Bovine Neutrophil Responses to Parenteral Vitamin E1
`
`J. S. HOGAN, W. P. WEISS, D. A. TODHUNTER, K. L. SMITH,
`and P. S. SCHOENBERGER
`Department of Dairy SCience
`Ohio Agricultural Research and Development Center
`The Ohio State University
`Wooster 44691
`
`ABSTRACT
`
`Twenty-eight Holsteins were tested to
`detennine effects of dietary and paren(cid:173)
`teral vitamin E supplementation during
`the dry period on plasma a-tocopherol
`and in vitro neutrophil functions at calv(cid:173)
`ing. Cows were assigned to one of four
`experimental groups
`receiving either
`supplemental dietary vitamin E,
`injec(cid:173)
`tions of vitamin E, both dietary and in(cid:173)
`jections of vitamin E, or neither source
`of supplemental vitamin E during the dry
`period in a 2 x 2 factorial arrangement.
`Cows receiving parenteral vitamin E
`were injected subcutaneously with 3000
`IU of vitamin E (dl-a-tocopherol) at 10
`and 5 d prior to anticipated calving.
`Cows not receiving parenteral vitamin E
`were injected with a placebo. Experi(cid:173)
`mental groups receiving dietary vitamin
`E during the dry period were supple(cid:173)
`mented with 1040 IU/d compared with
`none for controls. Cows injected with
`vitamin E had
`greater
`plasma a(cid:173)
`tocopherol concentration 5 d after the
`first injection, at calving, and 1 wk after
`calving than did cows injected with
`placebo. Plasma a-tocopherol concentra(cid:173)
`tions did not differ between dietary vita(cid:173)
`min E treatment groups from calving
`through 4 wk postpartum. No interaction
`was found between dietary and paren(cid:173)
`teral supplementation of vitamin E on
`plasma a-tocopherol concentration. Neu(cid:173)
`trophils from cows injected with vitamin
`
`Received September 9, 1991.
`l'ccep~ October 7, 1991.
`Salaries and research support were provided by state
`and federal funds appropriated to the Ohio Agricultural
`Research and Development Center, The Ohio State Uni(cid:173)
`versity. Manuscript Number 185-91.
`
`E had greater intracellular kill of bacteria
`atc~ing than ~~oo~hils from
`placebo-injected cows. Neither phagocy(cid:173)
`tic index nor percentage of neutrophils
`phagocytizing differed between vitamin
`E-injected and placebo-injected cows.
`Dietary vitamin E during the dry period
`had no effect on neutrophil fimction at
`calving. Intracellular kill and plasma a(cid:173)
`tocopherol were correlated at calving.
`(Key words: vitamin E, neutrophils,
`parenteral)
`Abbreviation key: HBSS = Hanks balanced
`salt solution.
`
`INTRODUCTION
`
`Dietary deficiencies in vitamin E were asso(cid:173)
`ciated with increased prevalence of mastitis
`(13). Plasma vitamin E concentrations in dairy
`cows are normally lowest when rates of IMI
`are highest and when neutrophil functions are
`depressed. Rates of IMI and clinical mastitis
`are highest during the first 7 d after calving
`compared with other stages of lactation (12,
`21~. !he hi~ rate of new IMI at calving
`comcldes WIth reported immunosuppression
`and decreased bactericidal activity by neu(cid:173)
`trophils during wk 1 of lactation (15). Plasma
`vitamin E concentrations in multiparous cows
`also decrease approximately 7 d prior to calv(cid:173)
`ing and remain depressed for 7 to 14 d after
`calving (22, 23). The relationship among these
`occurrences may be that vitamin E is critical in
`protecting neutrophils from the destructive ac(cid:173)
`~on of toxic ?xygen molecules necessary for
`mtracellular kill of ingested pathogens (l 14
`18).
`'
`,
`Decreased plasma a-tocopherol during the
`periparturient period is due in part to decreased
`feed intake during this period. Plasma a(cid:173)
`toc~herol concentrations
`are
`sensitive
`to
`changes in consumption of vitamin E in dairy
`
`1992 I Dairy Sci 75:399--40S
`
`399
`
`Par Pharm., Inc.
