J Dairy Sci 942557-2565 doi103168jds2010~3980

copy American Dairy Science Associationreg 2011

Effects of changing milk replacer feedings from twice to once daily on Holstein calf innate immune responses before and after weaning 1

L E Hulbertt C J Cobb J A Carrollt and M A Ballou2 Animal and Food Sciences Texas Tech University Lubbock 79409 tUSDA-ARS Livestock Issues Research Unit Lubbock TX 79403

ABSTRACT

The objectives of this study were to determine the effects of witching Holstein calves to once-daily feedshying during the fourth week of life (24 plusmn 23 d of age once-fed 11 twice-fed n = 22) OIl innate immune responses and to evaluate whether carry-over effects occurred when the calves were weaned during the sevshyenth week of life Peripheral blood samples were taken immediately before the change in feeding strategy (24 d of age) and at 27 31 45 48 52 and 66 d of age and were analyzed for circulating cortisol haptoglobin total leukocvte counts neutrophilmononuclear cells and hemato~rit percentage Heparinized whole blood was also stimnlated with lipopolysaccharide (LPS) for 24 h and the concentration of tumor necrosis factor-a (TNF-a) in the supernatant was analyzed Neutrophil L-selectin ami l2-integTin expression were analyzed by flow cytometry Simultaneous neutrophil phagocytic and oxidative burst responses to a heat-killed Escherishychia coli were quantified by dual-color flow-cytometry Treatment (once-daily or twice daily feeding) had no effect on pre- or postweaning performance Once-fed calves tended to have more circulating neutrophils at 27 d of age greater expression of L-selectin on n~utroshyphUs at 31 and 45 d of age and mtensity of phagocytosis at 45 d of age Once-fed calves secreted Ipss TF-n in LPS-stilllulted whole hlood cultures at 45 cl of age compared with twice-fed calves and this tended to persist through the immediate postweaning period None of the other immune parameters differed after weaning between the preweaning feeding strate-

Consolidating calf milk replacer into one feeding during the fourth week of life was li~ely a mild ~r~d acute stresor as evidenced by tranSIent neutrophIlIa in the absenc~ of suppressed functional capacities of

Hcceived Novcmber 2 2fllO Febrllarv 2 2011 of trad~ namcs or commercial in this article

is solely for the purpose of providing information and does not illlply recommendation Or endorsement by the US Department of Agriculture ~

CoITcoponding iluthor michadballou(91ttuedu

neutrophils Future research should address the mechashynism and immunological significance of the persistent decreased TNF-o response in once-fed calves Key words feeding frequency innate immunity stress

INTRODUCTION

Feeding milk or milk replacer twice daily to dairy calves is labor intensive A management that decreases calf-rearing costs due to reductions in time and labor is to feed milk or milk replacer only once daily Galton and Brakel (1976) reported a 39 deshycrease in labor when calves were fed once daily versus twice daily Moreover calves fed once daily performed as well as calves fed twice daily Feed intake BV gain body composition feed efficiency and morbidity did not differ between calves fed once versus twice daily (Ackerman et al 1969 Galton and Brake 1976 Keshyhoe et aI 2007)

Changes in feeding strategies including frequency and composition can be perceived as a tressor animals This perceived stress could modulate the innate immune response thereby decreasing the resistance to disease No research has determined the influence of changing feeding frequency to once daily on the innate immune responses of preweaning dairy calves Therefore the objectives of this study were to determine the effects of switching Holstein calves to once-daily feeding during

the fourth week of life on innate immune responses and to evaluate whether carry-over effects existed when the calves were weaned during the seventh week of life

MATERIALS AND METHODS

The experiment wa conducted from March to April 2010 All animal procedures were reviewed and apshyproved by the Texas Tech University Animal Care and Use Committee Forty-four Holstein bull calves (24 to 48 h after birth) were purchased from 2 local comshymercial dairies over a 7-d period Seventeen additional bull calves purchased from the same farms in the ame period were also used in this study these calves w~re weaned early (at 24 plusmn 23 d of age) and used as b1Oshy

2557

2558 HULBERT ET AL

Table 1 Calf starter ingrcltii(llt aud chemical compositions

Item AlllOllllt

Ingredient of DM SWalIl-flakld com 673 Soybean meal 18 CP 131 Cot tOlls(~ccl Jlwal 113 1Ioias 51 Calf mincralvitamin 21

Chcmical composition mI 882 CP 189 ~IE Mcalkl Wfi

Prcmix cuntainc(l (DM basis) 5~6 limestone 399 soybean meal 6 salt OHi I ~inc sulfftw n41lt bdeuium selenite 02 0267 11111gI1(8C oxide Olllii vitamin E SOO IUg 0157 copper sulfate 142 lllg(kg vltal~lU A LOUO kIU12 IV mgkg of ethylenediftmine dlhyltirOlochdc 8 I mgkg cobalt carbonfttc

logical control during the weaning stage of the experishyment All calves were fed 38 L of pooled first-milking colostrum from their respective dairy within 12 h of birth and all calve were trarmported approximately 60 km to the Hilmar Cheese Calf Research Facilitv at Texas Tech University (New Deal TX) Calves ~ere housed with straw bedding in commercial polyethylene calf hutches (Agri-Plastics Tonawanda NY)

Feeding and Weaning Strategies

Before assignment to feeding strategy treatments all calves were fed 227 g on an as-fed basis of a 20 proshytein-20 fat milk replacer (Herd ~Iaker Land OLakes Animal Protein Co Shoreview MN) in 2 L of water twice daily at 0800 and 1600 h After the first week all calves were offered ad libitum access to a calf starter (Table 1) and water for the remainder of the study The quantity of calf tarter was adjusted daily for approxishymately 15 refusals At 24 23 d of age all calves (476 plusmn 41 kg of BW) were completely randomized to 1 of 2 feeding-strategy treatment once-fed (n = 22) or twice-fed (n = 22) Beginning on this day once-fed calves were given 454 g as fed of the milk replacer in 4 L of water at the 0800 h feeding whereas twice-fed calves had no changes to their original feeding regimen Although previous research (Ackerman et aL 1969 Galton and Brakel 1976) reported that dairy calves can be started on a once-daily feeding regimen at 3 d of age without influencing performance during the preweaning period compared with calves fed twice daily we decided to first evaluate switching to the once-daily regimen during the wk1 life because the risk of enteric disease is reduced at this age compared with during the first 3 wk of life (Ballou and DePeters 2008) Future research should determine whether switching to onceshydaily feeding during the first week of life is stressful and suppresses innate immune responses

Journal of Dairy Science Vol 94 No 5 2011

Once-fed and twice-fed calves were all weaned 21 d after treatment enrollment 45 plusmn 23 d of age) Early-weaned calves were weaned 21 d earlier (24 plusmn 21 d of age) ~Weaning began by restricting the intake of lIlllk replacer by 50 Twice-fed calves had their 1600 h milk replacer withdrawn and ollce-fed calves had 50( of ~heir milk replacr withdrawn at the 0800 h feeding to 227 g (as~fed baSIS) Calves were completelv weaned ~hen they consumed 900 g of calf starter (s-fed bashySlS~ for 2 consecutive days Calves were individuallv weIghed at arrival and at 31 45 52 and 66plusmn 23 d of age

Blood Collection and Analyses

~ine milliliters of peripheral blood was collected via jugular venipuncture immediately before assignment of treatments (age 24 plusmn 23 d) and at 27 31 52 and 66 plusmn 23 d of age Samples had to be collected into 2 blocks on 2 consecutive days to accOlIDnoclate the logistics of running the immune function assays The block (A or B) was assigned randomly to calves within their treatment (11 = 11 twice-fed and n 11 once-fed per block) Blood was collecttd into 2 evacuated blood ~ollection tubes (6 and 3 mL) containing heparin and ImmedIately placed on ice Vithin 1 h after collection the 3-mL Vacntainers from each calf were analyzed for hematocrit total leukocyte counts and differential analyses of neutrophils aud mononuclear cells using a Cell Dyn 3700 with automated 50-sample loader and vet-package software (Abbott Laboratories Abbott IL) In addition the neutrophilmoIlonllclear cell ratio (NM) was calculated Plasma was collected after a 15-min centrifugation at 1200 x 9 and stored at~80degC until analyzed for cortisol and haptoglobin concentrashytions All plasma samples were analyzed in duplicate Circulating concentrations of cortisol were determined using a commercially available competitive-binding Chemiluminescence-ELISA kit (Searchlight-Allshon BioSystems Inc Billerica vIA) The illtra- and iushyterassay coefficients of variation were 58 and 72 respectively for the cortisol assay Plasma haptoglobin concentrations were determined by measuring haptoshyglobinhemoglobin complex by the estimation of difshyferences in peroxidase activity (Makimura and Suzuki 1982 Arthington et aL 20(3) Results were expressed in arbitrary units resulting from the absorption reading at 450 nm x 100 The intra- and interastmy coefficients of variation were 18

