Relationships Among Vitamin E, Selenium, and Bovine Blood Neutrophils1

NUTRITION, FEEDING, AND CALVES Relationships Among Vitamin E, Selenium, and Bovine Blood Neutrophils1 J. S. HOGAN, K. L. SMITH, W. P. WEISS, D.A.TODHUNTER, and W. L.SCHOCKEY Department of Dairy ScIence The Ohio Stale University Ohio Agricultural Research and Development center Wooster 44691

ABSTRACT

Effects of vitamin E and selenium supplementation on in vitro phagocytosis and intracellular kill of bacteria by bovine neutrophils were investigated. Diets were not supplemented with vitamin E and selenium during the dry period and first 21 d of lactation. Cows were then assigned to one of four treatment diets for 30 d. Treatment diets were either unsupplemented or supplemented with vitamin E, selenium, or both vitamin E and selenium, in a 2 x 2 factorial arrangement. Peripheral blood neutrophils were is0lated from each cow on lactation d 51. Vitamin E supplementation of diets increased intracellular kill of Staphylococcus aureus and Escherichia coli by neutrophils. Intracellular kill of S. aureus was greater in neutrophils isolated from selenium supplemented cows than in neutrophils from cows without supplemental selenium. Intracellular kill of E. coli did not differ between neutrophils from selenium supplemented and selenium unsupplemented cows. Ability of neutrophils to phagocytize either S. aureus or E. coli was independent of vitamin E and selenium. (Key words: vitamin E, selenium, and neutrophils) INTRODUCTION

Research has shown that incidence and severity of mastitis are related to vitamin E and

Received 1uly 26,1989. Accepted March 7,1990. 1Salaries and research support were provided by state and federal funds appropriated 10 the Ohio Agriculture Research and Deveiopment Center, 1be Ohio State University. Manuscript Number 213-89. 1990 1 Dairy Sci 73:2372-2378

Se status of a dairy herd. Vitamin E and Se supplementation of dairy cows resulted in reduced rates and duration of intramammary infections and incidence of clinical mastitis (23, 24). Field surveys concurred with results of these controlled studies. Erskine et al. (11) reported a negative correlation between percentage of quarters infected with major mastitis pathogens and mean herd glutathione peroxidase (GSHpx; E.C. 1.11.1.9) activity in whole blood. A subsequent survey of herds with low bulk tank milk sec revealed negative correlations between dietary vitamin E intake and rates of clinical mastitis, plasma Se and rates of clinical mastitis, and plasma Se and bulk tank. milk sec (28). Vitamin E and Se appear to enhance host defenses against infections by improving phagocytic cell function. Both vitamin E and GSHpx are antioxidants that protect phagocytic cells and surrounding tissues from oxidative attack by free radicals produced by the respiratory burst of neutrophils and macrophages during phagocytosis (1,2,3). Neutrophils from cows fed Se-deficient diets had reduced intracellular killing of Candida albicans (5, 6), Staphylococcus aureus (14, 15), and Escherichia coli (14). Vitamin E has also been reported to enhance neutrophil function in mice (17) and humans (2, 7, 8). However, data are limited on the effects of dietary supplementation of vitamin E on function of bovine neutrophils. The purpose of the present study was to determine the effects of vitamin E and Se supplementation on in vitro phagocytosis and intracellular kill of S. aureus and E. coli by bovine neutrophils. MATERIALS AND METHODS Experimental Design

Experimental cows were 21 multiparous Holsteins in the Ohio Agricultural Research