`Exhibit 1009
`Page 001
`
`

`
`400
`
`HOGAN ET AL.
`
`cows. Concentration of plasma a-tocopherol
`and dietary intake both decreased during the
`periparturient period even though the amount
`of dietary vitamin E offered to animals was
`constant throughout this period (22). Adminis(cid:173)
`tration of vitamin E to late gestation cows in a
`manner other
`than in feed may maintain
`plasma concentrations of vitamin E. Parenteral
`administration of vitamin E elevated plasma
`vitamin E successfully during early lactation
`(11). The purpose of this study was to deter(cid:173)
`mine whether injections of vitamin E prior to
`calving maintain a-tocopherol concentration in
`plasma and prevent suppression of neutrophil
`function at calving.
`
`MATERIALS AND METHODS
`
`Experimental Design
`
`Twenty-eight Holsteins in the Ohio Agri(cid:173)
`cultural Research and Development Center
`herd were assigned to one of four experimental
`groups balanced by parity, milk production in
`the previous lactation, and IMI status at drying
`off. Cows were dried off 60 d before antici(cid:173)
`pated calving by abrupt cessation of milking,
`and all four quarters were infused with a dry
`cow antibiotic product containing 300 mg of
`cephapirin (Tomorrow, Franklin Laboratories,
`Amarillo, TX).
`Experimental groups received either supple(cid:173)
`mental dietary vitamin E (n = 7), injections of
`vitamin E (n = 9), both dietary and injected
`vitamin E (n = 6), or neither source of supple(cid:173)
`mental vitamin E (n = 6) during the dry period
`in a 2 x 2 factorial arrangement. Only cows
`that calved within ± 3 d of anticipated calving
`were used. Cows receiving parenteral vitamin
`E were injected (total 12 ml) ~th 3000 IU of
`vitamin E (dl-a-tocopherol) in a 20% ethyl
`alcohol and 1% benzyl alcohol emulsifiable
`solution (Rocavit-E, Hoffman-LaRoche, Nut(cid:173)
`ley, NJ) at 10 and 5 d prior to anticipated
`calving. Cows not receiving parenteral vitamin
`E were injected at 10 and 5 d prior to antici(cid:173)
`pated calving with a placebo (12 ml) contain(cid:173)
`ing the alcohol emulsifiable solution void of
`vitamin E. Injections were subcutaneous on the
`upper part of the rib cage just posterior to the
`scapula.
`Dry cows were fed approximately 6 kg of
`late vegetative grass silage,
`.9 kg of concen-
`
`Journal of Dairy Science Vol. 75, No.2, 1992
`
`trate, and 3.5 kg of mature grass hay daily
`(OM basis). The concentrate mix was 96.5%
`com, 1.5% limestone, 1.5% trace-mineralized
`salt, and .5% vitamin premix. Each kilogram
`of concentrate provided 60,000 IU of vitamin
`A and 20,000 IU of vitamin D. All cows
`received a basal amount of 300 IU/d of vita(cid:173)
`min E. Rations
`for
`the two experimental
`groups receiving dietary vitamin E during the
`dry period were supplemented with an addi(cid:173)
`tionall040 IU/d Dry cow diets were adequate
`in all other nutrients,
`including .3 ppm of
`selenium. All cows were fed the same diet
`following parturition. The lactating cow diet
`was adequate as specified by the NRC (16),
`including .3 ppm. of selenium and 500 mg of
`supplemental vitamin E. Feeds were analyzed
`monthly for a-tocopherol and a-tocopherol
`acetate (23). Cows were group fed during the
`dry period until 10 d prior to anticipated calv(cid:173)
`ing. Cows were fed individually from 10 d
`prior to calving until 4 wk after calving.
`Blood samples were collected from all cows
`at drying off, 30 d after drying off, 10 d and 5
`d prior to anticipated calving, within 48 h after
`calving, and I, 2, and 4 wk postpartum to
`detennine a-tocopherol concentrations. Sam(cid:173)
`ples 10 and 5 d prior to calving were collected
`before cows received injections. Plasma was
`collected, and a-tocopherol was analyzed as
`described previously (14, 23). Glutathione per(cid:173)
`oxidase activity (17) was measured from
`whole blood samples collected at drying off,
`calving, and 4 wk into lactation.