Phagocytosis and Oxidative Burst Capacity of Neutrophils

The simultaneous phagocytic (PG) and oxidative burst (OB) capacities 01 peripheral bloo(l neutrophils

2559 EFFECT OF ONCE- VERSUS TWICE-DAILY MILKING ON IMMUNE RESPONSES

in response to enteropathogpnic Escherichia coli isolatshyed from the spleen of a tlepticemic calf were analyzed The E coli were grown overnight in tryptic soy broth and quantified by serial dilution and spread plating on tryptic soy agar The bacteria were heat-killed at 60degC for 30 min (killing was confirmed by spread plating on tryptic soy qgar) washed and resuspended at 109 cfn mL and incubated for 2 h under constant agitation at room temperature with an equal volume of 100 JigmL propidium iodide (Invitrogen Carlsbad CA) Killed propidium iodide-labeled bacteria were washed twice and resuspended at 109 cfllmL in 1 x PBS Bacteria were aliquoted into I-mL volumes and stored at -80degC Two hundred microliters of whole blood from the 6-mL Va(utainer per animal was incubated in an ice bath for 15 min Forty microliters of a 100 ~dvf working conshycentration of dihydrorhodamine (Invitrogen) and the nuorescently labeled E coli were added to each sample vortexed thoroughly and then placed in a 385degC water bath and incubated for 10 min After completion of incubation the samples were immediately placed in an ice bath for 5 min to stop the reaction at a conshystant rate Erythrocytes were hypotonically lysed and washed and the leukocytes were analyzed by dualshycolor now cytometry using a Cell Lab Quanta SC flow cytometer (Beckman Coulter Fullertoll CA) Using flow cytometer analysis software (QuantaSC 1IPL Beckman Coulter) neutrophils were gated on the scatshyterplot of electric volume x side scatter Neutrophils that were positive for both oxidative burst (OB+) and phagocytosis (PG +) were then gated using the FL-l by FL-3 scatterplot The OB+PG+ cell percentage and geometric mean nuorescence intensity of the FL-l and FL-3 were analyzed for OB and PG respectively Flow cytometry compensation settings were determined using E coli that were not nuorescently labeled with propidium iodide

Determination of Neutrophil Leukocyte Adhesion Molecules

Two hundred microliters of whole blood from the 6-mL Vacutainer for each animal was incubated at a final dilution of 5 I1gmL of anti-bovine CD62L (monoshyclonal antibody IgG1-isotype made in mouse VMRD Pullman WA) or 25 ~~gmL of anti-bovine CD18 (the i3Tintegrin chain of CDlla b and c heterodimers monoclonal antibody IgGl-isotype made in mouse VMRD) for 1 h in an ice bath Erythrocytes were hyshypotonically lysed and then rinsed once The leukocyte pellet wac resuspended in fluorescein-labeled secondshyary antibody at a 1AOO dilution [F(ab)2 anti-monse IgGFITC AbD Serotec Raleigh NC] Samples were

incubated on ice for an additional 1 h Samples ere then washed once using 1 x PBS and then analyzcd bv single-color flow cytometry Using the now cytomet~r analysis software neutrophils were on he scatshyterplot diagram of electric volume x side scatter The total geometric mean fluorescence intensity (FL-l) for L-selectin or p-rintegrin was analyzed

LPS Stimulation of Whole Blood

Whole blood was diluted 15 with RPMI 1640 (Inshyvitrogen) containing 1 antibiotics (Gibco AntihioticshyAntimycotic Invitrogen) Whole blood was stimulated at a final concentration of 1 [1grnL of LPS (E coli OlllB4 Sigma-Aldrich) Samples were incubated for 24 h in a humidified CO2 chamber The cell culture plates were centrifuged for 10 min at 1200 x 9 at 4degC The supernatant fraction was collected and tored in a _tonc freezer until analyzed for bovine TNF-oc usshying a commercially available ELISA (DY2270E RampD Systems Minneapolis MN) Standards were diluted in RPMI with 10 pooled bovine plasma and 1 antibishyotics The intra- and interassay coefficients of variation were 32 and respectively and the sensitivity of the assay was 125 pgmL

General Statistical Analysis

All data were analyzed by REML ANOVA using the MIXED procedure of SAS (vO2 SAS lnst Inc Cary NC) The ante-regressive (1) covariance structure for the within-subject measurement was used for all models Repeated data were tested for normality of the residuals evaluating the Shapiro-~Wilk statistic using the UNIVARIATE procedme of SAS (vO2 SAS lust Inc) Data that were not normally distributed were log-transformed before mixed model analysis Pairwise differences were performed at each time llsing a slicedshyeffect multiple comparison approach with a TukeyshyKramer adjustment Leac)t squares means (plusmnSEM) are reported throughout A treatment difference of P 005 was considered significant and P 010 was conshysidered a tendency

Statistical Analysis for the Acute Effects of Once- Versus Twice-Daily Feeding

Data to test the acute effects of once- versus twiceshydaily feeding included data from 24 27 31 and middot15 plusmn 23 d of age Baseline measurements taken illlmediately before assigning feeding strategy treatments (d 24) were tested as covariates in all statistical models For this model only the data from once-fed and twice-fed calves

Journal of Dairy Science Vol 94 No5 2011

2560 HULBERT ET AL

Table 2 Pcrfonnallcc data for calves fed once or twice daily from d 24 to 151

Largest hell) Once Twice SEN P-vaillc

ADG d 2middotj to 0637 0616 00596 0802 d 31 to 15 004 0628 00275 O5() d24t045 0615 0624 00218 0764 d 45 to 52 0993 0859 00629 OUl d 52 to 66 0662 OS86 00522 0300 d45to66 O77 0677 00420 0109

Starter intake kg of D)l d 24 to 1 121 123 061 O78J d45to66 307 2J6 104 0468

Fccdain kg of DIfkg of BW d 24 to middotj5 lG5 162 0043 0764 d 45 to (i6 206 231 0115 01gt2

Calve were fed 227 of milk replacer twice daily umil 24 plusmn 0 5 d of age After 24 d of age 22 calves (ol1eeshyfed) had the evening milk feeding withdravll and all 454 g of milk repliteer Willi fed in the morning whoreas twice-fed calves (11 20) had 110 dumge in f(odin strategy All calvos were wcaned at 45 d of age

were used For all metabolic and immune response data excluding data analyzed by flow cytometry a linear mixed model with the fixed effects of treatment time and the interactions of treatment x time and block x treatment x time was fitted The random effect Wl13

calf tested within treatment x block Before statistically analyzing all the fJow cytometry

data the data from the once-fed calves were divided by the block mean of the data from the twiee-fed calves to account for daily variation resulting from flow cytomshyetry settings All flow cytometry data for the ollce-fed calves are presented as the percentage of twice-fed calf means The fixed effects of time and the interaction of time x block were fitted The random effect was calf nested within block

Statistical Analysis for Carry-Over Effects on Weaning

Data that were used to test the carry-over effects of once-fed versus twice-fed on innate immule responses at weaning included measures from 15 48 52 and 66 plusmn 23 d of age For all metabolic and immune response data excluding data analyzed by flow cytometry a linear mixed model with the fixed effects of treatment time and the interactions of treatment x time and block x treatment x time was fitted The random efshyfect was calf tested within treatment x block

Similarly to the acute-effect analysis flow cytornetry data from the once-fed and twice-fed calves were dishyvided by the block mean from the control calf data (early-weaned calves) and are presented as the percentshyage of control calf means The fixed effects were time and the interaction of time x block and the random effect was calf nested within block