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VITAMIN E, SBLBNIUM, AND NBUTROPHILS

and Development Center dairy herd. Diets prior to drying off were Se (.3 ppm total diet) and vitamin E (400 to 600 mg/cow per d) supplemented. During the dry period and first 21 d of lactation, diets for experimental groups were void of supplemental vitamin E and Se. Dry cows were fed approximately 6 kg of late vegetative grass silage DM, .9 kg of concentrate DM, and mature grass hay ad libitum daily. The concentrate mix was 96.5% com, 1.5% limestone, 1.5% trace-mineralized salt, and .5% vitamin premix. Each kilogram of concentrate provided 15,000 IU vitamin A and 10,700 IU vitamin D. From calving to lactation d 21, cows were fed diets containing 30% corn silage, 24% alfalfa hay, and 45% concentrate (Table 1) on a DM basis. Cows were randomly assigned to one of four vitamin E, or Se, or vitamin E plus Se supplementation groups on lactation d 21. Cows were assigned to diets in a 2 x 2 factorial arrangement. Cows were maintained on diets for 30 d Diets were those fed from calving to lactation d 21 with the exception of vitamin E or Se supplementation (Table 1). Cows fed Sesupplemented diets were injected subcutaneously on lactation d 21 with 50 mg sodium selenite suspended in 680 ill dl-toeopherol acetate (MuSe, Schering Animal Health, Union, NJ). Vitamin E, selenium, and Glutathione Peroxidase

Blood samples were collected from each cow on the day of drying off, calving, and lactation d 21 and 51 for vitamin E, Se, and GSHpx analyses. Plasma a-tocopherol was determined by injecting a hexane extract of plasma into a HPLC equipped with a 4 mm x 25 em reverse phase column (Val-U-Pak. Regis Chemical Co., Morton Grove, IL) and UV detector set at 285 nm. The solvent used was initially 80% methanol, 20% butanol, and changed linearly to 50% methanol, 50% butanol over 5 min. Flow rate was 1.1 ml/min. Selenium concentrations were determined in plasma and whole blood (20). Glutathione peroxidase activity in whole blood was measured by the procedures outlined by Pagalia and Valentine (21). Preparation of Blood Neutrophlls

Blood samples were collected on lactation d 51 for isolation of neutrophils. Blood samples

TABLE 1. Ingredient composition of concentrate mix fed to lactating cows.

Bar com, ground Soybean meal Dehydrated alfalfa

59.8

26.0 8.0 2.2 2.0

Dicalcium phosphate Molasses Trace mineral salt Urea

.6

.4

Potassium sulfate Magnesium oxide Yitamin premixl Trace mineral premix2

.3 .3 .3 .1

lYitamin premix provided 12,000 IU vitamin A, 900 IU vitamin D. and either 0 or 90 mg dl-a-tocopherol acetate! kg concentrate DM.

en.

2Mineral premix provided 55 mg and either 0 or .45 mg Se (from sodium selenate)/kg concentrate DM

were collected as described by Carlson and Kaneko (9). Samples were centrifuged and erythrocytes lysed as detailed by Shuster (22). The final cell preparations were washed twice in Hanks' balanced salt solution (HBSS; pH 7.2). Viable cells were determined by trypan blue exclusion and counted with a hemocytometer. A portion of each final cell preparation was stained (Diff-Quik; AHS del Caribe, Inc., Aguada, Puerto Rico) for differential counts. Cell preparations averaged (mean ± SD) 87 ± 6% neutrophils, 11 ± 6% mononuclear cells, and 96 ± 1% viability. Cell concentrations were adjusted to 8 x 1rF viable neutrophils/ml of HBSS. Bacteria

Bacteria tested were S. aureus ATCC 29740 and E. coli McDonald 487. Prior to testing, bacteria were stored separately in tryticase soy broth (BBL Microbiology Systems, Cockeysville, MD) containing 20% glycerin at -70·C. A total of .1 ml of thawed stock culture was inoculated into 12 ml of trypticase soy broth and incubated overnight at 37'C on a gyratory shaker at 200 rpm. Cells were washed twice in .15 M phosphate-buffered saline (PBS) and resuspended in HBSS. Bacterial cultures were diluted in HBSS to percentage transmission of 74% for S. aureus and 70% for E. coli at 540 om (Beckman DU-50 Spectrophotometer, Beckman Instruments, Fullerton, CA) and incubated in 20% homologous serum for 20 min at Journal of Dairy Science Yol. 73,

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HOGAN ET AL.

