J. Anim. Sci. 2005. 83:1274-1286
© 2005 American Society of Animal Science
Influence of supplementation of all-rac-
-tocopheryl acetate preweaning and vitamin C postweaning on -tocopherol and immune responses of piglets1
C. Lauridsen2 and
S. K. Jensen
Department of Animal Health, Welfare and Nutrition, Research Centre Foulum, Denmark
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Abstract
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This study was designed to test whether dietary maternal supplementation of all-rac-
-tocopheryl acetate during lactation and dietary vitamin C supplementation after weaning could increase the -tocopherol status pre- and postweaning and the immune responses of piglets postweaning. The experiment involved 12 crossbred sows that were fed increasing levels of all-rac--tocopheryl (70, 150, and 250 IU/kg, as-fed basis) during lactation. After weaning (d 28 of age), litters were divided into two groups that were supplemented with or without vitamin C (500 mg/kg of feed, as-fed basis). Milk and blood samples were obtained from the sows during lactation. Pigs were bled at 4, 16, 28, 35, 42, and 49 d of age. Liver, heart, muscle, and s.c. adipose tissues were collected (on 28, 35, 42, and 49 d of age) and analyzed for -tocopherol. On the same days, alveolar macrophages of the lungs were collected, and analyzed for the concentration of -tocopherol and its stereoisomer composition, fatty acid composition, and release of prostaglandin E2, leukotriene B4, and thromboxane B2. Increasing dietary all-rac--tocopheryl acetate concentration increased the concentration of -tocopherol in plasma (P = 0.02) and milk (P = 0.007) of sows, and the sow milk concentrations of -tocopherol and vitamin A were greater on d 2 of lactation than later on during lactation. The plasma concentration of -tocopherol in piglets decreased from d 4 to later on during suckling (P < 0.001) and again as the postweaning period progressed (P < 0.001). When lipid-standardized, plasma -tocopherol was increased in piglets of sows fed 250 IU of all-rac--tocopheryl acetate compared with other sow-groups (P = 0.005). At 28 d of age, -tocopherol concentrations in tissues were increased with supplementation of the high dietary all-rac--tocopheryl acetate levels to the sows; however, after weaning, a decrease in -tocopherol concentration in most tissues (except liver) was observed, but the decrease tended to be less in the muscle (P = 0.099) and adipose tissue (P = 0.11) of piglets suckling sows fed 150 and 250 IU of all-rac--tocopheryl acetate. Vitamin C supplementation after weaning increased liver -tocopherol (P = 0.01) and serum immunoglobulin M concentration (P = 0.04), and vitamin C supplementation increased the proportion of the RRR--tocopherol (P = 0.03) at the expense of the RRS-stereoisomer form (P = 0.05) of -tocopherol in alveolar macrophages of the piglets. In conclusion, this study on maternal all-rac--tocopheryl acetate and postweaning vitamin C supplementation suggests a nutritional strategy for increasing -tocopherol status and immune responses of weaned piglets.
Key Words: Immune Response • Pigs • Tocopherol • Vitamin E • Vitamin C • Weaning
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Introduction
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Two of the more critical stages for dietary vitamin E as a nutrient for growth and health status in pigs are immediately after birth and after weaning. Previous reports (Hidiroglou et al., 1993
; Pehrson et al., 2001) have shown that supplementation of sow diets with vitamin E increases the vitamin E status of their progeny during suckling, but little is known with regard to the subsequent effect on the vitamin E status of the progeny after weaning. As tocopherols (vitamin E) are absorbed in the small intestine as free alcohols alone or in combination with emulsified fat products, the commercially available all-rac--tocopheryl acetate must be hydrolyzed before absorption, a process that may be limited in weaned pigs (Chung et al., 1992; Hedemann and Jensen, 1999; Lauridsen et al., 2001).
Vitamin C is not routinely added to pig feed because pigs are capable of synthesizing vitamin C; however, during stressful situations such as weaning, the presence of L-gulono-
-lactone oxidase (GLO), a necessary enzyme for the biosynthesis of vitamin C, might be low (Ching et al., 2001). The interaction between vitamin E and C is interesting due to the reported sparing action of vitamin C on vitamin E, or synergism between the two vitamins as reviewed by Burton et al. (1990). Increased intake of vitamin C might, therefore, be one strategy to improve the vitamin E status of piglets after weaning.
Dietary vitamins E and C increased the cellular and humoral immunity in pigs (Jensen et al., 1988; Hayak et al., 1989; Schwager and Schulze, 1998). Vitamin E deficiency also has been found to predispose pigs to different diseases, including Escherichia coli infection (Ellis and Vorhies, 1976). The objective of this investigation was to test whether maternal supplementation of all-rac--tocopheryl acetate during suckling and postweaning vitamin C supplementation could increase the -tocopherol status and the cellular and humoral immune responses of piglets.
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Materials and Methods
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Animals
Twelve sows (Danish Landrace x Danish Yorkshire) were selected randomly from the herd at the Danish Institute of Agricultural Sciences. The sows were arranged in a randomized complete block design, with four blocks of three sows, corresponding to three treatments per block. Within each block, sows had a common sire breed, the same parity number, and were mated with a Duroc boar. At weaning (28 d of age), piglets within each litter were divided into two diet groups. Piglets were provided the experimental diets until 49 d of age (end of experiment). The experiment complied with the guidelines of the Danish Ministry of Justice with respect to animal experimentation and care of animals under study.
Housing
The sows were housed individually in pens (2.2 x 2.4 m) with partly slatted floors of solid concrete combined with slats of iron grates. The farrowing crate was 2.4 m long and flexible in width (0.6 to 0.8 m). Sows and piglets were provided moderate quantities of straw bedding. The creep area for piglets (1.0 x 1.3 m) was partly covered, and heating lamps were installed during the first week after birth. After weaning, piglets within a litter were housed in 1.5 x 1.8 m pens. The floor comprised two parts: a concrete, heated floor (located just behind the trough) and a manure area with a plastic net. Because each litter was divided into two groups, the pen was divided into two parts by a galvanized plate.
Diets
The three experimental diets for sows fed from 108 d of gestation until weaning (d 28 d after farrowing) were based on soybean meal, barley, and wheat, and were supplemented with all-rac--tocopheryl acetate (Rovimix E 50-Ads, provided by Roche a/s, Basel, Switzerland) to provide 70, 150, or 250 IU of -tocopherol/kg feed (as-fed basis, Table 1). Piglets were not offered creep feed during the suckling period. The postweaning diets for the pigs consisted of the basal diet containing 70 IU/kg of all-rac--tocopheryl acetate, or this diet supplemented with 500 mg/kg of vitamin C in a stabilized form (Rovimix STAY-C 35, provided by Roche a/s, Basel, Switzerland). All experimental diets were mixed at Research Centre Foulum, Denmark, and were provided as mash.