RESULTS

Performance

Two twice-fed calves died and were removed before data analysis No differences (P lt 0139 Table 2) in performance between once- and twice-fed calves were observed before or after implementing the feeding stratshyegy All calves consumed enough calf starter to have milk replacer completely removed at 2 plusmn 08 d EuLer the weaning process was initiated on d 45 plusmn 23 d of age (Pgt 010)

Acute Effects of Once- Versus Twice-Daily Milk Replacer

No treatment or treatment x time effects were obshyserved for peripheral total leukocyte counts hematocrit percentages cortisol concentrations or haptoglobin concentrations Among all calves peripheral total leukocytes and cortisol concentrations were greater on 31 and 45 d of age (P lt 001 Table 3) Plasma hapshytoglobin concentrations decreased with increasing age in all calves (P lt 005 Table 3) At 27 d of age NM ratios tended (log-transformed P 007 Figure 1) to be greater among once-fed than twice-fed calves

Concentrations of TNF-o froUl LPS-stimulated whole blood increased with increasing age (P lt 001 Table 3) and were lower from once-fed calves than twice-fed calves at 45 d of age (P lt 005 Figure 2) A tendency (P = 007) was observed for neutrophils froIll once-fed calves to express more L-selectin on 31 and 45 d of age when compared with twice-fed calves (Table No differences in neutrophil rh-integrin expression or OB responses were observed between once-fed and twiceshyfed calves however ollce-fed calves tended (P 006)

Journal of Dairy Science Vol 94 No5 2011

2561

c

EFFECT OF ONCE- VERSUS TWICE-DAILY MILKING ON IMMUNE RESPONSES

09

o 08 1 ~ ~ OJ

o g 06

]c ~ OS ~

Z 0

OJ 24 31 4S

Aged

Figure 1 Acute dfeds on change in fceding frequency for llcntrophilmononudcar cdl mtios for twice-fed and once-fed calves Trclttmcnt x time sliced dkct log-trausfonncd lip 010 Calves were f(~1 227 g of milk replacer twice daily until 24 plusmn 05 d of age After 24 ] of 22 calves (once-fed) had the evening milk fcooing withdmwn awl g of milk was fed in the morning whereas twice-fed calves (n = 20) uo change in feediug strategy

to have greater neutrophil phagocytosis intensity at 45 d of age (Table 4)

Previous Feeding Strategy Effects on Weaning

ro significant treatment or treatment x time effects were observed for peripheral total leukocyte cOllnL NM ratio hematocrit percentage cortisol concentration or haptoglobin concentration After weaning all calves had decreased hematocrit (P lt 001 Table 5) on 52 and 66 d of age The NM cell ratios were greater (P lt 005 Table 5) at 66 d of age compared with d 52 of age

in all calves whereas plasma haptoglobin concentrashytions (P lt 001 Table 5) were d(creased

Twice-fed calves had greater (P lt 005) TNF-o from LPS-stimulated whole blood compared with once-fed calves at d 15 of age which was immediately before weaning (Figure In addition a treatment tondency (P = 009) wa observed for once-fed calves to secrete less TNF-a than twice-fed calves during the p()~Lweanshying period (Figure 3) All calves had decreased TNF-a concentrations in LPS-stimnlated whole blood after weaning (P lt 001 Table 5) but this was not likelyasshysociated with weaning because the early-weaned calves also bad less TrF-n secreted froIll LPS-stinllliated whole blood on that day

No treatment x time or treatment effects were observed for neutrophil adhesion molecule expression between once-fed and twice-fed calves after weaning (P gt CU~) however all calves had increased r3 T illtegrin expression 3 d after weaning (P lt 001 Tableuro 6) In addition all calves had a decrease in the percentage of neutrophils that produced both OB and PG as well as their intensities 3 d after weaning (P lt 001 Table 6)

DISCUSSION

Consolidating milk replacer feedings into one feeding during wk 4 of life would decrease labor and subsequent calf-rearing costs therefore the objectives of this study were to determine the effects of switching Holstein calves to once-daily feeding during wk L1 of life on inshynate immune responses and to evaluate any carry-over effects when the calves were weaned during the seventh week of life In agreement with other data (Ackerman et aL 1969 Galton and Brakel 1976 Kehoe et aL 2007) no differences in performance were observed between

Table 3 Daily means of hlood measurements of all calves (11 42) after once-fed calves had milk replacer consolidated iuto 1 fcediugmiddot2

Time relative to age d l-value Largest

Item 24 27 31 15 SEM Time Trt Trt x Time

WBC x lOmL 72 73 82 8 r h 035 002 OGS 078 Nlll OG6 D55 05] 044 006 OS6 OA1 OlO Hematocrit Cortisol ngmL Haptoglouin OD5

TNF-o pgmL

326 272 211

1134

127 252

191 13784

321 nh

1611gt 1590

3011gt 397

15211c

19611

04 36 O]S

123

lt001 ltlUll

002 lt001

DS OU 028 014

014 090 0l2 006

middotMeans within a row witb different superscripts differ significantly P lt 005 lCalves were fed 227 g of milk replacer twice until 24 plusmn 05 d of age After 2middot1 d of ago 22 calves (once-feel) had the evellillg milk feeding withdrawn aud all 454 g of milk replacer was the morning whereas twice-fed calves (n 20) had no change in feedillg strategy Data for 24 d of age was nsed as a covariate in the model therefore comparisons between LS means were only made for d 27 n and 45 of age Trt treatment Circulating tohl )cukocytc middotcounts iNeutrophi1lIlononllc1car ratios in peripheral blood log-transformed l-valucs

Optical density (x 100) root-transformed P-ValllCS

Whole blood was timuiated with 1 ~gmL of LPS for 24 h and the liupenmtant was analyzed for tnmor necroliis factor-o (TNF-o)

Journal of Dairy Science Vol 94 No5 2011

2560 HULBERT ET AL

Table 2 Pcrfonnallcc data for calves fed once or twice daily from d 24 to 151

Largest hell) Once Twice SEN P-vaillc

ADG d 2middotj to 0637 0616 00596 0802 d 31 to 15 004 0628 00275 O5() d24t045 0615 0624 00218 0764 d 45 to 52 0993 0859 00629 OUl d 52 to 66 0662 OS86 00522 0300 d45to66 O77 0677 00420 0109

Starter intake kg of D)l d 24 to 1 121 123 061 O78J d45to66 307 2J6 104 0468

Fccdain kg of DIfkg of BW d 24 to middotj5 lG5 162 0043 0764 d 45 to (i6 206 231 0115 01gt2

Calve were fed 227 of milk replacer twice daily umil 24 plusmn 0 5 d of age After 24 d of age 22 calves (ol1eeshyfed) had the evening milk feeding withdravll and all 454 g of milk repliteer Willi fed in the morning whoreas twice-fed calves (11 20) had 110 dumge in f(odin strategy All calvos were wcaned at 45 d of age

were used For all metabolic and immune response data excluding data analyzed by flow cytometry a linear mixed model with the fixed effects of treatment time and the interactions of treatment x time and block x treatment x time was fitted The random effect Wl13

calf tested within treatment x block Before statistically analyzing all the fJow cytometry

data the data from the once-fed calves were divided by the block mean of the data from the twiee-fed calves to account for daily variation resulting from flow cytomshyetry settings All flow cytometry data for the ollce-fed calves are presented as the percentage of twice-fed calf means The fixed effects of time and the interaction of time x block were fitted The random effect was calf nested within block

Statistical Analysis for Carry-Over Effects on Weaning

Data that were used to test the carry-over effects of once-fed versus twice-fed on innate immule responses at weaning included measures from 15 48 52 and 66 plusmn 23 d of age For all metabolic and immune response data excluding data analyzed by flow cytometry a linear mixed model with the fixed effects of treatment time and the interactions of treatment x time and block x treatment x time was fitted The random efshyfect was calf tested within treatment x block

Similarly to the acute-effect analysis flow cytornetry data from the once-fed and twice-fed calves were dishyvided by the block mean from the control calf data (early-weaned calves) and are presented as the percentshyage of control calf means The fixed effects were time and the interaction of time x block and the random effect was calf nested within block