20·C. Serum was collected from nine lactating cows, pooled, and heated to 56"C for 30 min to inactivate complement. Bacteria were diluted to approximately 120 x 1()6 cfulml.

30

c

Neutrophil Assays

Phagocytosis and intracellular kill of bacteria by neutrophils were measured by modifications of the fluorochrome assay described by Goldner et al. (13) and Grasso (14). Briefly, suspensions of neutrophils and opsonized bacteria were added to incubation tubes in a ratio of 15:1 (bacterial cfu:neutrophils) and incubated at 37·C at 100 rpm on a gyratory shaker for 90 min. Viable bacteria counts were confirmed by removing a portion of assay suspension prior to incubation, serially diluting bacteria, and plating bacteria on Trypticase soy agar (19). Following incubation, samples were removed and diluted 2:1:1 as assay suspension: acridine orange (14 mg/1oo ml PBS):crystal violet (50 mg/loo ml PBS). Wet mount slides were prepared and number of live (green bacterial cells) and dead (red bacterial cells) bacteria counted in the first 50 neutrophils visible under 1,OOOx oil immersion magnification while the microscope (Nikon Fluorescence Microscope, Nikon Inc., Garden City, NJ) stage was moved horizontally from the left to right edges of the cover slip. Staphylococcus aureus and E. coli were tested separately. Phagocytic ability was calculated as average number of bacteria phagocytosed per neutrophil Percentage intracellular kill was determined as (number of dead phagocytosed bacteria/number of live + number of dead phagocytosed bacteria) detailed by Grasso (14). Percentage intracellular kill data include all phagocytosed bacteria, because dead bacteria that were phagocytosed could not be distinguished from bacteria that were killed intracellularly. Mean percentage of dead bacterial cells in control assays incubated without neutrophils were determined subsequently as number of dead bacteria per total bacteria counted. All assays were in duplicate. Assays were conducted blind: laboratory personnel had no prior knowledge of cow or experimental group identification. Statistical Analyses

Differences among vitamin E, Se, and GSHpx values were tested by analysis of variance. Main effects were day of sample collecJournal of Dairy Science Vol. 73,

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10 L.

.

Drying Ofr

Calving

Day

Day

21

51

Figure 1. Whole blood Se in cows fed diets void of Se supplementation from drying off to lactation d 21. Cows were fed diets either supplemented (Se+; n = 9) or unsupplemented (Se--; n = 12) with Se from d 21 to 51 of lactation.

tion and experimental groups. Phagocytic ability and percentage intracellular kill were tested by analysis of covariance. Main effects were vitamin E, Se, and vitamin E x Se interaction. Covariants for phagocytic ability and intracellular kill were colony-forming units per milliliter of bacteria in neutrophil assays. Phagocytic ability and percent intracellular kill data were normalized by loglo and arcsine transformations, respectively. Relationships among blood and plasma values with phagocytosis and intracellular kill were tested by Pearson's correlation coefficient (25). Arithmetic means are presented for clarity. RESULTS Vitamin E, Selenium, and Glutathione Peroxidase

Cows injected with sodium selenite and fed diets supplemented with Se (Se+) had greater (P<.05) concentrations of Se in whole blood (Figure 1) and plasma (Figure 2) on lactation d 51 than did cows that were neither injected nor fed supplemental Se (So-). Selenium concentrations in whole blood and plasma of group Sowere unchanged between lactation d 21 and 51 (b.05) compared with an 18% (P<.05) increase in whole blood and 73% (P<.05) increase in plasma Se concentrations in Se+ cows during this period. Glutathione peroxidase activity was also greater in whole blood (P<.05)

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VITAMIN E, SELENIUM, AND NEUTROPHll..S

90 .

