micellized (Treatment 4 vs Treatment 61 DL-a-tocopheryl acetate products, nor was there a difference (P > ,051 between emulsifiable and micellized (Treatment 5 vs Treatment 7) DL-atocopherol products. Micellization of DL-atocoperol or DL-a-tocopheryl acetate in this study did not increase a-tocopherol in serum concentration over that of other product forms tested. Sheep fed the vitamin E alcohol product had a higher ( P c .011 serum a-tocopherol concentration than did sheep fed the acetate when it was supplied in the emulsifiable form (Treatment 4 vs Treatment 51. In micellized form, there was no difference (P > .051 in serum a-tocopherol between alcohol and acetate products (Treatment 6 vs Treatment 71. Overall, serum a-tocopherol concentration across all treatments reached peak concentration on d 3 of the experiment (Figure 11. Animals from Treatment 5 reached peak a-tocopherol concentration on d 1, those from Treatment 6 on d 2, and those from the remaining treatments by d 3 (Figure 11. Serum a-tocopherol concentration had the tendency to stabilize by d 6 of the trial, whereas the control group did not vary ( P > ,051 throughout the trial. Tissue a-tocopherol concentrations in animals for all treatments were higher ( P e .051 than those of C except for the pancreas and heart. For sheep heart tissue, a-tocopherol concentration of the C group was not different from that of Treatment 3; all other treatments were higher than C (P c .051. None of the treatment groups was different from C for a-tocopherol in pancreas tissue. The tissue with the highest a-tocopherol concentration was liver, followed by pancreas (Figure 2).
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C T2 T3
Vitamin E content, IU/kgb
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VITAMIN E IN SERUM AND TISSUES OF SHEEP
Discussion
L
-
0 0
.6
r-
L Q
0 0
.I
0
bts
o 0
1
2
3
4
5
6
7
Days on trial Figure 1. Effect of time and supplemental vitamin E product form on sheep blood serum a-tocopherol concentration over 7 d. Treatment designation: C (A) = control, no supplemental vitamin E; T2 ( 0 ) = emulsifiable DL-a-tocopherylacetate-dry; T3 ( + ) = nonemulsifiable DL-a-tocopheryl acetate-dry; T4 ( * ) = emulsifiable DL-atocopheryl acetate-liquid; T5 (0) = emulsifiable DL-a-tocopherol-liquid;T6 (x) = micellized DL-a-tocopheryl acetate-liquid; T7 (0) = micellized DL-a-tocopherol-liquid.The SE for the 7 and 56 d were .010 and .006, respectively.
Downloaded from https://academic.oup.com/jas/article-abstract/70/8/2568/4632065 by Washington University in St. Louis user on 20 September 2018
with the highest a-tocopherol concentration in serum (Figure 1). The time required for serum a-tocopherol to It may be suggested that after blood was reach its peak level depends on vitamin source saturated with the vitamin at d 3, a-tocopherol and method of administration. Orally adminisstarted to be deposited in the tissues, which might tered, emulsifiable DL-a-tocopherol liquid (Treat- explain the decrease in concentration of a-tocophment 5) and micellized DL-a-tocopherol(Treatment erol in serum after d 3 (Figure 1). It was observed 7)product forms reached peak concentration on d that the tissue with the highest a-tocopherol 1 and 2, respectively. These data are in agreement concentration was liver, followed by pancreas. with those of Hidiroglou et al. (1989) obtained with Previous results are inconsistent in regard to cattle but are in disagreement with those of which tissue has the highest a-tocopherol concenHidiroglou et al. (19871 obtained with sheep, in tration. Hidiroglou et al., (19871 reported that in which serum a-tocopherol reached maximum peak sheep the tissues with the highest a-tocopherol on d 11. After a single i.m. injection of DL-a- concentration after oral administration followed tocopherol, time of serum maximum peak a- the order pancreas, liver, and spleen. However, tocopherol concentration was 6 d after administraHidiroglou and McDowell (1987) reported that tion (Hidiroglou and McDowell, 1987). When adadrenal tissue was highest, followed by adipose, ministered intraperitoneally, vitamin E peak level pancreas, and liver after a n intramuscular single was observed at d 1 (Hidiroglou et al., 1990). The dose of the vitamin. Hidiroglou et al., (19901 biological activity of a-tocopherol administered to observed that 28 d after i.p. administration of DLsheep was dependent on the method of administraa-tocopherol, a-tocopherol concentration was tion, following the order intravenous, muscular, greatest in adrenal gland, followed by pancreas, and oral (Hidiroglou and Karpinski, 19871, liver, and lung. In this experiment, the treated The data from the current study indicate a sheep exhibited a large variation in a-tocopherol treatment x day interaction for serum a-tocopherol concentration in kidney, adrenal gland, heart, and pancreas. This variation is most likely the reason concentration. However, emulsifiable a-tocopherol why treatment differences could not be detected in liquid (Treatment 5 ) was the highest at d 1 of the pancreatic a-tocopherol Concentration. experiment, and remained in the treatment groups
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OCHOA ET AL.