RESULTS

Performance

Two twice-fed calves died and were removed before data analysis No differences (P lt 0139 Table 2) in performance between once- and twice-fed calves were observed before or after implementing the feeding stratshyegy All calves consumed enough calf starter to have milk replacer completely removed at 2 plusmn 08 d EuLer the weaning process was initiated on d 45 plusmn 23 d of age (Pgt 010)

Acute Effects of Once- Versus Twice-Daily Milk Replacer

No treatment or treatment x time effects were obshyserved for peripheral total leukocyte counts hematocrit percentages cortisol concentrations or haptoglobin concentrations Among all calves peripheral total leukocytes and cortisol concentrations were greater on 31 and 45 d of age (P lt 001 Table 3) Plasma hapshytoglobin concentrations decreased with increasing age in all calves (P lt 005 Table 3) At 27 d of age NM ratios tended (log-transformed P 007 Figure 1) to be greater among once-fed than twice-fed calves

Concentrations of TNF-o froUl LPS-stimulated whole blood increased with increasing age (P lt 001 Table 3) and were lower from once-fed calves than twice-fed calves at 45 d of age (P lt 005 Figure 2) A tendency (P = 007) was observed for neutrophils froIll once-fed calves to express more L-selectin on 31 and 45 d of age when compared with twice-fed calves (Table No differences in neutrophil rh-integrin expression or OB responses were observed between once-fed and twiceshyfed calves however ollce-fed calves tended (P 006)

Journal of Dairy Science Vol 94 No5 2011

2561

c

EFFECT OF ONCE- VERSUS TWICE-DAILY MILKING ON IMMUNE RESPONSES

09

o 08 1 ~ ~ OJ

o g 06

]c ~ OS ~

Z 0

OJ 24 31 4S

Aged

Figure 1 Acute dfeds on change in fceding frequency for llcntrophilmononudcar cdl mtios for twice-fed and once-fed calves Trclttmcnt x time sliced dkct log-trausfonncd lip 010 Calves were f(~1 227 g of milk replacer twice daily until 24 plusmn 05 d of age After 24 ] of 22 calves (once-fed) had the evening milk fcooing withdmwn awl g of milk was fed in the morning whereas twice-fed calves (n = 20) uo change in feediug strategy

to have greater neutrophil phagocytosis intensity at 45 d of age (Table 4)

Previous Feeding Strategy Effects on Weaning

ro significant treatment or treatment x time effects were observed for peripheral total leukocyte cOllnL NM ratio hematocrit percentage cortisol concentration or haptoglobin concentration After weaning all calves had decreased hematocrit (P lt 001 Table 5) on 52 and 66 d of age The NM cell ratios were greater (P lt 005 Table 5) at 66 d of age compared with d 52 of age

in all calves whereas plasma haptoglobin concentrashytions (P lt 001 Table 5) were d(creased

Twice-fed calves had greater (P lt 005) TNF-o from LPS-stimulated whole blood compared with once-fed calves at d 15 of age which was immediately before weaning (Figure In addition a treatment tondency (P = 009) wa observed for once-fed calves to secrete less TNF-a than twice-fed calves during the p()~Lweanshying period (Figure 3) All calves had decreased TNF-a concentrations in LPS-stimnlated whole blood after weaning (P lt 001 Table 5) but this was not likelyasshysociated with weaning because the early-weaned calves also bad less TrF-n secreted froIll LPS-stinllliated whole blood on that day

No treatment x time or treatment effects were observed for neutrophil adhesion molecule expression between once-fed and twice-fed calves after weaning (P gt CU~) however all calves had increased r3 T illtegrin expression 3 d after weaning (P lt 001 Tableuro 6) In addition all calves had a decrease in the percentage of neutrophils that produced both OB and PG as well as their intensities 3 d after weaning (P lt 001 Table 6)

DISCUSSION

Consolidating milk replacer feedings into one feeding during wk 4 of life would decrease labor and subsequent calf-rearing costs therefore the objectives of this study were to determine the effects of switching Holstein calves to once-daily feeding during wk L1 of life on inshynate immune responses and to evaluate any carry-over effects when the calves were weaned during the seventh week of life In agreement with other data (Ackerman et aL 1969 Galton and Brakel 1976 Kehoe et aL 2007) no differences in performance were observed between

Table 3 Daily means of hlood measurements of all calves (11 42) after once-fed calves had milk replacer consolidated iuto 1 fcediugmiddot2

Time relative to age d l-value Largest

Item 24 27 31 15 SEM Time Trt Trt x Time

WBC x lOmL 72 73 82 8 r h 035 002 OGS 078 Nlll OG6 D55 05] 044 006 OS6 OA1 OlO Hematocrit Cortisol ngmL Haptoglouin OD5

TNF-o pgmL

326 272 211

1134

127 252

191 13784

321 nh

1611gt 1590

3011gt 397

15211c

19611

04 36 O]S

123

lt001 ltlUll

002 lt001

DS OU 028 014

014 090 0l2 006

middotMeans within a row witb different superscripts differ significantly P lt 005 lCalves were fed 227 g of milk replacer twice until 24 plusmn 05 d of age After 2middot1 d of ago 22 calves (once-feel) had the evellillg milk feeding withdrawn aud all 454 g of milk replacer was the morning whereas twice-fed calves (n 20) had no change in feedillg strategy Data for 24 d of age was nsed as a covariate in the model therefore comparisons between LS means were only made for d 27 n and 45 of age Trt treatment Circulating tohl )cukocytc middotcounts iNeutrophi1lIlononllc1car ratios in peripheral blood log-transformed l-valucs

Optical density (x 100) root-transformed P-ValllCS

Whole blood was timuiated with 1 ~gmL of LPS for 24 h and the liupenmtant was analyzed for tnmor necroliis factor-o (TNF-o)

Journal of Dairy Science Vol 94 No5 2011

2561

c

EFFECT OF ONCE- VERSUS TWICE-DAILY MILKING ON IMMUNE RESPONSES

09

o 08 1 ~ ~ OJ

o g 06

]c ~ OS ~

Z 0

OJ 24 31 4S

Aged

Figure 1 Acute dfeds on change in fceding frequency for llcntrophilmononudcar cdl mtios for twice-fed and once-fed calves Trclttmcnt x time sliced dkct log-trausfonncd lip 010 Calves were f(~1 227 g of milk replacer twice daily until 24 plusmn 05 d of age After 24 ] of 22 calves (once-fed) had the evening milk fcooing withdmwn awl g of milk was fed in the morning whereas twice-fed calves (n = 20) uo change in feediug strategy

to have greater neutrophil phagocytosis intensity at 45 d of age (Table 4)

Previous Feeding Strategy Effects on Weaning

ro significant treatment or treatment x time effects were observed for peripheral total leukocyte cOllnL NM ratio hematocrit percentage cortisol concentration or haptoglobin concentration After weaning all calves had decreased hematocrit (P lt 001 Table 5) on 52 and 66 d of age The NM cell ratios were greater (P lt 005 Table 5) at 66 d of age compared with d 52 of age

in all calves whereas plasma haptoglobin concentrashytions (P lt 001 Table 5) were d(creased

Twice-fed calves had greater (P lt 005) TNF-o from LPS-stimulated whole blood compared with once-fed calves at d 15 of age which was immediately before weaning (Figure In addition a treatment tondency (P = 009) wa observed for once-fed calves to secrete less TNF-a than twice-fed calves during the p()~Lweanshying period (Figure 3) All calves had decreased TNF-a concentrations in LPS-stimnlated whole blood after weaning (P lt 001 Table 5) but this was not likelyasshysociated with weaning because the early-weaned calves also bad less TrF-n secreted froIll LPS-stinllliated whole blood on that day

No treatment x time or treatment effects were observed for neutrophil adhesion molecule expression between once-fed and twice-fed calves after weaning (P gt CU~) however all calves had increased r3 T illtegrin expression 3 d after weaning (P lt 001 Tableuro 6) In addition all calves had a decrease in the percentage of neutrophils that produced both OB and PG as well as their intensities 3 d after weaning (P lt 001 Table 6)

DISCUSSION

Consolidating milk replacer feedings into one feeding during wk 4 of life would decrease labor and subsequent calf-rearing costs therefore the objectives of this study were to determine the effects of switching Holstein calves to once-daily feeding during wk L1 of life on inshynate immune responses and to evaluate any carry-over effects when the calves were weaned during the seventh week of life In agreement with other data (Ackerman et aL 1969 Galton and Brakel 1976 Kehoe et aL 2007) no differences in performance were observed between