Se-

...... -

------+9 Se-

L Drying Off

_ Calving

30~­

Day

Day

21

51

Figure 2. Plasma Se in cows fed diets void of Se supplementation from drying off to lactation d 21. Cows were fed diets either supplemented (Se+; n = 9) or unsupplemented (Se-; n = 12) with Se from d 21 10 51 of lactation.

of Se+ cows than Se- cows. Mean whole blood GSHpx activity in Se+ cows remained constant between lactation d 21 and 51 (P>.05) compared with a 16% (P<005) decrease in activity in Se- cows (Figure 3). Selenium and GSHpx values did not differ (P>.05) between Se+ and Se- cows at either drying off, calving, or lactation d 21. Mean (±SE) micrograms of a-tocopherol per milliliter plasma at lactation d 51 for cows fed diets supplemented with vitamin E (E+) was 5.0 ± .6 compared with 3.2 ± .3 (P<.05) in cows fed diets without supplemental vitamin E (E-). Plasma concentrations of a-tocopherol did not differ (P>.05) between E+ and E- cows at drying off, calving, or lactation d 21 (Figure 4). Vitamin E in the sodium selenite injection did not increase plasma a-tocopherol concentration in Se+E- cows compared with SE-E+ cows at lactation d 51 (P>.05).

Drying Off

Vitamin E and Se supplementation of diets increased percentage intracellular kill of S. aureus (Figure 5). Mean (±SE) percentage intracellular kill by neutrophils from E+ cows was 60.5 ± 4.1 compared with 51.7 ± 2.1 by neutrophils from E- cows (P.05). The

Day

Day

21

51

Figure 3. Whole blood glutathione peroxidase activity in cows fed diets void of Se supplementation from drying

off to lactation d 21. Cows were fed diets either supple>mented (Se+; n = 9) or unsupplemented (Se-; n = 12) with Se from d 21 to 51 of lactation.

correlation coefficient between percentage intracellular kill of S. aureus and whole blood selenium at d 51 of lactation was r = .49 (P<.05). Correlations between percentage intracellular kill of S. aureus and plasma vitamin E, Se, and whole blood GSHpx were not significant (P>.05). The colony-forming units per milliliter in assays did not influence intracellular kill of S. aureus (P>005). Cows fed E+ diets had greater intracellular kill of E. coli than did cows fed E- diets (Figure 6). Vitamin E-supplemented cows averaged 60.1 ± 4.3% intracellular kill of E. coli

_~ :::j ~

Neutrophil Assays

Cah'ing

1:

E+

a

t

~ 4.00

Drying Off

Calving

Dey Zl

Day ~l

Figure 4. Plasma a-toeopherol in cows fed diets void of vitamin E supplementation from drying off to lactation d 21. Cows were fed diets either supplemenled (E+; n =9) or nnsupplemented (E-; n = 12) with vitamin E from d 21 to 51 of lactation. Journal of Dairy Science Vol. 73, No.9, 1990

2376

HOOAN BT AL.

"f;

-:::

:::l ~

::J

=c

<:Q

'%J

''-

--::

d

Oil CJ

~

Q

:::l

'-

-'"'"

oS

~

--'"-..

~

'"

~

'l;

so

f:

E+

(11=9)

E-

(11=121

Se+

Se-

(11=91

(11=121

E+

E-

111=91

(11=1.2)

Se+ (n=9)

Se-

(n=l:~)

Figure 5. Percentage of dead Staphylococcus aureus in blood polymorphonuclear leucocytes isolated from cows fed diets supplemented (+) or unsupplemented (-) with vitamin E (E), Se, or both.

Figure 6. Percentage of dead Escherichia coli in blood polymorphonuclear leucocytes isolated from cows fed diets supplemented (+) or unsupplemented (-) with vitamin E (E), Se, or both.