`
`Neutrophil Assay
`
`Blood samples were collected from all cows
`within 48 h after calving and I, 2, and 4 wk
`into lactation to detennine in vitro neutrophil
`function. Blood samples were collected as
`described by Carlson and Kaneko (6). 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 for
`differential
`counts
`(Oiff-Quik; AHS
`del
`Caribe, Inc., Aguada, Puerto Rico). Cell prepa(cid:173)
`rations averaged (X ± SD) 94.6 ± 2.8% neu(cid:173)
`trophils and 95.9 ± 1.4% viability. Cell con(cid:173)
`centrations were adjusted to 25 x 1()6 viable
`neutrophils/ml of HBSS.
`
`Par Pharm., Inc.
`Exhibit 1009
`Page 002
`
`

`
`PARENTERAL VITAMIN E
`
`401
`
`50 t4.5
`
`4.0
`
`::: II
`2.5
`
`2.0
`
`•
`9
`
`INJECTED E
`CONTROL
`
`1.5
`1.0 --'-'_~_----L_---L_-'--_----'---_---"-_
`D-O C-l0
`C-5 C+O 1 WK 2 WK 4 WK
`
`Figure 1. Mean (± SE) plasma concentrations of vita(cid:173)
`min E in cows injected either with 3000 IU of vitamin E
`(n = 15) or placebo (n = 13) at 10 and 5 d prior to
`anticipated calving. Samples were collected at drying off
`(0-0); 10 d (C-lO) and 5 d (C-5) prior to anticipated
`calving; within 48 h after calving (C+O); and I, 2, and 4
`wk after calving.
`
`tested were Escherichia
`Bacteria
`coli
`(McDonald 487). Escherichia coli 487 is a ~
`strain originally isolated from a clinical case of z
`i
`bovine mastitis. Prior to testing, bacteria were
`stored in trypticase soy broth containing 20% E:
`glycerin at -70·C. A total of .1 rol of thawed >:
`stock culnrre was inoculated into 12 ml of
`<r:
`trypticase soy broth and incubated overnight at ~
`if)
`j
`37·C on a gyratory shaker at 200 rpm. A .2-ml
`portion of the overnight culture was inoculated
`(J....
`into 24 ml of fresh trypticase soy broth and
`incubated for 2.5 h at 37·C and 50 rpm. Bacte-
`ria were centrifuged and resuspended in HBSS.
`Bacterial cultures were diluted in HBSS to
`70% transmission at 540 nm (Beckman DU-50
`Spectrophotometer, Beckman
`Instruments,
`Fullerton, CA). Bacteria were opsonized in
`20% serum for 20 min at 20·C. Serum for
`opsonization was collected from 9 lactating
`cows, pooled, and heated to 56"C for 30 min to
`inactivate complement. Bacteria were diluted
`to approximately 75 x 1()6 cfo/ml.
`Phagocytosis and intracellular kill of bacte(cid:173)
`ria by neutrophils were measured by modifica(cid:173)
`tions of the fluorochrome assay described by
`Goldner et aI. (9). Briefly, suspensions of neu(cid:173)
`trophils and opsonized bacteria were added to
`incubation tubes in a ratio of 3:1 (bacteria:
`neutrophils) and incubated at 37·C at 100 rpm
`on a gyratory shaker for 90 min. Bacterial
`numbers were confirmed by removing a por(cid:173)
`tion of assay suspension prior to incubation,
`serially diluting bacteria, and plating bacteria
`on trypticase soy agar. Following incubation,
`samples were removed and diluted 2: 1: I as
`assay suspension:acridine orange (14 mg/loo
`ml of PBS):crystal violet (50 mg/loo ml of
`PBS). Wet mount slides were prepared, and
`the number of live (green bacterial cells) and
`dead (red bacterial cells) bacteria were counted
`in the first 50 neutrophils visible under 1000 x
`oil immersion magnification while the stage of
`the fluorescence microscope (Nikon Fluores(cid:173)
`cence Microscope, Nikon Inc., Garden City,
`NJ) was moved horizontally from the left to
`right edges of the cover slip. Phagocytic index
`was calculated as average number of bacteria
`phagocytosed per neutrophil. Intracellular kill
`was determined as (number of dead phagocy(cid:173)
`tosed bacterialnumber of live + number of
`dead intracellular bacteria x 100). Percentage
`of neutrophils phagocytizing was calculated.