Table 3 Daily means of hlood measurements of all calves (11 42) after once-fed calves had milk replacer consolidated iuto 1 fcediugmiddot2

Time relative to age d l-value Largest

Item 24 27 31 15 SEM Time Trt Trt x Time

WBC x lOmL 72 73 82 8 r h 035 002 OGS 078 Nlll OG6 D55 05] 044 006 OS6 OA1 OlO Hematocrit Cortisol ngmL Haptoglouin OD5

TNF-o pgmL

326 272 211

1134

127 252

191 13784

321 nh

1611gt 1590

3011gt 397

15211c

19611

04 36 O]S

123

lt001 ltlUll

002 lt001

DS OU 028 014

014 090 0l2 006

middotMeans within a row witb different superscripts differ significantly P lt 005 lCalves were fed 227 g of milk replacer twice until 24 plusmn 05 d of age After 2middot1 d of ago 22 calves (once-feel) had the evellillg milk feeding withdrawn aud all 454 g of milk replacer was the morning whereas twice-fed calves (n 20) had no change in feedillg strategy Data for 24 d of age was nsed as a covariate in the model therefore comparisons between LS means were only made for d 27 n and 45 of age Trt treatment Circulating tohl )cukocytc middotcounts iNeutrophi1lIlononllc1car ratios in peripheral blood log-transformed l-valucs

Optical density (x 100) root-transformed P-ValllCS

Whole blood was timuiated with 1 ~gmL of LPS for 24 h and the liupenmtant was analyzed for tnmor necroliis factor-o (TNF-o)

Journal of Dairy Science Vol 94 No5 2011

2562 HULBERT ET AL

Table 4 Neutnlhilmmns of blood llIcasuremcllts of ollcc-fcc calves after they had milk r(piaur cUllsolielatcd mto OIl feecimg shy

Time relative to d

Item 24 27 31 15 P-va)uc

L-s([ectin 761 763 1058 1058 168 OJ)7 i1 T intcgriil GCIFI 837 J(i 1028 923 8 067 OB+PG+ 1017 1025 1008 1022 19 017 OB GIFI 1029 flU 100l 1025 42 018 PG GMFI 1073 1099 1045 1143 35 (l06

All flow cytonwtrie measures arc prestmed as a percentage of twice-fcc calf day and block means After 2middot1 d of age samples were taken once-fed calves hael evening milk replacer feeltiing consolidated into OllC

morning f(ding Age 24 1 served as a covariate in the moeleL

Geometric llicall fluorescence intcllsity Percentage of totalncutrophib that produced both oxidative burst (OB) and phagocytosis (PG)

Originally measured as GRIFf presented as log-transformed P-values

treatmellts Once-fed calves consumed similar amounts of starter as twice-fed calves and it took less than 3 d to completely wean all calves from milk replacer Thereshyfore weaning was a similar experience for all calves and was all abrupt weaning from milk replacer

Three days after milk replacer was consolidated into one feeding for ollce-fed calves their NM cell ratios tended to be than those for twice-fed calves Toshytal leukocytes counts did not that the change in the ratio was a result of infection Once-fed calves likely experienced an acute stress as peripheral neutrophilia is commonly observed in cattle following many types of

2500 Oncemiddotfed

2400

2200

2000 E

i 1800

1 1600

i 140()

1200

lOoo

-~- Twice-fed

SOD

24 27 31 45

Asd

Figure 2 Acute effects on in feeding frequency for tumor llEltTois factor tTNF)-n fcffted LPS-stinmlated whole blood for twice-fed and once-fed calves from 24 to 45 d of age Treatment x tillle sliced effect ltP ()()5 Calves were fed 227 g of milk replacer twice nntil 24 plusmn (J5 d of age After 24 d of 22 calves (once-fed) had evening milk feeding cithdrawn and g of milk replacer was fed ill the morning whereas twice-fed cakeH 20) had uo strategy Whole blood was at a final of LPS for 24 h and the supernatant fraction was analyzed for

Journal of Dairy Science VoL 94 No 5 2011

stressors (Murata et al 1987 Buckham Sporer et Ill 2007 Gupta et aL 2007) In addition increased NM ratios are commonly observed immediately following weaning stress (Hulbert et a1 2011) Furthermore a transient increase in NM was also observed among all calves when they were weaned in the current study

In addition to changes in circulating leukocyte proshyportions functional capacities of neutrophils are respOHshysive to stress including suppression in phagocytic and oxidative burst capacities al well as adhesion molecule expression (Burton et al 2005 Buckharn Sporer et al 2007) However the type and iutensity of the stressor likely plays a role in the effect it has on the fUllctional response of neutrophils (Sorrells and Sapolsky 2007) The change in feeding frequency during the fourth week of life did not have an effect on neutrophil phagocytic or oxidative burst responses to E coli which suggests that the stress associated with the change in feeding frequency was either acute or less intense than other stressors Pang et al (2009) observed no changes in neutrophil phagocytic or oxidative burst capacities when Holstein bull calves were acutely challenged with hydrocortisone Furthermore no change in neutrophil phagocytic or oxidative burst capacities were observed when Holstein bull calves were castrated (Pang et al 2009) These data suggest that acute stress might not impair neutrophil phagocytic and oxidative burst cashypacities In contrast exposing 5-wk-old Holstein calves to exercise and cold stress (Henricks et al 1987) or weaning Holstein (Hulbert et al 2011) and beef (Lynch et aI 2010) calves caused a transient decrease in the phagocytic and oxidative burst capacities of neushytrophils Once-fed calves were able to cope with the change in feeding frequency rather rapidly ai evident by a lack of suppression of neutrophil phagocytic aIld oxidative burst capacities 3 d after consolidating to ollce-a-day feeding The lack of neutrophil L-selectin suppression further supports the finding that Holstein

2563 EFFECT OF ONCE- VERSUS TWICE-DAILY MILKING ON IMMUNE RESPONSES

Table 5 Dttiy mean of blood parameters of all calwo (n 42) after weaning

Time relativc to agc d P-valnc

ItCll 5 48 52 66 Largest

SEM Time Tn Trt Time

WBC x 1O(imL 1[ HClllatocrit l7r

()

Cortiol nglllL Haptoglobin OD J

TNF-et

84

87 l5(i

1978

87 018

307 198

Vi LlOn

82 DAO

29 128 170

l7DSmiddot

78 05

lOti 1782(

039 (L06 05 3 (lll

178

018 DOmiddot

lt001 lt001 lt001 lt001

OJ2 092 nA9 OlG 085 (W7

OJH 05 011 072 OS3 OJ])

llcalls wit hill a row with differcllt superscripts differ significantly l lt 005 Calves were fed 227 of milk r(placer twice daily umil 24 1 D5 d of age After 24 d of age 22 calves (ol1ct~fed) had the enniug milk itedillg withdrawn alld all g of milk replacer Wampi fed in the morning wilcrtas twice-fed calves (n = 20) had no change in feeding strategy Trt = tfcatUICnt

Circulating totalleukoeytc COlUltS

NcutrupllihuonOlmccar ratios in peripheral blood log-transformed P-valucs

JOptical density (x JOO) root-tranfoTmcd 1-vallJes Vholc blood wamp stimulated with 1 flgmL of LPS for 24 h and the bupernatant was analyzed for tumor-necrosis faetor-C (TNF-C)

calves are able to cope rapidly with the change in feed-frequency during wk 4 of life At n and 15 d of

age Once-fed calves actually expressed more L-selectin on their nentrophils In a companion paper Holstein calves weaned early had a transient decrease in Lshyselectin 3 d after initiating weaning but compensated with greater expression 7 d after weaning (Hulbert et al 2011) Additionally when Holstein bull calves were transported neutrophil L-seleetin expression was decreased 45 h after transport however L-selectin expression was greater 975 h after transport than at baseline (Buckham Sporer et aL 2007) In contrast neither intravenous challenge with hydrocortisone nor castration caused change in neutrophil expression of L-selectin however parturition stress in Holstien cows caused a decreased neutrophil L-selectin expression (Weber et aL 2001) Collectively these data suggest that mild or aCllte stressors do not or only transiently suppress neutrophil L-selectill expression and that neushytrophils may be primed following this type of stress Current data suggest that the mild stress of switching from twice-a-day feeding to once-a-day feeding during the fourth week of life stimulated neutrophil L-selectill expression Further supporting that a mild stress inshycreases expression of adhesion molecules on neutrophils is the observation that all calves had higher expression of 3T integrin 3 d after weaning The exact immunologishycal significance of the elevated expression of an adhesion molecule following a mild stressor is not known but this could be a compensatory mechallism that might improve resistance to disease

