Associations among behavioral and acute physiologic responses to lipopolysaccharide-induced clinical mastitis in lactating dairy cows

Jennifer L. Zimov Department of Animal Sciences, Ohio Agricultural Research and Development Center, College of Food, Agricultural, and Environmental Sciences, The Ohio State University, Wooster, OH 44691.

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Naomi A. Botheras Department of Animal Sciences, Ohio Agricultural Research and Development Center, College of Food, Agricultural, and Environmental Sciences, The Ohio State University, Wooster, OH 44691.

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William P. Weiss Department of Animal Sciences, Ohio Agricultural Research and Development Center, College of Food, Agricultural, and Environmental Sciences, The Ohio State University, Wooster, OH 44691.

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Joseph S. Hogan Department of Animal Sciences, Ohio Agricultural Research and Development Center, College of Food, Agricultural, and Environmental Sciences, The Ohio State University, Wooster, OH 44691.

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Abstract

Objective—To examine behavioral and physiologic effects of lipopolysaccharide (LPS)-induced mastitis in lactating dairy cows.

Animals—20 Holstein cows.

Procedures—Cows were assigned to 5 blocks (4 cows/block) on the basis of parity and number of days in lactation. Intramammary infusion and IV treatments were assigned in a 2 × 2 factorial arrangement. Cows within each block were assigned to receive intramammary infusion with 25 μg of LPS or sterile PBS solution 3 hours after milking, and treatment with flunixin meglumine or sterile PBS solution was administered IV 4 hours after intramammary infusion. Video monitoring was continuously performed during the study.

Results—LPS-infused cows spent less time during the first 12 hours after infusion lying, eating, and chewing cud, compared with results for PBS solution-infused cows. Behavioral responses were correlated with physiologic responses for the first 12 hours after intramammary infusion. Flunixin meglumine administration after intramammary infusion mitigated some behavioral and clinical systemic responses.

Conclusions and Clinical Relevance—Intramammary infusion of LPS caused changes in both behavioral and physiologic variables in lactating dairy cows. Time spent lying, eating, and chewing cud were negatively correlated with physiologic responses in cows. Evaluation of behavior patterns may provide an ancillary measure, along with evaluation of physiologic variables, for monitoring well-being, clinical responses, and recovery from acute clinical mastitis.

Abstract

Objective—To examine behavioral and physiologic effects of lipopolysaccharide (LPS)-induced mastitis in lactating dairy cows.

Animals—20 Holstein cows.

Procedures—Cows were assigned to 5 blocks (4 cows/block) on the basis of parity and number of days in lactation. Intramammary infusion and IV treatments were assigned in a 2 × 2 factorial arrangement. Cows within each block were assigned to receive intramammary infusion with 25 μg of LPS or sterile PBS solution 3 hours after milking, and treatment with flunixin meglumine or sterile PBS solution was administered IV 4 hours after intramammary infusion. Video monitoring was continuously performed during the study.

Results—LPS-infused cows spent less time during the first 12 hours after infusion lying, eating, and chewing cud, compared with results for PBS solution-infused cows. Behavioral responses were correlated with physiologic responses for the first 12 hours after intramammary infusion. Flunixin meglumine administration after intramammary infusion mitigated some behavioral and clinical systemic responses.

Conclusions and Clinical Relevance—Intramammary infusion of LPS caused changes in both behavioral and physiologic variables in lactating dairy cows. Time spent lying, eating, and chewing cud were negatively correlated with physiologic responses in cows. Evaluation of behavior patterns may provide an ancillary measure, along with evaluation of physiologic variables, for monitoring well-being, clinical responses, and recovery from acute clinical mastitis.

Intramammary infusion of sterile LPS causes a cascade of local and systemic inflammatory responses in dairy cows similar to those observed during intramammary infections caused by gram-negative bacteria.1,2 The LPS intramammary challenge-exposure method has historically been beneficial for use in delineating cytologic, hormonal, humoral, and acute-phase protein responses of dairy cows that result from mammary gland inflammation.3–6 The effects of LPS challenge exposure on behavioral changes in dairy cows have been investigated to determine aberrant traits that could be used to differentiate diseased animals.7 Abnormal behaviors may be expressed more often during times of stress and pain. Behaviors displayed by ill animals are part of a coordinated complex of adaptations used to fight disease.8 Understanding behavior of dairy cows may improve identification of diseased animals and improve well-being of diseased cows. Resting and chewing cud are behaviors reportedly altered by LPS intramammary challenge exposure.7