compared with 41.9 ± 4.4% for cows fed Ediets (P<.OI). The correlation coefficient between percentage intracellular kill of E. coli and plasma vitamin E at d 51 of lactation was r = .43 (P<.05). Mean intracellular kill of E. coli by neutrophils from Se+ cows (53.0 ± 4.4) did not differ (P>.05) from that for neutrophils from So- cows (47.3 ± 4.2). Interactions between vitamin E and Se supplementation on intracellular kill of E. coli were not significant (P>.OS). Correlations between intracellular kill of E. coli and whole blood GSHpx, plasma Se and whole blood Se were not significant (P>.05). The colony-forming units per milliliter in assays did not influence intracellular kill of E. coli (P>.05). The ability of neutrophils to phagocytize either S. aureus or E. coli were independent of vitamin E and Se supplementation of diets (fable 2). Mean phagocytic abilities of neutrophils isolated from all groups was 9.1 ± .3 for S. aureus and 5.1 ± .3 for E. coli. Colony-forming units per milliliter did not influence phagocytic ability in either S. aureus or E. coli assays (fable 2; P>.05). Mean percentage dead bacteria in control assays without neutrophils was 13% S. aureus and S% E. coli.

DISCUSSION

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The respiratory burst by neutrophils is characterized by marked changes in oxygen metabolism that result in increased production of superoxide and hydrogen peroxide (1). Though neutrophil-generated oxygen metabolites are necessary in antimicrobial defense mechanisms, these free radicals can also damage the neutrophil and surrounding tissues (29). Vitamin E and GSHpx are both cellular antioxidants that protect against the cytotoxic capabilities of oxygen metabolites. Vitamin E inhibits autoxidation of polyunsaturated fatty acids in neutrophil membranes (2, 7) and enhances neutrophil function (2). Dietary supplementation of vitamin E did increase intracellular kill by bovine neutrophils, but did not influence phagocytic ability in the present study. These results differ from those in which dietary supplementation of vitamin E increased phagocytosis and decreased intracellular kill by human neutrophils (2). The bovine neutrophil responses to vitamin E in the present study were more comparable to bovine neutrophil responses to dietary supplementation of Se (5, 6, 14, 15) than human neutrophil responses to vitamin E. Selenium deficiencies in cows were associated with decreased blood concentrations of

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VITAMIN E, SELENIUM, AND NEUTROPIllLS

TABLE 2. Phagocytic ability of blood neuttophils from cows fed diets supplemented (+) or unsupplemented (-) with vitamin E (E) and Se.

Staphylococcus aureus E+ Phagocytic ability 1

X

cfu/ml in assay2

± SE X ± SE

.92 .05 8.21

.10

E-

Se+

Se-

E+

B-

.98 .05

.94 .04 8.23 .08

.71 .06 8.27

.70 .06 8.43 .II

.99 .04 8.21 .06

Escherichia coli

8.19 .08

.13

Se+

Se-

.72

.69 .05 8.33 .10

.06

8.41 .16

INumber of bacteria phagocytosed (logI0)/neutrophil. 2colony-forming units of bacteria pet milliliter in each assay. Neutrophil counts were adjusted to a final concentration of 7.12 10glO/lIll.

GSHpx and intracellular kill by neutrophils (14, 15). Selenium supplementation also maintained whole blood GSHpx activity and increased in~ tracellular kill of S. aureus by neutrophils in the present experiment. Although means did not differ between groups, neutrophils from Sesupplemented cows also tended to have greater intracellular kill of E. coli than did neutrophils from cows receiving no Se supplementation. The Se containing enzyme GSHpx can protect neutrophils by detoxifying peroxides in the cytosol (4). The GSHpx activity in both supplemented and unsupplemented cows in this trial was greater than GSHpx activity reported for cows that had increased intracellular kill of microbes in a previous study following .1 ppm Se supplementation (14). These data lend further support to the recommendation that cows be supplemented with .3 ppm Se in the diet compared with .1 ppm supplementation (10). Phagocytic ability of neutrophils was not altered by Se supplementation in the present or previous studies (5, 6, 14). The effects of vitamin E and Se supplementation on intracellular kill were not additive. Supplementation with both vitamin E and Se did not result in greater intracellular kill of bacteria by neutrophils than did supplementation with either one of the nutrients alone. These data suggest that vitamin E and GSHpx have sparing effects on the requirements for one another relative to intracellular killing of bacteria. The protection afforded cellular membranes by vitamin E may spare the requirement for GSHpx by oxidizing free radicals at the membrane, thereby preventing leakage of free radicals into the cytosol and maintaining intracellular killing capacity of the cell. Conversely,