`All assays were in duplicate and were con-
`
`ducted blind: laboratory personnel did not have
`prior knowledge of cow, day of lactation, or
`experimental group identification.
`
`Statistical Analyses
`
`blood
`whole
`a-tocopherol,
`Plasma
`glutathione peroxidase, and neutrophil function
`data were analyzed using least squares analysis
`of variance. Models included the main effects
`of injection, diet, and the interaction between
`injection and diet. Ratio of bacteria:neutrophil
`in each neutrophil assay was unrelated to treat(cid:173)
`ment but was a significant (P < .05) effect on
`phagocytic index and intracellular kill. Ratio of
`bacteria:neutrophil was included as a covariate
`to adjust for neutrophil assay variability un(cid:173)
`related to treatment. Data were analyzed within
`sample period. Relationships among plasma
`concentrations of a-tocopherol and in vitro
`neutrophil functions were quantified using lin(cid:173)
`ear and multiple regression (20).
`
`RESULTS
`
`Cows injected with vitamin E had greater
`plasma a-tocopherol concentration 5 dafter
`the first injection, at calving, and 1 wk after
`calving than did cows injected with placebo
`(Figure 1; P < .05). Differences between
`plasma a-tocopherol concentrations in vitamin
`E and placebo-injected cows were not signifi(cid:173)
`cant 2 and 4 wk after calving. Cows fed diets
`
`Journal of Dairy Science Vol. 75, No.2, 1992
`
`Par Pharm., Inc.
`Exhibit 1009
`Page 003
`
`

`
`402
`
`HOGAN ET AL.
`
`of vitamin E on plasma a-tocopherol concen(cid:173)
`tration. Whole blood glutathione peroxidase
`was adequate in all cows and unrelated to
`either dietary or parenteral vitamin E (P > .05).
`Mean (± SD) glutathione peroxidase (millien(cid:173)
`zyme units per milligram of hemoglobin)
`among all cows was 77.8 ± 15.1, 79.5 ± 12.2,
`and 75.2 ± 13.7 at drying off, calving, and 4
`wk into lactation, respectively.
`Cows injected with vitamin E had greater
`intracellular kill of bacteria at calving than did
`placebo-injected cows (P < .05). Mean (± SE)
`intracellular kill was 80.1 ± 3.1 for vitamin E(cid:173)
`injected cows and 70.8 ± 4.4 for placebo(cid:173)
`injected cows (Figure 3). Differences in in(cid:173)
`tracellular kill between injection groups were
`not significant at 1, 2, and 4 wk after calving.
`Intracellular kill was greater at wk 2 and 4 of
`lactation than at calving within placebo(cid:173)
`injected cows (P < .05). Intracellular kill did
`not differ among sampling periods within vita(cid:173)
`min E-injected cows. Neither phagocytic index
`(Figure 4) nor percentage of neutrophils
`phagocytizing (Figure 5) differed between vita(cid:173)
`min E-injected and placebo-injected cows.
`Cows fed supplemental vitamin E during the
`dry period had greater intracellular kill at 4 wk
`after calving than cows
`fed control diets
`(Figure 6). Intracellular kill did not differ be(cid:173)
`tween dietary groups at either calving, 1 wk, or
`
`3.0
`>< 2.8
`2.6
`..........
`2.4
`U 2.2
`..........
`E--<
`>-< 2.0
`U 1.8
`0
`c.J
`1.6
`<r:::r:
`1.4
`1.2
`1.0
`
`~
`
`QZ
`
`0...