The content of DM, CP, crude fat, and ash in the diets was determined according to the AOAC (1990) procedures. Dietary lipids were extracted by the method of Stoldt (1952) using petroleum ether, and analysis of AA (cystine, lysine, methionine, and threonine) was performed according to Mason et al. (1980). Amino acid concentrations were not corrected for incomplete recovery resulting from hydrolysis. Analyzed composition of the basal diets is presented in Table 1
. The -tocopherol content of the diets was determined by HPLC after saponification and extraction into heptane as described by Jensen et al. (1999a). Analysis of vitamin C in the weaner diet was determined by HPLC by Roche a/s.
Sows were fed twice daily and had free access to water. From d 108 to 111 of pregnancy, 3.1 kg of feed was provided to supply 25.3 MJ of NE (as-fed basis). From d 112 of gestation until d 1 after farrowing, 19.6 MJ of NE/d was provided, followed by 23.2 and 27.0 MJ of NE/d on d 2 and 3 after farrowing, respectively. Thereafter, feed was offered semi-ad libitum during the next 25 d of lactation. Daily allocations were individually adjusted; thus, sows were able to empty the trough between feedings. After weaning, piglets had free access to feed and water.
Protocol and Laboratory Analyses
Milk and blood samples were obtained from each sow throughout the lactation (on d 108 of gestation [only blood], and on d 2, 16, and 28 after farrowing). Milk samples were obtained by hand-milking of four to six teats after i.v. injection (10 IU) of oxytocin (Leo Pharmaceutical Products, Ballerup, Denmark). Blood samples of sows were collected from the jugular vein by puncture into heparinized vacuum tubes. Sampling and treatment of blood from piglets was performed on d 4, 16, 28, 35, 42, and 49 after birth of three piglets per litter (before weaning) or per diet group (after weaning) into vacuum tubes (heparinized or serum). After blood sampling, plasma and serum were obtained after centrifugation at 3,000 x g and stored at –80°C until analysis.
-Tocopherol and vitamin A in plasma and -tocopherol in milk samples were analyzed by HPLC according to Jensen et al. (1999b). Vitamin A in milk was analyzed by HPLC according to Jensen (1994). Analyses of the plasma concentration of triglycerides were based on enzymatic determination of free glycerol after liberation from triglyceride using lipoprotein lipase. The analyses consequently did not differentiate between mono-, di-, or triglyceride origin. Plasma-free cholesterol and total cholesterol were determined before and after hydrolyses of cholesterol esters using cholesterol esterase. The free cholesterol in the presence of oxygen and cholesterol oxidase produces hydrogen peroxide, which in turn forms a quinoneimine dye that is determined colorimetrically at 500 nm. Determination of triglyceride and cholesterol concentrations were both performed with an autoanalyzer (OpeRA Chemistry System, Bayer Corp., Tarrytown, NY), and the procedure was standardized by Technicon RA Systems (Tarrytown, NY).
The concentrations of immunoglobulins G, M, and A (IgG, IgM, and IgA, respectively) were measured in serum of piglets using commercial kits (pig ELISA quantitation kit; Bethyl Laboratories, Montgomery, TX). Measurement of the content of specific E. coli 0149 K88ac antibodies in serum was done by indirect ELISA as follows: microtiter plate wells were coated and incubated overnight at 4°C with carbonate-bicarbonate coating buffer containing E. coli 0149 K88ac that had been disrupted by ultrasound. After washing the wells with PBS-Tween (0.05% Tween) and adding blocking buffer (PBS-Tween with 0.1% [wt/vol] gelatin), dilutions of serum were added to wells in triplicate. The plates were incubated for 1 h at 37°C and were then washed with PBS-Tween (with 0.1% gelatin). Anti-pig IgG alkaline phosphatase conjugate (Sigma A1192) was added to each well and allowed to incubate for a further 30 min at 37°C. After washing, phosphatase substrate in diethanol amine buffer was added to each well, and after incubation for 30 min in the dark at 37°C, NaOH was added to each well to stop the reaction. The absorbance was read at 405 nm. Titer values are reported as arbitrary values (i.e., the last dilution [x 102] that gave a positive reaction).
Piglets (one per litter on d 28, and three per treatment group at d 35, 42, and 49 of age, respectively) were killed by an injection of approximately 10 mL of pentobarbital solution (containing 200 mg/L, produced by The Royal Veterinary and Agricultural University, Copenhagen, Denmark). Blood samples were obtained during bleeding of the animals. After exsanguination of the carcasses, the liver, the heart, and samples of upper hip muscle (biceps femoris and/or gluteus biceps) and s. c. adipose tissue from this muscle area were obtained. Organs and tissue samples were frozen at –20°C for later analysis. Cellular membranes (microsomes and mitochondria) were isolated from muscle samples according to the procedures of Lauridsen et al. (2000). The concentration of vitamin C was determined in plasma according to the method of Zannoni et al. (1974). Determination of the plasma ferric reducing (antioxidant) power (FRAP) was performed using a modification of the FRAP assay (Benzie and Strain, 1999). L-Gulonolactone oxidase activity in the liver of piglets was based on a colorimetric procedure as described by Hooper et al. (2000), and protein in liver was determined according to Lowry and Passonneau (1972). Concentration of -tocopherol in organs, tissue samples, and muscle membranes were analyzed by HPLC according to Jensen et al. (1999a).
Isolation and Analysis of Alveolar Macrophages
Lungs from piglets of sows fed 70 or 250 mg of all-rac--tocopheryl acetate were filled with approximately 60 mL of ice-cold Hanks’ balanced salt solution (Gibco, Grand Island, NY) through the trachea. The lungs were gently shaken, and the content was filtered through gauze into containers on ice. This process was repeated twice (total recovery was approximately 100 mL). The tubes containing the alveolar macrophages were spun down (500 x g for 15 min), and washed twice with minimal essential medium (MEM; Gibco), with 2 mM L-glutamine, 10 mM HEPES (Gibco), penicillin (100,000 U/L; Gibco), and streptomycin (0.17 mmol/L; Gibco), pH 7.4. The cells were then spun at 500 x g for 10 min and resuspended in 1 mL of MEM. The cells were colored with trypan blue, counted on a hemocytometer, and resuspended at a final concentration of 5 x 106 cells/mL of MEM. The alveolar macrophage preparations were verified by Wright-Giemsa staining (Strober, 1992), and adherence properties. Samples of the cell suspension were resuspended in 10 mM EDTA and stored at –80°C until analysis of fatty acid composition and -tocopherol.