GSHpx activity in the cytosol may have spared the requirement for vitamin E in the membranes. Another factor that may have influenced this sparing effect is the relative rates of metabolism for vitamin E and Se. Fischer and Whanger (12) reported the vitamin E was metabolized more rapidly in Se-deficient rats than in Se-supplemented rats. Such a relationship in cows could explain why vitamin E effects on neutrophil function were more evident than Se effects, because all cows were fed Se deficient diets during the dry period and first 21 d of lactation. However, the transfer and retention rates of vitamin E and Se into bovine neutrophils are unknown. Although intracellular kill by neutrophils differed among experimental groups, correlations with blood and plasma vitamin E, Se, and GSHpx explained no more than 24% of variability among cows in intracellular kill. Harrison and Conrad (16) reported different rates of change in Se content and GSHpx activity among various tissues, plasma, and blood of dairy cows after short-term feeding. The rapid increase in plasma Se compared with whole blood Se in supplemented cows during the present study was indicative of blood prof'tles after short-term Se supplementation (26, 27). Similarly, intraperitoneal injection of dl-a-tocopherol resulted in different transfer rates into milk: and plasma (18). Selective absorption of vitamin E and Se by tissues suggest that neutrophil GSHpx activity, Se, and vitamin E content may be more closely related to neutrophil function than plasma or blood concentrations. A greater understanding of vitamin E and Se adsorption by bovine neutrophils is needed to optimize dietary supplementation of dairy cows. Journal of Dairy Science Vol. 73,

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HOGAN ET AL.

REFERENCES 1 Baboir, B. M.I984.1be respiratory burst ofphagocytes. 1. Clin. Invest 73:599. 2 Baehner, R. L., L. A. Boxer, 1. M Allen, and 1. Davis. 1977. Autoxidation as a basis for altered function by polymorphonuclear leukocytes. Blood 50:327. 3 Baker, S. S., and H. 1. Cohen. 1983. Altered oxidative metabolism in selenium-deficient rat granulocytes. 1. Immunol. 130:2856. 4 Bass, D. A., L. R. DeChaIelet, R. F. Burk, P. Shirley, and P. Szejda. 1977. Polymorphonuclear leukocyte bactericidal activity and oxidative metabolism during glutathione peroxidase deficiency. Infect. Immun. 18:78. 5 Boyne, R., and 1. R. Arthur. 1979. Alterations of neutrophil function in selenium-deficient cattle. 1. Compo Pathol. 89: 15 I. 6 Boyne, R., and 1. R. Arthur. 1981. Effects of selenium and copper deficiency on neutrophil function in cattle. 1. Compo Pathol. 91:271. 7 Boxer, L. A. 1986. Regulation of phagocyte function by a-tocopherol. Proc. Nutr. Soc. 45:333. 8 Boxer, L. A., 1. M Oliver, S. P. Speilberg, 1. M. Allen, and J. D. Schulman. 1979. Protection of granulocytes by vitamin E in glutathione synthetase deficiency. New England 1. Med. 301:901. 9 Carlson, G. P., and 1. 1. Kaneko. 1973. Isolation of leukocytes from bovine peripheral blood. Proc. Soc. Exp. BioI. Med. 142:853. 10 Erskine, R.I., R.I. Eberhart, P.I. Grasso, and R. W. Scholz. 1989. Selenium and mammary resistance to infectious disease. Page 119 in Am. Assoc. Bovine Pract, West Lafayette, IN. 21stAnnu. Conv. Proc., Am. Assoc. Bovine Pract., Calgary, AB, Can. 11 Erskine, R. I.,R. 1. Eberhart, L.I. Hutchinson, andR. W. Scholz. 1987. Blood selenium concentrations and glutathione peroxidase activities in dairy herds with high and low somatic cell counts. 1. Am. Vet. Med. Assoc. 190: 1417. 12 Fischer, W. C., and P. D. Whanger. 1977. Effects of selenium deficiency on vitamin E metabolism in rats. 1. Nutr. Sci. Vitaminol. 23:273. 13 Goldner, M, H. Farkas-Himsley, A. Kormendy, and M. Skinner. 1983. Bacterial phagocytosis monitored by fluorescence and extracellular quenching: ingestion and intracellular killing. Lab. Med. 5:29 I. 14 Grasso, P. 1. 1987. Phagocytosis, bactericidal activity, and oxidative metabolism of mammary neutrophils from selenium-adequate and selenium-deficient dairy cows. M.S. Thesis, The Pennsylvania State Univ., University Park. 15 Gyang, E. 0., 1. B. Stevens, W. G. Olson, S. D. Tsitsamis, and E. A. Usenik. 1984. Effects of seleniumvitamin E injection on bovine polymorphonucleated leukocytes phagocytosis and kiI1ing of Staphylococcus