`
`CALVING 1 WK
`2WK 4WK
`Figure 4. Mean (± SE) phagocytic index of neutrophils
`isolated from cows injected with either 3000 IU of vitamin
`E (n =15) or placebo (n =13) 10 and 5 d prior to calving.
`Neutrophils were collected within 48 h after calving and I,
`2, and 4 wk after calving.
`
`4.5
`
`4.0
`
`3.5
`
`3.0
`
`1.5
`
`1:£1
`
`ZS
`
`if)
`
`S
`<r:
`E-<
`.......>-
`2.5
`<r:
`::E 2.0
`j
`P-.
`
`DIETARY E
`•
`V CONTROL
`
`1. 0 L - ' - -_ ' - -_ ' - -_ ' - -_ ' - -_ ' - - - - - ' ' - -
`D-O C-10 C-5 C+O 1 WK 2 WK 4 WK
`
`Figure 2. Mean (± SE) plasma concentrations of vita(cid:173)
`min E in cows fed diets either supplemented (n = 13) or
`nnsupplemented (n = 15) with vitamin E during the dry
`period. Samples were collected at drying off (D-O); 10 d
`(C-IO) and 5 d (C-S) prior to anticipated calving; within
`48 h after calving (C+O); and 1,2, and 4 wk after calving.
`
`supplemented with vitamin E during the dry
`period had greater plasma a-tocopherol lad
`prior to anticipated calving than did cows fed
`unsupplemented diets (Figure 2; P < .05).
`Plasma a-tocopherol did not differ between
`dietary vitamin E treatment groups at 5 d prior
`to anticipated calving, at calving, or during
`early lactation. No interaction was found be(cid:173)
`tween dietary and parenteral supplementation
`
`• INJECTED E
`
`CONTROL
`
`v
`
`90
`
`80
`
`70 ,
`
`.......4
`.......4
`..........
`~
`
`~
`
`60 I
`4 WK
`2 WK
`CALVING 1 WK
`Figure 3. Mean (± SE) in vitro intracellular 1::iIl of
`bacteria by neutrophils isolated from cows injected with
`either 3000 IU of vitamin E (n =15) O£ placebo (n =13)
`10 and 5 d prior to calving. Neutrophils were collected
`within 48 h alta calving and 1,2, and 4 wk after calving.
`
`Journal of Dairy Science Vol. 75, No.2, 1'992
`
`Par Pharm., Inc.
`Exhibit 1009
`Page 004
`
`

`
`PARENTERAL VITAMIN E
`
`403
`
`80
`
`70
`
`60
`
`• INJECTED E
`
`CONTROL
`
`7
`
`90
`
`80
`
`70
`
`......:1
`......:1
`,.......
`~
`
`~
`
`0
`Z,.......
`N,.......
`Eo-<
`>-<
`U
`0
`0
`
`-<:r::
`
`0...
`~
`
`50 L------'-__--'-
`CALVING 1 WK
`
`' - -__--'----_
`2 WK
`4 WK
`
`60 L---L__----..L__-----.J'---__-'--_
`CALVING 1 WK
`2 WK
`4 WK
`
`Figure 5. Mean (± SE) percentage of neutrophils
`pbagocytizing from cows injected with either 3000 IU of
`vitamin E (n = 15) or placebo (n = 13) 10 and 5 d prior to
`calving. Neulrophils were collected within 48 h after
`calving and I, 2, and 4 wk after calving.
`
`Figure 6. Mean (± SE) in vitro intracellular kill of
`bacteria by neutropbils isolated from cows fed dry period
`diets either supplemented (n = 13) or unsupplemented (n =
`15) with vitamin E. Neutrophils were collected within 48 h
`after calving and I, 2, and 4 wk after calving.
`
`2 wk after calving. Dietary vitamin E treat(cid:173)
`ments had no effect on either phagocytic index
`or percentage of neutrophils
`(Figure 7)
`phagocytizing (Figure 8). No interactions were
`found between dietaIy and parenteral supple(cid:173)
`mentation of vitamin E on neutrophil func(cid:173)
`tions.