To each well of a 12-well cell culture plate, 0.5 mL (2.5 x 106) of the alveolar macrophage suspension was added along with 0.5 mL of MEM (with 10% calf serum/FBS; Gibco). Plates were placed in an incubator at 37°C with 5% CO2 for 4 h to allow cells to adhere. After washing the adhered cells three times with MEM, the number of adhered cells per well was estimated by counting the cells not adhered (dead and alive). Then, 0.5 mL of MEM with 10% serum/FBS was added to each well, and 0.5 mL/well of MEM alone (controls) or containing calcium ionophore A231857 (2 µg/mL; Sigma-Aldrich, Vallensbaek Strand, Denmark) was added to wells in order to stimulate eicosanoid production. After 1 h, supernatant fractions of controls and calcium ionophore were collected in 1.5-mL polypropylene microcentrifuge tubes, spun (9,000 x g; 3 min), transferred to a new tube, and stored at –80°C.
Cell supernatant fractions were analyzed for concentrations of eicosanoids (prostaglandin E2, PGE2), leukotrienes B4 (LTB4), and thromboxane B2 (TXB2) using commercially available immunoassay kits for each eicosanoid (Caymann Chemicals Co., Ann Arbor, MI). Analysis of FAME in alveolar macrophage suspensions was performed by GLC as methyl esters (Jensen and Nyholm, 1996) after extraction of the lipids according to the method of Bligh and Dyer (1959). The concentration of -tocopherol was determined as described above for tissues, whereas the relative distribution of the stereoisomers of -tocopherol was determined in the remaining extract. Briefly, this heptane extract containing 1 to 2 µg of -tocopherol in 9 mL of heptane was evaporated to exact dryness under a N2 stream. Then the -tocopherol extract was derivatized to its methyl ether following the method described by Drotleff and Ternes (2001). The methyl ester derivative was extracted with 1,000 µL of heptane. From this heptane extract, 100 µL was injected into the HPLC. Chromatographic separation was achieved on a Chiralcel OD-H column (25 x 0.46 cm; 5-µm particle size, cellulose tris [3,5-dimethylphenylcarbamate]; Daicel Chemical Industries, Ltd., Tokyo, Japan) with heptane modified with 0.01% isopropanol as a modifier. This method allows for the separation of the eight stereoisomers of -tocopherol into five peaks. Peak 1 contains all four 2S forms (2SR/SR/S); Peak 2 contains the 2RSS--tocopherol; Peak 3 contains 2RRS--tocopherol; Peak 4 contains 2RRR--tocopherol (= natural -tocopherol); and Peak 5 contains 2RSR--tocopherol.
Statistical Analyses.
The statistical analysis of data was accomplished using the MIXED procedure of SAS (SAS Inst., Inc., Cary, NC). Analysis of data obtained on sows and piglets during suckling was based on the following model:
where Ysdbi is the dependent blood or milk response variable, µ the overall mean, s the systematic effect of dietary all-rac--tocopheryl acetate (70, 150, 250 IU), ßd the systematic effect of time (of blood or milk sampling), and (ß)sd is the corresponding interaction. Usb refers to the effect of sow/block (i.e., the dependency between sow and block) and 
sdbi denotes the error term. In this model, the pen was the experimental unit.
Analysis of data obtained on piglets after weaning (d 28 of age) was performed according to the following model:
where Ysdci is the dependent blood or tissue response variable, µ the overall mean, s the systematic effect of dietary all-rac--tocopheryl acetate supplemented to the sows, ßd the systematic effect of age (at which blood or tissue sampling was performed), c the effect of dietary vitamin C, and (ß)sd, ()sc, (ß)dc, and (ß)sdc are the corresponding interactions. The value 
xsdci denotes the covariant (i.e., the measurement of the given response variable at d 28), and Usb refers to the effect of block (i.e., the dependency between sow and block). For data on cell-mediated immune response, the covariant was omitted from the model. As shown, the full model included the interactions of the three responses, but no statistically significant effects were detected; therefore, this effect is not further described in the results. In this model, the piglet group was the experimental unit.
The statistical models were verified by plotting the residuals against the predicted values and by use of quantile plots of the residuals. Results of statistical analysis are given as least squares means and SE. Differences among dietary treatments and time of sampling were tested by differences between least squares means using the LSMEANS option of SAS. Probability values 
0.05 were considered significant.
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Results
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Feed Analyses
The analyzed concentrations of -tocopherol in the diets were very close to the formulated amounts as Diet 1 contained 75 mg (SD = 7 mg), Diet 2 contained 145 mg (SD = 10 mg), and Diet 3 contained 265 mg (SD = 8 mg) of -tocopherol/kg feed. In addition, the -tocopherol concentration of the weaner diet was in the range 83 to 91 mg of -tocopherol/kg of feed, which agreed with the expected value. The concentration of vitamin C in the weaner diet supplemented with 500 mg of vitamin C/kg of diet was slightly lower (411 to 456 mg/kg of diet) than stipulated.
Sow Plasma and Milk
Increasing dietary all-rac--tocopheryl acetate concentration increased the concentration of -tocopherol in the plasma (P = 0.02) and milk (P = 0.007) of the sows, but had no influence on the vitamin A concentration in plasma or milk (Table 2). Regardless of dietary all-rac--tocopheryl acetate concentration, the milk concentration of both -tocopherol (P < 0.001) and vitamin A (P < 0.001) was notably higher on d 2 of lactation compared with sampling later on during lactation. Time of sampling also influenced the plasma concentration of -tocopherol (P < 0.001) and vitamin A (P = 0.048), as the concentrations were lower at d –8 (d 108 of gestation) and d 2 of lactation than later on during lactation (Table 2).
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Table 2. Effect of supplemental dietary (as-fed basis) all-rac--tocopheryl acetate to sow feed on the concentration of -tocopherol and vitamin A in plasma and milk (after storage at –20°C) from 1 wk before expected farrowing (d-8) until weaninga
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Piglets During Suckling
The plasma -tocopherol concentration of piglets during suckling was influenced by age (P < 0.001), as the concentration of -tocopherol in plasma was higher at 4 d of age than later on during the suckling period (Table 3). To determine whether the influence of age on the -tocopherol plasma concentration could be ascribed to the content of plasma lipids, the plasma concentration of -tocopherol was expressed relative to the total content of lipids (triglycerides and cholesterol). When lipid-standardized in this manner, the influence of dietary treatments of sows on -tocopherol concentration of piglets was highly significant (P = 0.005).