The efficacy and mode of action of anti-inflammatory drugs as therapeutic agents in treating clinical mastitis have also been tested by use of an LPS challenge-exposure method.9,10 Flunixin meglumine suppresses the febrile response and maintains rumen activity in lactating cows following LPS challenge exposure.11 However, the possible effects of flunixin meglumine on behavior of cows with clinical mastitis have not been characterized. Therefore, the purposes of the study reported here were to use an LPS challenge-exposure method to determine changes in behavioral variables during acute mastitis, to evaluate effects of flunixin meglumine on behavioral responses to acute clinical mastitis, and to identify correlations between physiologic and behavioral variables during acute mastitis.

Materials and Methods

Animals—Twenty Holstein cows in the herd at the Ohio Agricultural Research and Development Center dairy farm were used in the study. Cows were assigned to 5 blocks (4 cows/block) on the basis of parity and number of days in lactation. One block was composed of primiparous cows, and the other 4 blocks were composed of multiparous cows. Number of days in lactation ranged from 65 to 110 days. All cows were housed in tie stalls for the duration of the study. Stalls had a concrete base with 170 × 150 × 2-cm inlaid rubber mats. Each stall was bedded twice daily with 10 kg of dried sawdust to cover the rubber mats with sawdust to a depth of approximately 3 cm. Cows remained in the tie stalls except during milking in a parlor at 3:00 am and 3:00 pm. Mean ± SD milk production of each cow was 40.5 ± 5.0 kg for the 7 days before intramammary infusion. Cows were fed a total-mixed ration each day at 3:30 am. Treatment and husbandry of cows were conducted in a manner to avoid unnecessary discomfort by use of proper management and experimental procedures that were approved by The Ohio State University Agricultural Animal Care and Use Committee.

Experimental treatments were assigned in a 2 × 2 factorial arrangement. Cows within each block were assigned via a randomization procedure (ie, blinded, sequential selection of cow identification numbers within a block from a hat) to receive intramammary infusion with LPS followed by treatment with flunixin meglumine IV, intramammary infusion with LPS followed by treatment with PBS solution IV, intramammary infusion with PBS solution followed by treatment with flunixin meglumine IV, or intramammary infusion with PBS solution followed by treatment with PBS solution IV.

Intramammary infusion—Intramammary infusions were administered in a front mammary gland of each cow 3 hours after milking (6:00 am). Only front mammary glands were selected for infusion to minimize possible unknown differences between front and hind mammary glands in response to LPS challenge exposure. Infected mammary glands were not infused. The infection status of mammary glands was determined by use of bacteriologic analyses of foremilk samples obtained from each front mammary gland 7,5, and 3 days before intramammary infusion. Briefly, milk samples were collected by use of aseptic techniques,12 placed on ice, and plated on the surface of blood-esculin agar (0.01 mL of milk sample) and MacConkey agar (0.1 mL of milk sample) within 3 hours after collection. Plates were incubated aerobically at 37°C, and growth was recorded after 24 and 48 hours. Only mammary glands with no bacterial growth in 2 of the 3 preinfusion samples were eligible for intramammary infusion. Cows with only 1 eligible front mammary gland were included in the study. If both front mammary glands of a cow were eligible for infusion, the gland to be infused was selected via a randomization procedure (ie, determined by the flip of a coin).

Cows within each block received an intramammary infusion on the same day. Each cow in a block received an intramammary infusion in 1 front mammary gland via the teat canal by use of a 33.8-mm sterile teat infusion cannula.a Concentrated LPS (Escherichia coli 026:B6b) was used; this LPS was diluted in sterile PBS solution (pH, 7.2) and sterilized by filtration through a 0.2-μm filter.c The challenge inoculate was 25 μg of LPS in 10 mL of PBS solution, which was selected on the basis of results of another study4 and preliminary titration data (data not shown) that indicated this concentration approaches the minimal dose needed to ensure a repeatable systemic response in dairy cows in early lactation. Control cows were infused with 10 mL of filter-sterilized PBS solution.