`The correlation coefficient between intracel(cid:173)
`lular kill and plasma <X-tocopherol was r = .41
`at calving (P < .05). Correlation coefficients
`between intracellular kill
`and plasma a(cid:173)
`tocopherol at wk 1, 2, and 4 of lactation were
`not significant. Phagocytic index and percent(cid:173)
`age of neutrophils phagocytizing were not cor(cid:173)
`related with plasma a-tocopherol.
`
`DISCUSSION
`
`concentrations
`a-tocopherol
`Plasma
`decrease dramatically during the periparturient
`period and may be sensitive to changes in
`consumption of vitamin E in dairy cows (22,
`23). Concentration of plasma a-tocopherol
`typically drops by approximately 50% and re(cid:173)
`mains low during the first weeks of lactation,
`even when dietary vitamin E offered to cows is
`constant throughout this period. Likewise, die(cid:173)
`tary supplementation of vitamin E during the
`dry period did not maintain plasma a-
`
`tocopherol at calving and during the fIrst
`weeks of lactation in the present study. Admin(cid:173)
`istration of vitamin E to late gestation cows in
`a manner other than in feed was tested as a
`means to maintain plasma concentrations of
`vitamin E. Parenteral administration of vitamin
`E successfully elevated plasma a-tocopherol
`during late gestation and early lactation peri(cid:173)
`ods.
`Vitamin E enhances host defenses against
`infections by improving phagocytic cell func(cid:173)
`tion. Vitamin E is an antioxidant that protects
`phagocytic cells and surrounding tissues from
`oxidative attack by free radicals produced by
`the respiratory burst of neutrophils and macro(cid:173)
`phages during phagocytosis (1, 2, 3). The res(cid:173)
`piratory burst by neutrophils is characterized
`by marked changes in oxygen metabolism that
`result
`in increased production of superoxide
`and hydrogen peroxide (1). Vitamin E is local(cid:173)
`ized in cellular membranes in close proximity
`to oxidase enzymes that initiate the production
`of free radicals. Polyunsaturated fatty acids
`located in the vicinity of the oxidase enzymes
`are protected from peroxidation by vitamin E
`(2). Vitamin E inhibits autoxidation of polyun(cid:173)
`saturated fatty acids in neutrophil membranes
`(4, 5) and enhances neutrophil function (2).
`Impaired neutrophil function in cows during
`
`Journal of Daily Science Vol. 75, No.2, 1992
`
`Par Pharm., Inc.
`Exhibit 1009
`Page 005
`
`

`
`404
`
`3.0
`:x:
`~ 2.8
`Cl
`2.6
`Z
`....... 2.4
`U 2.2
`........
`E-<>-- 2.0
`U
`1.8
`0
`0
`1.6
`-<
`::r:
`1.4
`0...
`1.2
`1.0
`
`HOGAN ET AL.
`
`• DIETARY E
`
`CONTROL
`
`V
`
`CALVING 1 WK
`
`2 WK
`
`4 WK
`
`0
`Z
`.......
`N
`.......
`E-<
`>--
`U
`0
`0
`-<
`:I:
`0...
`
`~
`
`80
`
`70
`
`60
`
`50
`
`• DIETARY E
`
`CONTROL
`
`V
`
`I
`
`CALVING 1 WK
`
`2 WK
`
`4 WK
`
`Figure 7. Mean (± SE) phagocytic index of neutrophils
`isolated from cows fed dry period diets either supplrr
`mented (n = 13) or unsupplemented (n = 15) with vitamin
`E. Neutrophils were collected witbin 48 h after calving
`and I, 2. and 4 wk after calving.
`
`Figure 8. Mean (± SE) percentage of neutrophils
`phagocytizing from cows fed dry period diets either sup(cid:173)
`plemented (n = 13) or unsupplemented (n = 15) with
`vitamin E. Neutrophils were collected witbin 48 h after
`calving and I, 2, and 4 wk after calving.
`
`the periparturient period is thoroughly docu(cid:173)
`mented (10, 15).
`Impainnent of neutrophil
`microbicidal mechanisms
`and membrane(cid:173)
`associated activities of chemokinesis and in(cid:173)
`gestion were reported in cows during wk 1
`after calving (15). Specifically, neutrophil
`functions associated with the oxidative burst of
`metabolism were altered. These neutrophil im(cid:173)
`pairments were similar to depressed neutrophil
`activity in vitamin E-deficient animals (5, 14).