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Table 3. Effect of supplemental dietary (as-fed basis) all-rac--tocopheryl acetate to sow feed on the concentration of -tocopherol in piglet plasma or in relation to total plasma lipids from 1 wk before expected farrowing (d –8) until weaninga
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Compared with feeding a level of 70 IU of all-rac--tocopheryl acetate/kg to the sows (Table 4), feeding of 150 and 250 IU/kg increased the -tocopherol concentration in adipose tissue (P = 0.04) and muscle mitochondrial membranes (P = 0.005), and feeding of 250 IU/kg increased the -tocopherol concentration in the heart (P = 0.04). In addition, a numerically increased -tocopherol concentration was observed in the liver (P = 0.09) and muscle (P = 0.18), reflecting dietary treatments of sows.
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Table 4. Effect of supplemental dietary (as-fed basis) all-rac--tocopheryl acetate to sow feed on concentration of -tocopherol in tissues, muscle mitochondria and microsomes of piglets on d 28a
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Piglets After Weaning
Plasma -tocopherol decreased with increasing age of the piglets (P < 0.001), but dietary sow or vitamin C treatments had no influence on this variable (Table 5). With regard to muscle (P = 0.099) and adipose tissue (P = 0.11), tendencies for an interaction between age and sow treatment were observed, as the decrease in -tocopherol concentration was less when 150 or 250 IU of all-rac--tocopheryl acetate/kg was provided. In addition, the concentration of -tocopherol in muscle mitochondria (P 0.01) was increased with increasing dietary all-rac--tocopheryl acetate (results not shown). As age progressed, -tocopherol status decreased in heart (P < 0.001) and muscle tissue (P < 0.001), and tended to decrease in adipose tissue (P = 0.07), whereas the concentration tended to increase in liver (P = 0.10). Only the concentration of -tocopherol in adipose tissue was influenced (P = 0.03) by its concentration on d 28 of age. Concentration of -tocopherol in muscle microsomes was not influenced by any of the responses (results not shown). Addition of vitamin C to the weaner diet increased the concentration of -tocopherol in the liver (P = 0.01), and an interaction between age and vitamin C was observed on the -tocopherol in the muscle (P = 0.015), with the concentration of -tocopherol being somewhat higher at 35 and 49 d of age but lower at 42 d of age in vitamin C-supplemented piglets than in unsupplemented piglets.
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Table 5. Effect of maternal dietary (as-fed basis) all-rac--tocopheryl acetate (All-rac) and weanling pig vitamin C supplementation on concentration of -tocopherol in plasma and tissues of piglets at 35, 42, and 49 d of agea,b
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The concentration of vitamin C in plasma increased from 42 to 49 d of age (P < 0.001, Table 6); however, no influence of addition of vitamin C to the weaner diet was observed on this variable. Although the FRAP values seemed to be influenced by the age of the piglets, no effect of any responses was obtained on this variable. An interaction between age of piglets and treatment of sows was observed on the activity of GLO in liver (P = 0.02), as the increase in activity (d 35 to d 42 of age) and decrease (d 42 to d 49 of age) was stronger for the unsupplemented piglets than for vitamin C-supplemented piglets. On d 28 of age, no influence of dietary treatments of sows was observed with regard to the concentration of vitamin C and FRAP values in plasma and the activity of GLO in liver (results not shown).
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Table 6. Effect of maternal dietary (as-fed basis) all-rac--tocopheryl acetate (All-rac) and weanling pig vitamin C supplementation on concentration of vitamin C and ferric reducing antioxidant power (FRAP) in plasma, and L-gulono--lactone oxidase (GLO) activity in liver of piglets at 35, 42, and 49 d of agea,b
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Humoral Immune Response
On d 28 of age, the concentration of immunoglobulins did not differ between dietary treatments of sows (results not shown). After weaning, the concentrations of IgG and IgA in serum of piglets were not influenced by the dietary all-rac--tocopheryl acetate provided the sows, age, or vitamin C added to the weaner diet (Table 7). The IgM concentration in serum of piglets provided vitamin C was greater (233 mg/dL serum; SE = 15) compared with control piglets (196 mg/dL serum; SE = 13; P = 0.04) and was not influenced by age of piglets or dietary treatment of sows. Escherichia coli antibodies in serum increased with increasing age (P < 0.001), and when analyzing the effect of dietary treatment of sows using a linear contrast, this effect was significant (P = 0.008; i.e., a decrease of E. coli antibodies with increasing dietary all-rac--tocopheryl acetate). The concentration of E. coli antibodies in serum at 28 d of age was 60, 44, and 25 (SE = 18) for piglets suckled of sows fed 70, 150, and 250 IU/kg of all-rac--tocopheryl acetate. The number of litters treated after weaning because of diarrhea was four out of four in sows provided with 70 IU, three out of four in sows provided 150 IU, and one out of four in sows provided 250 IU/kg of all-rac--tocopheryl acetate.
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Table 7. Effect of maternal dietary (as-fed basis) all-rac--tocopheryl acetate (All-rac) and weanling pig vitamin C supplementation on concentration of immunoglobulins (Ig) and antibodies against Escherichia coli in serum of piglets at 35, 42, and 49 d of agea,b
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Cell-Mediated Immune Response
Only piglets suckling sows provided 70 or 250 IU/kg of dietary all-rac--tocopheryl acetate were used in the study of the cell-mediated immune response (Table 8 and 9). Although large variation among piglets was observed with regard to the eicosanoid production of alveolar macrophages, a lower LTB4 production (P = 0.005) was observed in macrophages of piglets at 43 d of age compared with piglets at 35 or 49 d of age. In addition, an interaction (P = 0.05) between dietary treatment of sows and vitamin C addition was observed on the production of PGE2 of alveolar macrophages, with a higher concentration of PGE2 in alveolar macrophages of piglets fed vitamin C than in unsupplemented pigs at the 70 IU/kg all-rac--tocopheryl acetate level, whereas the opposite was observed at the 250 IU/kg level. The production of TXB2 was not affected by any treatments, and the eicosanoid production of alveolar macrophages on d 28 was not affected by dietary treatments of sows (results not shown).