Treatment with flunixin meglumine—Flunixin meglumined (solution, 50 mg/mL) was injected IV via jugular venipuncture 4 hours after intramammary infusion (10 am) at a dose of 50 mg/45 kg. Control cows were given sterile PBS solution (1 mL/45 kg) IV via jugular venipuncture 4 hours after intramammary infusion (10 am). The decision was made a priori to administer flunixin meglumine or PBS solution IV 4 hours after intramammary infusion on the basis of experiments that revealed local and systemic clinical signs of mastitis were evident within 3 hours after LPS challenge exposure.2,4 The interval between sample collections allowed minimal impact of the IV treatments on measured variables. The sample collection at 3 hours after intramammary infusion was completed before IV treatments were administered, and an interval of 2 hours elapsed before the sample collection at 6 hours after intramammary infusion.

Sample collection—Foremilk samples from the mammary glands and blood samples were collected immediately before intramammary infusion (time 0) and 3, 6, 9, 12, and 24 hours after intramammary infusion. Approximately 10 mL of foremilk/mammary gland was collected by use of aseptic techniques.12 Two milliliters of milk was frozen until analysis for concentrations of amyloid A.e The remainder of each milk sample was used for bacteriologic analyses and determination of the SCC.13 Blood samples (5 mL) were collected via coccygeal venipuncture by use of 20-gauge needles. Serum was harvested and stored at −20°C until analysis to measure concentrations of cortisol.f

Physiologic measurements obtained at the time of each sample collection included the number of rumen sounds, rectal temperature, and clinical mastitis score. Rumen sounds were measured by means of auscultation.14 Clinical mastitis score was measured on a 5-point scale (1 = clinically normal mammary gland and clinically normal milk, 2 = clinically normal mammary gland but small flakes in milk, 3 = clinically normal mammary gland but grossly abnormal milk, 4 = swollen mammary gland and grossly abnormal milk, and 5 = systemic signs of infection, swollen mammary gland, and abnormal milk).13

DMI and milk production—Cows were milked twice daily at 12-hour intervals throughout the experiment. Milk production was electronically measured at each milking. The DMI was recorded throughout the experiment. Postinfusion daily milk production and DMI were expressed as a percentage of the mean for the 7 days before intramammary infusion and were calculated as (b/a) × 100, where b is the daily mean value after intramammary infusion and a is the daily mean value for the 7 days before intramammary infusion.13

Recording for behavior analysis—Cows were videotapedg continuously in the tie stalls from time of intramammary infusion until 24 hours after intramammary infusion. Two cameras equipped with 24 infrared light—emitting diodesh were used to record each cow. One camera was located directly above the cow, and the other was positioned to record the front of the cow. The amount of time for each of the following activities was determined for the first 24 hours of the study: lying, eating, and chewing cud. The interdependency of eating and chewing cud led to the analyses of the amount of time spent performing these behaviors separately and in combination, as reported elsewhere.15 Amount of time spent lying was defined as the amount of time cows were recumbent in stalls. Amount of time spent eating was defined as the amount of time cows were standing with their head lowered into the feed bunk. Amount of time spent chewing cud was defined as the amount of time cows were masticating while lying or while standing with their head outside the feed bunk. Activities were measured in increments of minutes. The 24 hours after intramammary infusion were stratified into 3-hour segments whereby behavioral activities coincided with physiologic variables. Behavior data were compared among treatment groups in 3-hour increments corresponding with physiologic measurements and as cumulative amount of time in periods from 0 to 3 hours, 0 to 6 hours, 0 to 9 hours, 0 to 12 hours, and 0 to 24 hours after intramammary infusion.

Statistical analysis—Differences among groups with regard to SCC, rectal temperature, milk amyloid A concentration, serum cortisol concentration, rumen sounds, amount of time spent chewing cud, amount of time spent eating, amount of time spent lying, number of days in lactation, and milk production were compared by use of statistical software.i The model used was Y = mean + block + mammary gland infusion + IV treatment + (mammary gland infusion•IV treatment) + error, where block was included as a random effect, mammary gland infusion was LPS or PBS solution, IV treatment was flunixin meglumine or PBS solution, and mammary gland infusion•IV treatment was the interaction term. All analyses were conducted within specified time periods. Associations among behavioral variables (amount of time spent chewing cud, amount of time spent eating, combined amount of time spent eating and chewing cud, and amount of time spent lying) and physiologic variables (SCC, cortisol concentration, amyloid A concentration, and rectal temperature) during the 12 hours after intramammary infusion were tested by use of the Pearson correlation test. For all analyses, values of P < 0.05 were considered significant.

Results

SCC—Cows that received LPS had a significant (P < 0.001) increase in SCC at 3 through 24 hours after intramammary infusion, compared with results for control cows that received an intramammary infusion of PBS solution (Figure 1). Overall, no significant effects were detected for the flunixin meglumine treatment or the interaction effects between LPS and flunixin meglumine treatment after challenge exposure.