`Cows that received vitamin E injections main(cid:173)
`tained intracellular kill by neutrophils and
`plasma concentrations of vitamin E in the pres(cid:173)
`ent study. Subcutaneous injections of vitamin
`E approximately 10 and 5 d prior to calving
`negated the suppression of in vitro intracellular
`kill of E. coli by neutrophils at calving.
`In(cid:173)
`tracellular kill by blood neutrophils from cows
`receiving prepartum injections of vitamin E
`was constant from calving to wk 4 of lactation.
`Neutrophils
`from cows
`injected with the
`placebo had depressed intracellular kill at calv(cid:173)
`ing compared with those from cows 2 and 4
`wk postpartum. Parenteral vitamin E supple(cid:173)
`mentation
`had no effect on phagocytic
`parameters in this study.
`Vitamin E supplementation of diets in(cid:173)
`creased intracellular kill of E. coli and Staphy(cid:173)
`lococcus aureus by blood neutrophils collected
`
`Journal of Dairy Science Vol. 75, No.2, 1992
`
`from cows during peak milk production (14).
`Dietary vitamin E supplementation during the
`dry period had no effect on intracellular kill
`and phagocytosis by neutrophils collected dur(cid:173)
`ing the first 2 wk after calving in the present
`study. Cows fed supplemental vitamin E dur(cid:173)
`ing the dry period had greater intracellular kill
`at 4 wk after calving than cows fed control
`diets during the dry period. A physiological
`explanation for
`this difference is unknown,
`because all cows were fed the same supple(cid:173)
`mented diet from calving to wk 4 of lactation.
`In addition, there was no relationship between
`plasma vitamin E and intracellular kill at wk 4,
`whereas this relationship was significant at
`calving.
`
`CONCLUSIONS
`
`The coefficient of determination between
`intracellular kill and plasma a-tocopherol
`in
`cows at calving was r2 = .17. Unaccounted
`variability in intracellular kill possibly in(cid:173)
`cluded effects of other variables implicated as
`causes of suppressed neutrophil functions at
`calving, such as increased blood concentrations
`of ACTII and other stress-mediated factors
`associated with parturition (10, 19). Increased
`oxidation of membranes occurs during periods
`
`Par Pharm., Inc.
`Exhibit 1009
`Page 006
`
`

`
`PARENTERAL VITAMIN E
`
`405
`
`of stress (4, 8). Increasing the concentration of
`natural antioxidants in blood may be useful to
`maintain host defenses against disease at calv(cid:173)
`ing and early lactation. This is documented by
`the present study and by an earlier report (7)
`that
`in vitro supplementation of phagocytes
`collected during the periparturient period with
`13-carotene also resulted in enhanced intracellu(cid:173)
`lar kill. However, both a-tocopherol and 13(cid:173)
`carotene
`plasma
`concentrations
`usually
`decrease at calving. Prepartum subcutaneous
`injections of vitamin E may be an effective
`management practice to maintain plasma a(cid:173)
`tocopherol concentrations and to optimize neu(cid:173)
`trophil function during early lactation.
`
`REFERENCES
`
`1 Baboir, B. M. 1984. The respiratory burst of phago(cid:173)
`cytes. J. Coo. Invesl. 73:599.
`2 Baehner, R. L., L. A Boxer, J. M. Allen, and J. Davis.
`1977. Autoxidation as a basis for altered function by
`polymorphonuclear leukocytes. Blood 50:327.
`3 Baker, S. S., and H. J. Cohen. 1983. Altered oxidative
`metabolism in selenium-deficient rat granulocytes. J.
`ImmunoI. 130:2856.
`4 Bendich, A. 1990. Antioxidant micronutrients and im(cid:173)
`mune responses. Ann. New York Acad. Sci. 587:168.
`5 Boxer, L. A 1986. Regulation of phagocyte function
`by a-tocopherol. Proc. Nutr. Soc. 45:333.