At 45 d of age which was 21 d after consolidating to a single feeding in the once-fed calves secretion of TNF-(x from whole blood cultures stimulated with LPS was decreased in the once-fed calves Furthermore durshying the postweaning period a tendency was observed for

a carry-over effect wherein once-fed calves continued to secrete less TNF-0 froIll whole hlood clllt1m~s This response is not likely associated with the stressor of switching to once-a-day feeding because of the 21-d latency until the response was evident as well as its pershysistency into the postweaning period In pigs TNF-clt

2700

2500

2300

2100 ~

e 1900 a cshy 1700 15 Ishy 1500cent

1300

1100

900

700

Figure 3 Weaning cffltets on calves fed milk replacer once or twice for tmnor necrosis factor (T-rF)-rgt ~crrct(d from LPS-sIimlllat(yl blood from 15 to 66 d of age Treatmellt x tilllt slicod dkct

log-transformed P 005 and treatment effect P 009 Calves were fcyl 227 of milk twice daily until 21 (JJ d of age After 24 d of age calves had the evening milk fceding withdrawn and all 454 g of replacer was fed in tlle morning wheres twiccshyfed calves (n 20) had no change in fceding strategy Weaning was initiated by rCmOyillg 50 of the milk rltJplacer ami tlWll calve wcre completely weaned after consuming 900 g of as-f(n calf ~artcr for 2 consecutive davs vVholc Llood was stimulated at Gnal concmltratioll of 1 lLglIlL of ips for 24 hand tllC slJpernatant fraction was analyz(d for TNF-a

Journal of Dairy Science Vol 94 No5 2011

___Once-fed

-e- Twice-fed

45 48 52 66

Aged

2564 HULBERT ET AL

Table 6 NltrltlpltiJ means of flow cytomctry llWllSLlrcmcnts of all calves aftCr

Time relative to agC d P-value

-item 45 48 0 66 Time Txt Trt x Tillle

L-bclectin G IFf 949 94 1025 916 91 087 OW (UiG fjrilltqrill lll16 UnA 1009 1019 75 003 DGl 05i Ofl+PG+ lOiiO 998b 1015 IOU 08 lt001 US 09 OB G--IFI 1180 1OS3 1182 112G 48 OUj 075 nHI PG GtvIFI 1120 989 1122 1012 t8 lt001 021 O(a

a leans within a row with different superscripts differ significantly P OOii All How cytolllctric nlCasurcs arc prescutcd flS a percCutagc of control calf day and block means Txt = trcatment

G(Olll(trie lucan OUOrCHCCl1CC intcn~ity

Jlrcentagc of totalncntrophib that prodllcLu both oxidative burst (OB) and phagocytosis (PG)

measured as GMFI presented as 101-trandonned P- valmti

secretion from whole blood cultures stimulated with LPg decreased by 617 2 d after weaning (Carstensen et al 2(05) Lightweight early-weaned Holstein bull calves had decreased secretion of TF -0 at 3 rtud 7 d after weaning but TNF-(x concentrat ions returned to a level not different from that of nonshyweaned control calves by d 21 after weaning (Hulbert et aL 2(11) In human surgical patients low whole blood TXF-(x responses were associated with greater morbidity and mortality et aL 20(3) The imshymunological significance of the lower TXF-a response by once-fed calves is not known but it is conceivable that these calves may have decreased ability to activate an acute phase response and recruit effector cells to sites of infection It is tempting to speculate on the mechanism underlying the lower secretion of TNF-a in once-fed calves Early life experiences either in utero or during the neonatal period can influence a physiologishycal response later in life This phenomenon was coined pre-programming (Dorner 1975) It is not known whether feeding frequency early in life can have persisshytent effects on the pro-inflammatory responsiveness of leukocytes to pathogen-associated molecular patterns this warrants further research

CONCLUSIONS

COllsoliclating calf milk replacer into one feeding durshying the fourth week of life was likely a mild and acute stressor as evident by transient neutrophilia in the absence of suppressed functional capacities of neutroshyphils Once-fed calves did not have acute suppression of TNF-a secretion from whole blood cultures but 21 d after changing the feeding frequency they were pershysistently lower through the end of the study Whether this continues to persist later in life and the underlying mechanisms and resistance to disease implications of this are not known and warrant further research

ACKNOWLEDGMENTS

The authors thank Jeff Dailey (USDA-ARS) and Colshyton Cobb (Texas Tech University) for their assistance with animal husbandry Luke Schwertner (Texas Tech Cniversity) for his laboratory assistance and Land OLakes Animal Milk Products Co (Shoreview MN) for donating milk replacer

REFERENCES

Ackerman R A R O Thomas W V Thayne anG D F Hutcil(r 1969 Effects of oncc-a-day feeding of milk replac([ on hody weight

of dairy calves J Dairy Sci 52 186) 11-gt72 rtUtmj(totl J D S D Eicher V E KUllkk and F G Martin 200

of transportation and COlllllllnlling Oil the aCHte-phase prOshytein respOllRe growth and feed intake of newly weaned beef CHIves J Anim Sci 811120 1125

Ballou M A and E J DcPeters 2008 Supplementing milk replacer with omega-~ faHy acids from fL3h oil OIl immullocompetence and health of Jersey calves J Dairy Sci Jl 3488~~3500

Bnckham Sporer K R J 1 Burton B Earley and M A Crowe 2007 Transportation stress in young bulls alters expression of neutrophil genes important for the regulation of apoptosis tissue remodeling marginabon and anti-hacterial function Vet Tmmushyno Immunopathol lISll 29

Burton J L S A 1fadscn L C Chang P S Veber K R Bllckshyham R van Dorp gt1 C Hickey and B Earley 2005 Gcne cxshy

uu in neutrophilli exposed to glucocorticoids A to help (xplain neutrophil dysfunction in parturi shy

cows Vet Il1l111UllOl TmmUllopathol 1051l7219 Carstensen C M H(5ntved and 1- P Nielscn 2005 Dctcrrniuatioll

of tumor necrosis factor-alpha f(spOllsivcncss in pillets arollnd wcaning using an cx vivo whole blood stimulation flSsay Vet Illlshymuno Irrnllunopathol lO559 06

Domer G 1975 Perinatal llOnnollc levels and brain organization Anat Neurocndoerinol 1245252

Galton D 1 alld VJ Brakd 1976 Illfhwncc of fceding milk reshyplacer onc(- verSus twice- daily on growth1 organ lnplttsurmllcnts aud mineral content of tissues J Dairy Sci 59941 948

GuptaS B Earlcy and M A Crowe 20()7 Effect of 12-hour road naJlsportatiol1 ou physiological imlllunological md 11a[)nat010lishycal parameters in bulls housed at different space allowances Vet 1 173605 616

Heagy V K Nieman C --1 Cohen D DHniesoll and ]VI A West 2001 Lower levels of blood LPS-1illlulatcd cytokim release arc associated with poorer clinical outcome ill ~llrliea1 reu patients Snrg Infect (Larclnnt) (171~ 180

Journal of Dairy Science Vol 94 No5 2011

2563 EFFECT OF ONCE- VERSUS TWICE-DAILY MILKING ON IMMUNE RESPONSES

Table 5 Dttiy mean of blood parameters of all calwo (n 42) after weaning

Time relativc to agc d P-valnc

ItCll 5 48 52 66 Largest

SEM Time Tn Trt Time

WBC x 1O(imL 1[ HClllatocrit l7r

()