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Table 8. Effect of maternal dietary (as-fed basis) all-rac--tocopheryl acetate (All-rac) and weanling pig vitamin C supplementation on production of eicosanoids by alveolar macrophages (pg/106 cells) of piglets at 35, 42, and 49 d of agea,b
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Table 9. Effect of maternal dietary (as-fed basis) all-rac--tocopheryl acetate (All-rac) and weanling pig vitamin C supplementation on the concentration of -tocopherol and the stereoisomer profile percentages of alveolar macrophages of piglets at 35, 42, and 49 d of agea,b
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The percentage of living cells of the lavage fluid was 83% (SE = 4%) at 28 d of age, and 88% (SE = 13), 89% (SE = 9%), and 78% (SE = 24) at 35, 42, and 49 d of age for piglets suckled by sows fed 70 IU/kg of all-rac--tocopheryl acetate. Piglets suckling sows fed 250 IU/kg of all-rac--tocopheryl acetate had a numerically higher percentage of living cells after weaning; 83% (SE = 12), 95% (SE = 6), 91% (SE = 6%), 91% (SE = 3) at 28, 35, 42, and 49 d of age.
Relative Distribution of Stereoisomers of -Tocopherol in Alveolar Macrophages
No difference was obtained in the -tocopherol content in alveolar macrophages on d 28 of age (0.52 and 0.87 µg of -tocopherol per 107 cells of piglets suckled sows fed 70 or 250 IU/kg of all-rac--tocopheryl acetate, respectively; P = 0.21). After weaning, the concentration of -tocopherol in the alveolar macrophages decreased (P = 0.006) with increasing age of the piglets, whereas no difference between the dietary treatments of sows was observed (Table 9). The RRR--tocopherol was the predominant form of -tocopherol, and it contributed approximately one-third of the total -tocopherol concentration, followed by the RRS-, the RSR-, and the RSS-stereoisomers, whereas the 2S-forms contributed with less than 2% of the -tocopherol (Table 9). Addition of vitamin C to the feed increased the proportion of RRR--tocopherol (P = 0.03) and decreased the proportion of RRS--tocopherol (P = 0.05), whereas no effects were obtained on the proportion of the remaining stereoisomer forms.
Fatty Acids in Alveolar Macrophages
No clear treatment effects were observed with respect to the content of fatty acids in alveolar macrophages, and pooled results showed that the average content of n-3 fatty acids in alveolar macrophages was 16 (SD = 7) pg/107 cells of C22:5n-3 and 23 (SD = 13) pg/107 of C22:6n-3. Among the n-6 fatty acids, the content C20:4n-6 was by far the greatest, averaging 103 (SD = 29) pg/107, followed by C18:3n-6, 76 (SD = 41) pg/107, C18:2n-6, 49 (SD = 15) pg/107, and C22:5n-6, 13 (SD = 9) pg/107, with an approximate ratio between n-6 and n-3 fatty acids of 8.3.
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Discussion
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In this study, the effect of supplementing up to 250 IU/kg of all-rac--tocopheryl acetate in the diet for sows from 1 wk before farrowing and during lactation was followed with respect to the -tocopherol status of piglets before and after weaning. The dietary levels of all-rac--tocopheryl acetate were clearly reflected in both sow colostrum and milk -tocopherol concentration, as also reported before using up to 136 mg of all-rac--tocopheryl acetate (Babinszky et al., 1991; Mahan, 1994; Mahan et al., 2000). However, addition of 250/kg of IU all-rac--tocopheryl acetate to the diet for sows resulted in an increase in milk -tocopherol content by over 100% compared with addition of 70 IU/kg of all-rac--tocopheryl acetate to the sow diet, indicating the high potential of the sow to enrich the milk. According to previous studies (Mahan, 1991, 1994), plasma -tocopherol concentration of sows also increased due to supplementation with all-rac--tocopheryl acetate. Day of blood and milk sampling influenced both the concentration of -tocopherol and vitamin A. As shown previously (Csapo et al., 1996), the concentration of both vitamins is notably higher in colostrum compared with milk of sows. The lower plasma vitamin concentration observed 1 wk before parturition and on the second day after farrowing could be a consequence of the considerable amount of vitamins partitioned into the colostrum (Lauridsen et al., 2002).
The higher content of -tocopherol in colostrum compared with milk may explain the higher level of -tocopherol found in the plasma of piglets during the first days of life. Only when plasma -tocopherol concentration in piglets was lipid-standardized, was the high level of dietary all-rac--tocopheryl acetate given to the sows reflected in the plasma -tocopherol concentration of piglets during suckling. Intramuscular supplementation of 1.5 g of -tocopheryl acetate to sows on d 7 and 2 before farrowing increased the -tocopherol concentration in colostrum of sows and in the serum of piglets 2 and 5 d after birth (Pehrson et al., 2001). Plasma -tocopherol may be considered as a short-term pool as surplus -tocopherol is eventually transferred to the tissues (Jensen et al., 1990). This also was con-firmed by the tissue -tocopherol content on d 28 as a clear effect of supplementation of 150 and 250 IU/kg of all-rac--tocopheryl acetate was noted, especially with regard to the accumulation of -tocopherol in muscle mitochondria, adipose tissue, and heart. Thus, the -tocopherol status of piglets at weaning was improved by maternal all-rac--tocopheryl acetate supplementation during lactation.
The present study also was designed to determine whether an eventual increase in the -tocopherol status at weaning could prevent the frequently observed decrease in the -tocopherol status during the subsequent weaning period (Hedemann and Jensen, 1999; Moreira and Mahan, 2002). The drop in -tocopherol concentration after weaning has been ascribed to limited synthesis of the enzyme carboxylic ester hydrolase, which is needed to hydrolyze the -tocopheryl acetate present in the weaner diet before absorption of -tocopherol (Hedemann and Jensen, 1999). Another factor could be impaired absorption of lipids, and thereby lipid-soluble vitamins, such as -tocopherol. From our results, tissues responded differently depending on their metabolic activity. In the heart, which is an active muscle with a high oxidative capacity, the decrease in -tocopherol status after weaning was significant regardless of the concentration of all-rac--tocopheryl acetate in the diets for sows. In muscle (P = 0.099) and adipose (P = 0.11) tissue, however, the decrease in -tocopherol tended to be less pronounced in piglets suckling sows fed 150 and 250 IU/kg of all-rac--tocopheryl acetate than from piglets nursing sows fed 70 IU/kg, whereas a numerical increase was observed in liver -tocopherol concentration. Previous observations have shown that the porcine liver has a very high short-term storage capacity for -tocopherol (Jensen et al., 1990; Lauridsen et al., 2002). However, the decrease in the vitamin E status of the other tissues may indicate that the dietary level of all-rac--tocopheryl acetate in the weaner diet (70 IU/kg) or the bioavailability of the dietary all-rac--tocopheryl acetate source was insufficient to cause an export of -tocopherol from the liver to other tissues.