`6 Carlson, G. P., and J. J. Kaneko. 1973. Isolation of
`leukocytes from bovine peripheral blood. Proc. Soc.
`Exp. BioI. Med. 142:853.
`7 Daniel, L. R., B. P. Chew, T. S. Tanaka, and L. W.
`Tjoelker. 1991. f!-Carotene and vitamin A effects on
`bovine phagocyte function in vitro during the peripar(cid:173)
`tum period. J. Dairy Sci. 74:124.
`8 Duthie, G. G., J. R. Author, F. Nicol, and M. Walker.
`1989. Increased indices of lipid peroxidation in slress(cid:173)
`susceptible pigs and effects of vitamin E. Res. Vel
`Sci. 46:226.
`9 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. 5:291.
`
`10 Guidry, A J., M. J. Paape, and R. E. Pearson. 1976.
`Effects of parturition and lactation on blood and milk
`cell concentrations, corticosteriods, and neutrophil
`phagocytosis in the cow. Am. J. Vel. Res. 37:1195.
`11 Hidiroglou, M. 1989. Mammary transfer of vitamin E
`in dairy cows. J. Dairy Sci. 72:1067.
`12 Hogan, J. S., K. L. Smith, K. H. Hoble!, P. S. Schoen(cid:173)
`berger, D. A Todhunter, W. D. Hueston, D.
`E.Pritehard, G. L. Bowman, L. E. Heider, B. L.
`Brockett, and H. R. Conrad. 1989. Field survey of
`mastitis in low somatic cell count herds. J. Dairy Sci.
`72:1547.
`13 Hogan, J. S., K. L. Smith, and W. P. Weiss. 1989.
`Update on nutrition and mastitis. Page 38 in Proc.
`Nat!. Mastitis Counc., Louisville, KY. Nail. Mastitis
`Counc., Arlington, VA
`14 Hogan, J. S., K. L. Smith, W.P. Weiss, D. A. Todhun(cid:173)
`ter, and W. L. Shockey. 1990. Relationships among
`vitamin E, selenium, and bovine blood neutrophils. J.
`Dairy Sci. 73:2372.
`15 Kehrli, M. E., B. J. Nonnecke, and J. A. Roth. 1989.
`Alterations in bovine neutrophil function during the
`periparturient period. Am. J. Vet. Res. 50:207.
`16 National Research Council. 1988. Nutrient require(cid:173)
`ments of dairy cattle. 6th ed. Nat!. Acad. Sci., Wash(cid:173)
`ington, DC.
`17 Paglia, D. E., and W. N. Valentine. 1967. Studies on
`the quantitative and qualitative characterization of
`erythrocyte glutathione peroxidase. J. Lab. Clin. Med.
`70:158.
`18 Putnam, M. E., and N. Comben. 1987. Vitamin E.
`Vet. Rec. 121:541.
`19 Roth, J. A, M. L. Kaeberle, and W. H. Hsu. 1982.
`Effects of AClH administration on bovine polymor(cid:173)
`phonuclear leukocyte function and lymphocyte blasto(cid:173)
`genesis. Am. J. Vel. Res. 43:412.
`20 SAS® User's Guide: Statistics, Version 5 Edition.
`1985. SAS Inst., Inc., Cary, NC.
`21 Smith, K. L., D. A. Todhunter, and P. S. Schoenberg(cid:173)
`er. 1985. Environmental mastitis: cause, prevalence,
`prevention. J. Dairy Sci. 68:1531.
`22 Weiss, W. P., J. S. Hogan, K. L. Smith, and K. H.
`Hoblet. 1990. Relationships among selenium, vitamin
`E, and mammary gland health in commercial dairy
`herds. J. Dairy Sci. 73:381.
`23 Weiss, W. P., D. A Todhunter, J. S. Hogan, and K. L.
`Smith. 1990. Effect of duration of supplementation of
`selenium and vitamin E on periparturient cows. J.
`Dairy Sci. 73:3187.
`
`Journal of Dairy Science Vol. 75, No.2, 1992
`
`Par Pharm., Inc.
`Exhibit 1009
`Page 007