Cortiol nglllL Haptoglobin OD J

TNF-et

84

87 l5(i

1978

87 018

307 198

Vi LlOn

82 DAO

29 128 170

l7DSmiddot

78 05

lOti 1782(

039 (L06 05 3 (lll

178

018 DOmiddot

lt001 lt001 lt001 lt001

OJ2 092 nA9 OlG 085 (W7

OJH 05 011 072 OS3 OJ])

llcalls wit hill a row with differcllt superscripts differ significantly l lt 005 Calves were fed 227 of milk r(placer twice daily umil 24 1 D5 d of age After 24 d of age 22 calves (ol1ct~fed) had the enniug milk itedillg withdrawn alld all g of milk replacer Wampi fed in the morning wilcrtas twice-fed calves (n = 20) had no change in feeding strategy Trt = tfcatUICnt

Circulating totalleukoeytc COlUltS

NcutrupllihuonOlmccar ratios in peripheral blood log-transformed P-valucs

JOptical density (x JOO) root-tranfoTmcd 1-vallJes Vholc blood wamp stimulated with 1 flgmL of LPS for 24 h and the bupernatant was analyzed for tumor-necrosis faetor-C (TNF-C)

calves are able to cope rapidly with the change in feed-frequency during wk 4 of life At n and 15 d of

age Once-fed calves actually expressed more L-selectin on their nentrophils In a companion paper Holstein calves weaned early had a transient decrease in Lshyselectin 3 d after initiating weaning but compensated with greater expression 7 d after weaning (Hulbert et al 2011) Additionally when Holstein bull calves were transported neutrophil L-seleetin expression was decreased 45 h after transport however L-selectin expression was greater 975 h after transport than at baseline (Buckham Sporer et aL 2007) In contrast neither intravenous challenge with hydrocortisone nor castration caused change in neutrophil expression of L-selectin however parturition stress in Holstien cows caused a decreased neutrophil L-selectin expression (Weber et aL 2001) Collectively these data suggest that mild or aCllte stressors do not or only transiently suppress neutrophil L-selectill expression and that neushytrophils may be primed following this type of stress Current data suggest that the mild stress of switching from twice-a-day feeding to once-a-day feeding during the fourth week of life stimulated neutrophil L-selectill expression Further supporting that a mild stress inshycreases expression of adhesion molecules on neutrophils is the observation that all calves had higher expression of 3T integrin 3 d after weaning The exact immunologishycal significance of the elevated expression of an adhesion molecule following a mild stressor is not known but this could be a compensatory mechallism that might improve resistance to disease

At 45 d of age which was 21 d after consolidating to a single feeding in the once-fed calves secretion of TNF-(x from whole blood cultures stimulated with LPS was decreased in the once-fed calves Furthermore durshying the postweaning period a tendency was observed for

a carry-over effect wherein once-fed calves continued to secrete less TNF-0 froIll whole hlood clllt1m~s This response is not likely associated with the stressor of switching to once-a-day feeding because of the 21-d latency until the response was evident as well as its pershysistency into the postweaning period In pigs TNF-clt

2700

2500

2300

2100 ~

e 1900 a cshy 1700 15 Ishy 1500cent

1300

1100

900

700

Figure 3 Weaning cffltets on calves fed milk replacer once or twice for tmnor necrosis factor (T-rF)-rgt ~crrct(d from LPS-sIimlllat(yl blood from 15 to 66 d of age Treatmellt x tilllt slicod dkct

log-transformed P 005 and treatment effect P 009 Calves were fcyl 227 of milk twice daily until 21 (JJ d of age After 24 d of age calves had the evening milk fceding withdrawn and all 454 g of replacer was fed in tlle morning wheres twiccshyfed calves (n 20) had no change in fceding strategy Weaning was initiated by rCmOyillg 50 of the milk rltJplacer ami tlWll calve wcre completely weaned after consuming 900 g of as-f(n calf ~artcr for 2 consecutive davs vVholc Llood was stimulated at Gnal concmltratioll of 1 lLglIlL of ips for 24 hand tllC slJpernatant fraction was analyz(d for TNF-a

Journal of Dairy Science Vol 94 No5 2011

___Once-fed

-e- Twice-fed

45 48 52 66

Aged

2564 HULBERT ET AL

Table 6 NltrltlpltiJ means of flow cytomctry llWllSLlrcmcnts of all calves aftCr

Time relative to agC d P-value

-item 45 48 0 66 Time Txt Trt x Tillle

L-bclectin G IFf 949 94 1025 916 91 087 OW (UiG fjrilltqrill lll16 UnA 1009 1019 75 003 DGl 05i Ofl+PG+ lOiiO 998b 1015 IOU 08 lt001 US 09 OB G--IFI 1180 1OS3 1182 112G 48 OUj 075 nHI PG GtvIFI 1120 989 1122 1012 t8 lt001 021 O(a

a leans within a row with different superscripts differ significantly P OOii All How cytolllctric nlCasurcs arc prescutcd flS a percCutagc of control calf day and block means Txt = trcatment

G(Olll(trie lucan OUOrCHCCl1CC intcn~ity

Jlrcentagc of totalncntrophib that prodllcLu both oxidative burst (OB) and phagocytosis (PG)

measured as GMFI presented as 101-trandonned P- valmti

secretion from whole blood cultures stimulated with LPg decreased by 617 2 d after weaning (Carstensen et al 2(05) Lightweight early-weaned Holstein bull calves had decreased secretion of TF -0 at 3 rtud 7 d after weaning but TNF-(x concentrat ions returned to a level not different from that of nonshyweaned control calves by d 21 after weaning (Hulbert et aL 2(11) In human surgical patients low whole blood TXF-(x responses were associated with greater morbidity and mortality et aL 20(3) The imshymunological significance of the lower TXF-a response by once-fed calves is not known but it is conceivable that these calves may have decreased ability to activate an acute phase response and recruit effector cells to sites of infection It is tempting to speculate on the mechanism underlying the lower secretion of TNF-a in once-fed calves Early life experiences either in utero or during the neonatal period can influence a physiologishycal response later in life This phenomenon was coined pre-programming (Dorner 1975) It is not known whether feeding frequency early in life can have persisshytent effects on the pro-inflammatory responsiveness of leukocytes to pathogen-associated molecular patterns this warrants further research

CONCLUSIONS

COllsoliclating calf milk replacer into one feeding durshying the fourth week of life was likely a mild and acute stressor as evident by transient neutrophilia in the absence of suppressed functional capacities of neutroshyphils Once-fed calves did not have acute suppression of TNF-a secretion from whole blood cultures but 21 d after changing the feeding frequency they were pershysistently lower through the end of the study Whether this continues to persist later in life and the underlying mechanisms and resistance to disease implications of this are not known and warrant further research

ACKNOWLEDGMENTS

The authors thank Jeff Dailey (USDA-ARS) and Colshyton Cobb (Texas Tech University) for their assistance with animal husbandry Luke Schwertner (Texas Tech Cniversity) for his laboratory assistance and Land OLakes Animal Milk Products Co (Shoreview MN) for donating milk replacer

REFERENCES

Ackerman R A R O Thomas W V Thayne anG D F Hutcil(r 1969 Effects of oncc-a-day feeding of milk replac([ on hody weight

of dairy calves J Dairy Sci 52 186) 11-gt72 rtUtmj(totl J D S D Eicher V E KUllkk and F G Martin 200

of transportation and COlllllllnlling Oil the aCHte-phase prOshytein respOllRe growth and feed intake of newly weaned beef CHIves J Anim Sci 811120 1125

Ballou M A and E J DcPeters 2008 Supplementing milk replacer with omega-~ faHy acids from fL3h oil OIl immullocompetence and health of Jersey calves J Dairy Sci Jl 3488~~3500

Bnckham Sporer K R J 1 Burton B Earley and M A Crowe 2007 Transportation stress in young bulls alters expression of neutrophil genes important for the regulation of apoptosis tissue remodeling marginabon and anti-hacterial function Vet Tmmushyno Immunopathol lISll 29

Burton J L S A 1fadscn L C Chang P S Veber K R Bllckshyham R van Dorp gt1 C Hickey and B Earley 2005 Gcne cxshy

uu in neutrophilli exposed to glucocorticoids A to help (xplain neutrophil dysfunction in parturi shy

cows Vet Il1l111UllOl TmmUllopathol 1051l7219 Carstensen C M H(5ntved and 1- P Nielscn 2005 Dctcrrniuatioll

of tumor necrosis factor-alpha f(spOllsivcncss in pillets arollnd wcaning using an cx vivo whole blood stimulation flSsay Vet Illlshymuno Irrnllunopathol lO559 06