8
0 0 7
1
E) c-
2
+
c
a, 0
c
0
0 0 L
a,
c
a. 0 0
F 8
Serum
Liver
Kidney
Adrenal
Adipose
Heart
"L. Dors."
"G.Medi." Pancreas
TREATMENTS Figure 2. Effect of supplemental vitamin E product form on sheep tissue a-tocopherol concentration. Within a tissue, treatments are shown from left to right in this order: control, no supplemental vitamin E; Treatment 2, emulsifiable DL-a-tocopheryl acetate-dry; Treatment 3, nonemulsifiable DL-a-tocopherylacetate-dry; Treatment 4, emulsifiable DL-a-tocopheryl acetate-liquid; Treatment 5, emulsifiable DL-a-tocopherol-liquid; Treatment 6, micellized DL-a-tocopheryl acetate-liquid; and Treatment 7, micellized DL-a-tocopherol-liquid.The SE for each tissue was as follows: serum, .006; liver, .442; kidney, .51; adrenal, .196; adipose, .184; heart, .121; longissimus muscle, .064; Gluteus medius, .048; and pancreas, 248.
Hidiroglou et al. (1987) reported that sheep supplemented with 400 IU of vitamin E activity. animal-l.d-l for 28 d as DL-a-tocopherol had higher (P e .05) serum a-tocopherol concentrations than those supplemented with DL-a-tocopheryl acetate, but no difference (P c .05)was found in tissue a-tocopherol concentration. Furthermore, the same results were obtained by Hidiroglou et al. (1988) in cattle after a daily supplementation of 1,000 IU of vitamin E activity per animal for a 28-d period. After a single oral dose of 50 mg of vitamin E/kg of BW was given to cattle and 100 mg to sheep, DL-a-tocopherol had greater (P c .05) potency than its ester form (Hidiroglou et al., 19891. However, sources of vitamin E were administered on a n equal-weight basis rather than a n equalactivity basis (i.e., 10% more vitamin E activity administered with the alcohol source). Results of the current investigation would indicate that there was no difference in vitamin E availability from various vitamin E products tested based on tissue a-tocopherol accretion when administered on a n equal-activity basis; however, there was a tendency of the vitamin E alcohol product forms to maintain higher blood serum a-tocopherol concentration than vitamin E acetate product forms. No
difference in serum a-tocopherol concentration was seen among the supplemented groups by the last 2 wk of the trial.
Implications All vitamin E sources tested resulted in relatively similar tissue a-tocopherol concentrations, but there was a tendency for vitamin E alcohol forms to promote greater serum a-tocopherol concentrations than did acetate products. In general, all forms would serve as suitable supplemental sources of vitamin E.
Literature Cited Box, G.E.P. 1954. Some theorems on quadratic forms applied in the study of analysis of variance problems. 11. Effects of inequality of variance and correlation between errors in the two-way classification. Ann. Math. Stat. 25:484. Faustman, C., R. G. Cassens, D. M. Schaefer, D. R. Buege, S. N. Williams, and K. K. Scheller. 1989. Improvement of pigment and lipid stability in Holstein steer beef by dietary supplementation with vitamin E. J. Food Sci. 54:4. Geisser, S., and S. W. Greenhouse. 1958. An extension of Box's results on the use of the F distribution in multivariate
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0 .c.
VITAMIN E IN SERUM AND TISSUES OF SHEEP
Hidiroglou, N., and L. R. McDowell. 1987. Plasma and tissue level of vitamin E in sheep following intramuscular administration in a n oil carrier. Int. J. Vitam. Nutr. Res. 57:261. Hidiroglou, N., L. R. McDowell, and 0. Balbuena. 1989. Plasma tocopherol in sheep and cattle after ingesting free or acetylated tocopherol. J. Dairy Sci. 72:1793. Hidiroglou, N., L. R. McDowell, and R. Pastrana. 1987. Bioavailability of various vitamin E compounds in sheep. Int. J. Vitam. Nutr. Res. 58:189. SAS. 1988. SAS User’s Guide: Statistics. SAS Inst. Inc., Cary, NC.
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analysis. Ann. Math. Stat. 28:885. Hidiroglou, M., and K. Karpinski. 1987. Vitamin E kinetics in sheep. Br. J. Nutr. 58:113. Hidiroglou, N., G. Butler, and L. R. McDowell. 1990. Plasma and tissue vitamin E concentrations in sheep after administration of a single intraperitoneal dose of dl-a-tocopherol. J. Anim. Sci. 88:782. Hidiroglou, N., L. F. Laflamme, and L. R. McDowell. 1988. Blood plasma and tissue concentrations of vitamin E in beef cattle as influenced by supplementation of various tocopherol compounds. J. Anim. Sci. 86:3227.
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