Interestingly, the addition of vitamin C increased the -tocopherol concentration of the liver during the weaning period, and an interaction between the effect of vitamin C and age was observed with regard to the muscle -tocopherol status. These results are supportive of the putative vitamin C x vitamin E interaction demonstrated in many in vitro studies (Stoyanovsky et al., 1995; Halpner et al., 1998), but to date, this is unconfirmed in weaned piglets. The increase in -tocopherol status in the liver after vitamin C supplementation implies an in vivo interaction between these two antioxidant vitamins, and probably indicates an increased regeneration of -tocopherol (Hamilton et al., 2000). Contrary to, for example, the studies by Yen and Pond (1981) and Mahan and Saif (1983), no significant influence of vitamin C supplementation could be found with regard to the concentration of vitamin C in plasma of the piglets; however, plasma vitamin C decreased after weaning, and a remarkable increase was observed at 49 d of age. This postweaning decrease in plasma vitamin C has been reported before (Yen and Pond, 1981; Mahan and Saif, 1983), and it may reflect a decreased in vivo synthesis of the vitamin during this period. The activity of GLO, which is an enzyme known to reflect the synthesis of vitamin C (Ching et al., 2001), was influenced by both age of piglets and dietary all-rac--tocopheryl acetate treatment of sows. In addition, a numerically higher GLO activity was observed in the livers of piglets not supplemented with vitamin C, and it may be concluded that the synthesis of vitamin C by the piglet itself was the reason why no differences could be observed between dietary vitamin C supplementation on the vitamin C concentration in plasma. In agreement with the lack of influence of dietary treatments on the plasma concentrations of -tocopherol and C, FRAP-values were not influenced. Vitamins E and C are reported to contribute 5 to 10% and 10 to 15%, respectively, to the FRAP value of fasting plasma in humans (Vasankari et al., 1997; Benzie and Strain, 1999).
The borderline -tocopherol concentration distinguishing -tocopherol sufficiency from deficiency is considered to be 0.4 to 1.0 mg/L of plasma (Van Vleet, 1980; Jensen et al., 1988); for optimal immune response, the borderline -tocopherol concentration seems to be approximately 3 mg/L (Jensen et al., 1988). The plasma -tocopherol concentrations of the suckling piglets in all three dietary treatment groups of sows was between 3.8 to 7.4 mg/L, which should be sufficient for avoiding clinical symptoms of deficiency disease and reaching optimal immune function. However, during the weaning period, the plasma -tocopherol concentration decreased gradually from 1.7 µg/mL of plasma (35 d of age) to 1.1 µg/mL of plasma (49 d of age), which was close to a critical level. No clinical -tocopherol deficiency symptoms and no decrease in the concentration of the measured immunoglobulins in the serum of piglets were observed during the weaning period, but E. coli antibodies in serum increased with increasing age, which may indicate higher level of invasion of E. coli as age progressed, or even the difference between the development of a primary and a secondary antibody response. Escherichia coli may cause diarrhea and death in young piglets raised on commonly used commercial feed, and in accordance, the E. coli antibodies in serum and treatments for diarrhea decreased with increasing dietary levels of -tocopherol of the sows. Previous studies have shown a carryover effect of -tocopherol during lactation (Babinszky et al., 1991), as well as a positive effect of high levels (100 to 1,000 mg/kg of diet) of -tocopherol to piglets after weaning (Ellis and Vorhies, 1976; Peplowski et al., 1981; Hidiroglou et al., 1995) on immune responses of piglets.
In swine, vitamin C alters the proliferative response to mitogens and induces transient changes in the populations of peripheral blood T and B lymphocytes (Schwager and Schulze, 1998); however, it is generally assumed that pigs can synthesize vitamin C and thus do not require dietary vitamin C supplementation under most conditions (NRC, 1998). In our study, the addition of vitamin C to the weaner diet consistently increased the concentration of IgM throughout the weaner period compared with piglets with no added dietary vitamin C. This result confirms previous studies (Prinz et al., 1980), indicating that vitamin C exerts a positive influence on the immune functions. In addition, supplementation with vitamin C caused an increase in the relative contribution of the RRR--tocopherol in immune cells of the piglets on the expense of the RRS--tocopherol. It is well known that vitamin C accumulates in cells of the immune system (reviewed by Grimble, 1997), where it reverses deleterious effects of reactive oxygen intermediates like other antioxidants. Burton et al. (1990) concluded that the long-postulated "sparing" action of vitamin C on -tocopherol was of negligible importance in vivo in guinea pigs and claimed that there should be no reason why it should not also apply to other animals. Nonetheless, based on our results, we conclude that vitamin C is able to regenerate the RRR--tocopherol in alveolar macrophages of piglets after weaning, which is the form of -tocopherol showing the highest biological activity in animals (Weiser and Vecchi, 1982).
The variation among piglets with regard to the production of eicosanoids by alveolar macrophages was large, which agrees with the results of du Manoir et al. (2002), who observed that alveolar macrophage functions of pigs were variable both on an individual basis and over time. In their study, it was concluded that the oxidative burst responses of neutrophil and alveolar macrophages were lower in younger than in older pigs, which may have implications for the susceptibility of young pigs to primary bacterial pneumonia (du Manoir et al., 2002). In agreement, we observed a notably lower concentration of leukotriene B4 of macrophages at 42 d. -Tocopherol is known to modulate eicosanoid production via its influence on the availability, and/or metabolism of PUFA, their precursors, and substrates (Panganumala and Cornwell, 1982). The present numerical difference between the -tocopherol concentration in alveolar macrophages probably was not sufficiently high to produce any difference in the eicosanoid production ex vivo.
Although there are no known criteria to evaluate -tocopherol adequacy in the pig, it would seem that the minimum serum concentration needed would seem to be the level that would maintain a relatively constant balance between blood concentrations and tissue retention mechanisms, and thereby relatively constant blood concentrations during the feeding period (Moreira and Mahan, 2002). Except for the liver, a steady decrease in the -tocopherol status of plasma and tissues occurred in the present study, and it may therefore be concluded that a dietary concentration of 70 IU of all-rac--tocopheryl acetate/kg of feed was not sufficient to maintain the -tocopherol status of the piglets up to 3 wk after weaning, even with addition of 500 mg of vitamin C to the weaner feed or high dietary levels of all-rac--tocopheryl acetate during lactation.