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aureus. Am. 1. Vet. Res. 45:175. 16 Harrison, 1. H., and H. R. Conrad. 1984. Selenium content and glutathione peroxidase activity in tissues of the dairy cow after short-term feeding. 1. Dairy Sci. 67: 2464. 17 Heinzerling, R. H., R. P. Tenderdy, L. L. Wick, and D. C. Lueker. 1974. VitaminE protects mice against Diplococcus pneumoniae type I infection. Infect. Immun. 10: 1292. 18 Hidiroglou, M 1989. Mammary transfer of vitamin E in dairy cows. 1. Dairy Sci. 72:1067. 19 Nonnecke, B. 1., and K. L. Smith. 1984. Inhibition of mastitis bacteria by bovine apo-Iactoferrin evaluated by in vitro microassay of bacterial growth. 1. Dairy Sci. 67: 606. 20 Olson, O. E., I. Palmer, and E. Cary. 1975. Modification of the official flourometric method for selenium in plants. J. Assoc. Offic. Agric. Chern. 58:117. 21 Pagalia, D. E., and W. N. Valentine. 1967. Studies on the quantitative and qualitative characterization of erythr0cyte glutathione peroxidase. 1. Lab. Clin. Med. 70:158. 22 Shuster, D. E. 1985./n vitro effects of anti-inflammatory agents on bovine neutrophils stimulated with opsonized Staphylococcus aureus. M.S. Thesis, Univ. Kentucky, Lexington. 23 Smith, K. L., 1. H. Harrison, D. D. Hancock, D. A. Todhunter, and H. R. Conrad. 1984. Effect of vitamin E and selenium supplementation on incidence of clinical mastitis and duration of clinical symptoms. 1. Dairy Sci. 67:1293. 24 Smith, K. L., H. R. Conrad, B. A. Amiet, P. S. Schoenberger, and D. A. Todhunter. 1985. Effect of vitamin E and selenium dietary supplementation on mastitis in first lactation dairy cows. 1. Dairy Sci. 68(Suppl. 1):190. (Abstr.) 25 Sokal.,R. R., andF.I.Rohlf.1981. Biometry. 2nd ed. W. H. Freeman Co., San Francisco, CA. 26 Thompson, K. G., A. 1. Fraser, B. M. Harrop, and 1. A. Kirk. 1980. Glutathione peroxidase activity in bovine serum and erythrocytes in relation to selenium concentrations of blood, serum, and liver. Res. Vet. Sci. 28:321. 27 Weiss, W. P., V. F. Colenbrander, M D. Omningham, and C. 1. Callahan. 1983. Selenium/vitamin E: role in disease prevention and weight gain of neonataI calves. 1. Dairy Sci. 66:1101. 28 Weiss, W. P., 1. S. Hogan, K. L. Smith, and K. H. Hoblet. 1990. Relationships among selenium, vitamin E, and mammary gland health in commercial dairy herds. 1. Dairy Sci. 73:381. 29 Weiss, S. 1., and A. F. LoBuglio. 1982. Phagocyticgenerated oxygen metabolites and cellular injury. Lab. Invest. 47:5.