Domer G 1975 Perinatal llOnnollc levels and brain organization Anat Neurocndoerinol 1245252

Galton D 1 alld VJ Brakd 1976 Illfhwncc of fceding milk reshyplacer onc(- verSus twice- daily on growth1 organ lnplttsurmllcnts aud mineral content of tissues J Dairy Sci 59941 948

GuptaS B Earlcy and M A Crowe 20()7 Effect of 12-hour road naJlsportatiol1 ou physiological imlllunological md 11a[)nat010lishycal parameters in bulls housed at different space allowances Vet 1 173605 616

Heagy V K Nieman C --1 Cohen D DHniesoll and ]VI A West 2001 Lower levels of blood LPS-1illlulatcd cytokim release arc associated with poorer clinical outcome ill ~llrliea1 reu patients Snrg Infect (Larclnnt) (171~ 180

Journal of Dairy Science Vol 94 No5 2011

2564 HULBERT ET AL

Table 6 NltrltlpltiJ means of flow cytomctry llWllSLlrcmcnts of all calves aftCr

Time relative to agC d P-value

-item 45 48 0 66 Time Txt Trt x Tillle

L-bclectin G IFf 949 94 1025 916 91 087 OW (UiG fjrilltqrill lll16 UnA 1009 1019 75 003 DGl 05i Ofl+PG+ lOiiO 998b 1015 IOU 08 lt001 US 09 OB G--IFI 1180 1OS3 1182 112G 48 OUj 075 nHI PG GtvIFI 1120 989 1122 1012 t8 lt001 021 O(a

a leans within a row with different superscripts differ significantly P OOii All How cytolllctric nlCasurcs arc prescutcd flS a percCutagc of control calf day and block means Txt = trcatment

G(Olll(trie lucan OUOrCHCCl1CC intcn~ity

Jlrcentagc of totalncntrophib that prodllcLu both oxidative burst (OB) and phagocytosis (PG)

measured as GMFI presented as 101-trandonned P- valmti

secretion from whole blood cultures stimulated with LPg decreased by 617 2 d after weaning (Carstensen et al 2(05) Lightweight early-weaned Holstein bull calves had decreased secretion of TF -0 at 3 rtud 7 d after weaning but TNF-(x concentrat ions returned to a level not different from that of nonshyweaned control calves by d 21 after weaning (Hulbert et aL 2(11) In human surgical patients low whole blood TXF-(x responses were associated with greater morbidity and mortality et aL 20(3) The imshymunological significance of the lower TXF-a response by once-fed calves is not known but it is conceivable that these calves may have decreased ability to activate an acute phase response and recruit effector cells to sites of infection It is tempting to speculate on the mechanism underlying the lower secretion of TNF-a in once-fed calves Early life experiences either in utero or during the neonatal period can influence a physiologishycal response later in life This phenomenon was coined pre-programming (Dorner 1975) It is not known whether feeding frequency early in life can have persisshytent effects on the pro-inflammatory responsiveness of leukocytes to pathogen-associated molecular patterns this warrants further research

CONCLUSIONS

COllsoliclating calf milk replacer into one feeding durshying the fourth week of life was likely a mild and acute stressor as evident by transient neutrophilia in the absence of suppressed functional capacities of neutroshyphils Once-fed calves did not have acute suppression of TNF-a secretion from whole blood cultures but 21 d after changing the feeding frequency they were pershysistently lower through the end of the study Whether this continues to persist later in life and the underlying mechanisms and resistance to disease implications of this are not known and warrant further research

ACKNOWLEDGMENTS

The authors thank Jeff Dailey (USDA-ARS) and Colshyton Cobb (Texas Tech University) for their assistance with animal husbandry Luke Schwertner (Texas Tech Cniversity) for his laboratory assistance and Land OLakes Animal Milk Products Co (Shoreview MN) for donating milk replacer

REFERENCES

Ackerman R A R O Thomas W V Thayne anG D F Hutcil(r 1969 Effects of oncc-a-day feeding of milk replac([ on hody weight

of dairy calves J Dairy Sci 52 186) 11-gt72 rtUtmj(totl J D S D Eicher V E KUllkk and F G Martin 200

of transportation and COlllllllnlling Oil the aCHte-phase prOshytein respOllRe growth and feed intake of newly weaned beef CHIves J Anim Sci 811120 1125

Ballou M A and E J DcPeters 2008 Supplementing milk replacer with omega-~ faHy acids from fL3h oil OIl immullocompetence and health of Jersey calves J Dairy Sci Jl 3488~~3500

Bnckham Sporer K R J 1 Burton B Earley and M A Crowe 2007 Transportation stress in young bulls alters expression of neutrophil genes important for the regulation of apoptosis tissue remodeling marginabon and anti-hacterial function Vet Tmmushyno Immunopathol lISll 29

Burton J L S A 1fadscn L C Chang P S Veber K R Bllckshyham R van Dorp gt1 C Hickey and B Earley 2005 Gcne cxshy

uu in neutrophilli exposed to glucocorticoids A to help (xplain neutrophil dysfunction in parturi shy

cows Vet Il1l111UllOl TmmUllopathol 1051l7219 Carstensen C M H(5ntved and 1- P Nielscn 2005 Dctcrrniuatioll

of tumor necrosis factor-alpha f(spOllsivcncss in pillets arollnd wcaning using an cx vivo whole blood stimulation flSsay Vet Illlshymuno Irrnllunopathol lO559 06

Domer G 1975 Perinatal llOnnollc levels and brain organization Anat Neurocndoerinol 1245252

Galton D 1 alld VJ Brakd 1976 Illfhwncc of fceding milk reshyplacer onc(- verSus twice- daily on growth1 organ lnplttsurmllcnts aud mineral content of tissues J Dairy Sci 59941 948

GuptaS B Earlcy and M A Crowe 20()7 Effect of 12-hour road naJlsportatiol1 ou physiological imlllunological md 11a[)nat010lishycal parameters in bulls housed at different space allowances Vet 1 173605 616

Heagy V K Nieman C --1 Cohen D DHniesoll and ]VI A West 2001 Lower levels of blood LPS-1illlulatcd cytokim release arc associated with poorer clinical outcome ill ~llrliea1 reu patients Snrg Infect (Larclnnt) (171~ 180

Journal of Dairy Science Vol 94 No5 2011

2565 EFFECT OF ONCE- VERSUS TWICE-DAILY MILKING ON IMMUNE RESPONSES

Hcmicb P A G I Flinkhorst alld F P nIeJ HJH7 Stres diltlinish(s ill filtration and nll)V cells in calVfs Inflammation

Hulhert L E C 1 Cobb J A CarrolL 1Id1[ A Ballou 2011 The effects of tady wcaning on innate immune respOllses of Holstein Cillvcs J Dain Sci 9254 2556

Kehoe S 1 C D vechow and A J Heinrichs 2007 Effects of weallshying age and milk f(((liug freqneney on dairy calf growth health and rumen parameters Livest Sci 110267-272

Lynch E M B Earley 11 MeGec and S Doyle 2010 Effect of weaning at housin~ on leukocyte distribution fUllctional of l1(utrophihi and acuto pha~( protein respOl1se of bcpf

calws B1IC Vet ns 63947 lIakiltmra altd N Suzuki HJ82 Quantitativc determination of

and its elevation ill some inflammatory Zasshi 1521 ~

Murata H H Takahashi alld H Matsullloto llS7 The effects of road transportation on peripheral blood lymphocyte bpopnlashytions lymphocyte blastogenesis and neutrophil function in calve Bl Vet J 14U6617middotL

Pang W Y B Earley T SW1Cmey S Piralli V Gath and M A Crowe lOon Efftct~ of banding or burdizw castration of btdlli 1)11

neutrophil and respiratory burst CD62-L exprlssioll and serum cOllcentration J Anim Sci 87H87 5193

Sorrells S F and R M 2007 An inflalllllla Iory revw of glucocorticoid actiolls eNS Brain Bchav Imllullol 21 259-272

Weber P S S A t-ladscll G V SmithJ J Ireland and I L Burton 20(H Pre-translational regulation of lwutrophil Lmiddotsdedill

)cortlcOl1i-chlllCl1ge(j cattle Vet Ittlllllmo1 IllllllunopathoI

Journal of Dairy Science Vol 94 No5 2011