Piglets suckling sows fed 150 or 250 IU/kg of all-rac--tocopheryl acetate had a higher -tocopherol status at weaning (28 d of age) than did piglets suckling sows fed 70 IU/kg. In sow milk, piglet plasma, and in some of the tissues, 250 IU of all-rac--tocopheryl acetate/kg increased the -tocopherol status compared with 150 IU/kg. Regardless of the dietary all-rac--tocopheryl acetate level for sows, addition of 70 IU of all-rac--tocopheryl acetate/kg to the weaner diet was not enough to prevent a decrease in the -tocopherol status in the heart, muscle, and immune cells during the 3 wk post-weaning period. The addition of 500 mg of vitamin C/kg to the weaner diet increased the -tocopherol status of the liver after weaning and also caused some increase in the -tocopherol status of the muscle tissue and immune cells during the first week after weaning. In addition, supplementation with vitamin C increased the IgM concentration of the pigs after weaning, and increased the biological activity of -tocopherol present in immune cells.
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Footnotes
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1 For laboratory assistance, the authors thank E. L. Pedersen and A. Stouby. For financial support, the authors acknowledge the Danish Research Council, and Roche a/s. The authors also thank Senior Scientist S. Højsgaard for statistical advice. 
2 Correspondence: P.O. Box 50, DK-8830. Tjele (phone: + 45 89 99 12 38; fax: + 45 89 99 11 66; e-mail: Charlotte.Lauridsen{at}agrsci.dk).
Received for publication February 10, 2004.
Accepted for publication March 24, 2005.
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Literature Cited
|
|---|
AOAC. 1990. Official Methods of Analysis. 14th. ed. Assoc. Off. Anal. Chem., Washington, DC.
Babinszky, L., D. J. Langhout, M. W. A. Verstegen, L. A. den Hartog, P. Joling, and M. Nieuwland. 1991. Effect of vitamin E and fat source in sows’ diets on immune response of suckling and weaned piglets. J. Anim. Sci. 69:1833–1842.[Abstract]
Benzie, I. F. F., and J. J. Strain. 1999. Ferric reducing (antioxidant) power as a measure of antioxidant capacity: The FRAP assay. Pages 15–27 in Oxidants and Antioxidants: A Volume of Methods in Enzymology. L. Packer, ed. Academic Press, Orlando, FL.
Bligh, E. C., and W. J. Dyer. 1959. A rapid method of total lipid extraction and purification. Can. J. Biochim. Phys. 37:911–917.
Burton, G. W., U. Wronska, L. Stone, D. O. Foster, and K. U. Ingold. 1990. Biokinetics of dietary RRR--tocopherol in the male guinea pig at three dietary levels of vitamin C and two levels of vitamin E. Evidence that vitamin C does not "spare" vitamin E in vivo. Lipids 25:199–209.[Medline]
Ching, S., D. C. Mahan, J. S. Ottobre, and K. Dabrowski. 2001. Ascorbic acid synthesis in fetal and neonatal pigs and in pregnant and postpartum sows. J. Nutr. 131:1997–2001.[Abstract/Free Full Text]
Chung, Y. K., D. C. Mahan, and A. J. Lepine. 1992. Efficacy of dietary D--tocopherol and DL--tocopheryl acetate for weanling pigs. J. Anim. Sci. 70:2485–2492.[Abstract]
Cline, J. H., D. C. Mahan, and A. L. Moxon. 1974. Progeny effects of supplemental vitamin E in sow diets. J. Anim. Sci. 39:974. (Abstr.)
Csapo, J., T. G. Martin, Z. S. Csapo-Kiss, and Z. Hazas. 1996. Protein, fats, vitamin and mineral concentrations in porcine colostrum and milk from parturition to 60 days. Int. Dairy J. 6:881–902.
Drotleff, A. M., and W. Ternes. 2001. Determination of RS,E/Z-tocotrienols by HPLC. J. Chromatogr. A. 909:215–223.[Medline]
Ellis, R., and M. W. Vorhies. 1976. Effect of supplemental dietary vitamin E on serologic response of swine to an Escherichia coli bacterin. J. Am. Vet. Med. Assoc. 168:231–232.[Medline]
Grimble, R. F. 1997. Effect of antioxidative vitamins on immune function with clinical applications. Int. J. Vitam. Nutr. Res. 67:312–320.[Medline]
Hayak, M. G., G. E.. Mitchell, Jr., R. J. Hamon, T. S. Stahly, G. L. Cromwell, R. E. Tucker, and K. B. Barker. 1989. Porcine immunoglobulin transfer after prepartum treatment with selenium or vitamin E. J. Anim. Sci. 67:1299–1306.
Halpner, A. D., G. J. Handelman, C. A. Belmont, J. M. Harris, and J. B. Blumberg. 1998. Protection by vitamin C of oxidant-induced loss of vitamin E in rat hepatocytes. J. Nutr. Biochem. 9:355–359.
Hamilton, I. M. J., W. S. Gilmore, I. F. F. Benzie, C. W. Mulholland, and J. J. Strain. 2000. Interactions between vitamins C and E in human subjects. Br. J. Nutr. 84:261–267.[Medline]
Hedemann, M. S., and S. K. Jensen. 1999. Vitamin E status in newly weaned piglets is correlated to the activity of carboxyl ester hydrolase. Page 181 in Proc. Aust. Pig Sci. Assoc. Adelaide, Australia. (Abstr.)
Hidiroglou, M., T. R. Batra, E. R. Farnworth, and F. Markham. 1995. Effect of vitamin E supplementation on immune status and -tocopherol in plasma of piglets. Reprod. Nutr. Dev. 35:443–450.
Hidiroglou, M., E. Farnworth, and G. Butler. 1993. Vitamin E and fat supplementation of sows and the effect on tissue vitamin E concentrations in their progeny. Reprod. Nutr. Dev. 33:557–565.
Hooper, C. L., D. V. Maurice, S. F. Lightsey, and J. E. Toler. 2000. Factors affecting ascorbic acid biosynthesis in chickens. I. Adaptation of an assay and the effect of age, sex, and food deprivation. J. Anim. Phys. Anim. Nutr. 84:48–56.
Jensen, M., C. Fossum, M. Ederroth, and R. V. J. Hakkarainen. 1988. The effect of vitamin E in the cell-mediated immune response in pigs. J. Vet. Med. B. 35:549–555.
Jensen, M., A. Lindholm, and R. V. J. Hakkarainen. 1990. The vitamin E distribution in serum, liver, adipose and muscle tissues in the pig during depletion and repletion. Acta Vet. Scand. 31:129–136.[Medline]
Jensen, S. K. 1994. Retinol determination in milk by HPLC and fluorescence detection. J. Dairy Res. 61:233–240.[Medline]
Jensen, S. K., R. M. Engberg, and M. S. Hedemann. 1999a. All-rac--tocopherol-acetate is a better vitamin E source than all-rac--tocopherol-succinate for broilers. J. Nutr. 129:1355–1360.[Abstract/Free Full Text]
Jensen, S. K., A. K. B. Johansen, and J. E. Hermansen. 1999b. Quantitative secretion and maximal secretion capacity of retinol, beta-carotene and alpha-tocopherol into cow’s milk. J. Dairy Res. 66:511–522.[Medline]
Jensen, S. K., and K. N. Nyholm. 1996. Distribution of tocopherols, retinol, ß-carotene and fatty acids among fat globule membrane and fat globule core in cow’s milk. J. Dairy Res. 63:565–574[Medline]
Lauridsen, C., H. Engel, S. K. Jensen, A. M. Craig, and M. G. Traber. 2002. Lactating sows and suckling piglets preferentially incorporate RRR- over all-rac--tocopherol into milk, plasma and tissues. J. Nutr. 132:1258–1264.[Abstract/Free Full Text]
Lauridsen, C., M. S. Hedemann, and S. K. Jensen. 2001. Hydrolysis of tocopherol and retinyl esters by porcine carboxyl ester hydrolase is affected by their carboxylate moiety and bile acids. J. Nutr. Biochem. 12:219–224.[Medline]
Lauridsen, C., S. K. Jensen, L. H. Skibsted, and G. Bertelsen. 2000. Influence of supranutritional vitamin E and copper on -tocopherol deposition and susceptibility to lipid oxidation of porcine membranal fractions of M. psoas major and M. longissimus dorsi. Meat Sci. 54:377–384.
Lowry, O. H., and J. V. Passonneau. 1972. Pages 283–284 in A flexible system of enzymatic analysis. Academic Press, New York, NY.
Mahan, D. C. 1991. Assessment of the influence of dietary vitamin E on sows and offspring in three parities: Reproductive performance, tissue tocopherol, and effects on progeny. J. Anim. Sci. 69:2904–2917.[Abstract]
Mahan, D. C. 1994. Effects of dietary vitamin E on sow reproductive performance over a five-parity period. J. Anim. Sci. 72:2870–2879.[Abstract]
Mahan, D. C., Y. Y. Kim, and R. L. Stuart. 2000. Effect of vitamin E sources (RRR- or all-rac--tocopheryl acetate) and levels on sow reproductive performance, serum, tissue, and milk -tocopherol contents over a five-parity period, and the effects on the progeny. J. Anim. Sci. 78:110–119.[Abstract/Free Full Text]
Mahan, D. C., and L. J. Saif. 1983. Efficacy of vitamin C supplementation for weanling swine. J. Anim. Sci. 56:631–639.
du Manoir, J. M., B. N. Albright, G. Stevenson, S. H. Thompson, G. B. Mitchell, M. E. Clark, and J. L. Caswell. 2002. Variability of neutrophil and pulmonary alveolar macrophage function in swine. Vet. Immunol. Immunopathol. 89:175–186.[Medline]
Mason, V. C., S. Bech-Andersen, and M. Rudemo. 1980. Hydrolysate preparation for amino acid determinations in feed constituents. 8. Studies of oxidation conditions for streamlined procedures. Zeitschrift Tierphysiol., Tierernährg. Futterrmittelkde 43:146–164.
Moreira, I., and D. C. Mahan. 2002. Effect of dietary levels of vitamin E (all-rac-tocopheryl acetate) with and without added fat on weanling pig performance and tissue -tocopherol concentration. J. Anim. Sci. 80:663–669.[Abstract/Free Full Text]
NRC. 1998. Nutrient Requirements of Swine. 10th ed. Natl. Acad. Press, Washington, DC.
Panganumala, R. V., and D. G. Cornwell. 1982. The effects of vitamin E on arachidonic acid metabolism. Ann. N. Y. Acad. Sci. 393:376–391.[Medline]
Pehrson, B., N. Holmgren, and U. Trafikowska. 2001. The influence of parentally administered -tocopheryl acetate to sows on the vitamin E status of the sows and suckling piglets and piglets after weaning. J. Vet. Med. A 48:569–575.
Peplowski, M. A., D. C. Mahan, F. A. Murray, A. L. Moxon, A. H. Cantor, and K. E. Ekstrom. 1981. Effect of dietary and injectable vitamin E and selenium in weanling swine antigenically challenged with sheep red blood cells. J. Anim. Sci. 51:344–351.
Prinz, W., J. Bloch, G. Gilich, and G. Mitchell. 1980. A systematic study on the effect of vitamin C supplementation on the humoral immune response in ascorbate dependent mammals. Int. J. Vitam. Nutr. Res. 50:294–300.[Medline]
Schwager, J., and J. Schulze. 1998. Modulation of interleukin production by ascorbic acid. Vet. Immunol. Immunopathol. 64:45–57.[Medline]
Stoldt, W. 1952. Vorschlag zur Vereinheitlichung der Fettbestimmung in Lebensmitteln. Fette Seifen. 54:206–207.
Strober, W. 1992. Pages A.3.5.–A.3.7. in Current Protocols in Immunology. Vol. 2. J. E. Coligan, A. M. Kruisbeck, D. H. Margulies, E. M. Shevach, and W. Strober, ed. John Wiley & Sons, New York, NY.
Stoyanovsky, D. A., R. Goldman, R. M. Darrow, D. T. Organisciak, and V. E. Kagan. 1995. Endogenous ascorbic acid regenerates vitamin E in the retina directly and in combination with exogeneous dihydrolipoic acid. Curr. Eye Res. 14:181–189.[Medline]
Van Vleet, J. F. 1980. Current knowledge of selenium—Vitamin E deficiency in domestic animals. J. Am. Vet. Med. Assoc. 176:321–325.[Medline]
Vasankari, T. J., U. M. Kujala, T. M. Vasankari, T. Vuorimara, and M. Ahotupa. 1997. Increased serum and LDL antioxidant potential after supplementation in endurance athletes. Am. J. Clin. Nutr. 65:1052–1056.[Abstract/Free Full Text]
Weiser, H., and M. Vecchi. 1982. Stereoisomers of -tocopheryl acetate: Biopotencies of all eight stereosisomers, individually or in mixtures, as determined by rat resorption-gestation tests. Int. J. Vitam. Nutr. Res. 52:351–370.[Medline]
Yen, J. T., and W. G. Pond. 1981. Effect of dietary vitamin C addition on performance, plasma vitamin C and hematic iron status in weanling pigs. J. Anim. Sci. 5:1292–1296.
Zannoni, V., M. Lynch, S. Goldstein, and P. Sato. 1974. A rapid micromethod for the determination of ascorbic acid in plasma and tissues. Biochem. Med. 11:41–48.[Medline]
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Abstract
Introduction