Figure 1—
Figure 1—

Mean ± SEM SCC (A), clinical mastitis score (B), and milk amyloid A concentration (C) in mammary glands of cows (n = 5 cows/treatment group) after intramammary infusion of 25 μg of LPS followed 4 hours later by IV injection of PBS solution (1 mL/45 kg; black circles [A and C] and diagonal-striped bars [B]), intramammary infusion of 25 μg of LPS followed 4 hours later by IV injection of flunixin meglumine (50 mg/45 kg; white circles [A and C] and white bars [B]), intramammary infusion of 10 mL of PBS solution followed 4 hours later by IV injection of 10 mL of PBS solution (1 mL/45 kg; inverted black triangles [A and C] and cross-hatched bars [B]), or intramammary infusion of 10mL of PBS solution followed 4 hours later by IV injection of flunixin meglumine (50 mg/45 kg; white triangles [A and C] and black bars [B]). Time of intramammary infusion was designated as time 0. Clinical mastitis score was measured on a 5-point scale (1 = clinically normal mammary gland and clinically normal milk, 2 = clinically normal mammary gland but small flakes in milk, 3 = clinically normal mammary gland but abnormal milk, 4 = swollen mammary gland and abnormal milk, and 5 = systemic signs of infection, swollen mammary gland, and abnormal milk).

Citation: American Journal of Veterinary Research 72, 5; 10.2460/ajvr.72.5.620

Clinical mastitis score—During the first 24 hours after intramammary infusion, LPS-infused cows had a significantly greater clinical mastitis score than did cows that were infused with PBS solution (Figure 1). Each mammary gland infused with LPS resulted in clinical mastitis scores ≥ 3 by 3 hours after challenge exposure. Mammary glands infused with PBS solution did not have clinical mastitis during the 24 hours after infusion. No significant effects of flunixin meglumine treatment or interaction effects between LPS and flunixin meglumine treatment were found for clinical mastitis scores.

Milk amyloid A concentration—Cows challenge exposed with LPS had a significantly (P = 0.01) greater milk amyloid A concentration at 9 and 24 hours after intramammary infusion than did cows infused with PBS solution (Figure 1). Cows challenge exposed with LPS had milk amyloid A concentrations that were 3- to 7-fold greater than the concentrations for cows infused with PBS solution at 9 and 24 hours after intramammary infusion. No significant effects of flunixin meglumine treatment or interaction effects between LPS and flunixin meglumine treatment were found.

Rectal temperature—Rectal temperatures of cows challenged exposed with LPS increased significantly (P = 0.01) at 3, 6, and 9 hours after intramammary infusion, compared with rectal temperatures of cows infused with PBS solution (Figure 2). The IV treatment (ie, flunixin meglumine or PBS solution) did not significantly affect rectal temperatures throughout the first 24 hours after intramammary infusion; however, a significant interaction between LPS infusion and flunixin meglumine treatment was detected at 6 (P = 0.04) and 12 (P = 0.01) hours after intramammary infusion, but not at 9 hours (P = 0.07). Cows challenge exposed with LPS and treated with flunixin meglumine had a lower rectal temperature than did cows challenge exposed with LPS and treated with PBS solution.

Figure 2—
Figure 2—

Mean ± SEM rectal temperature (A), frequency of rumen contractions (B), and serum cortisol concentration (C) of cows (n = 5 cows/treatment group) that received intramammary infusion of LPS followed 4 hours later by IV injection of PBS solution (black circles), intramammary infusion of LPS followed 4 hours later by IV injection of flunixin meglumine (white circles), intramammary infusion of PBS solution followed 4 hours later by IV injection of PBS solution (inverted black triangles), or intramammary infusion of PBS solution followed 4 hours later by IV injection of flunixin meglumine (white triangles). Time of intramammary infusion was designated as time 0.

Citation: American Journal of Veterinary Research 72, 5; 10.2460/ajvr.72.5.620

Rumen sounds—At 6, 9, and 12 hours after intramammary infusion, cows challenge exposed with LPS had rumen sounds significantly less frequently, compared with results for cows infused with PBS solution (Figure 2). Frequency of rumen sounds from cows treated IV with flunixin meglumine did not differ significantly from that of cows treated IV with PBS solution. A nonsignificant interaction between LPS infusion and flunixin meglumine treatment was detected 6 (P = 0.06) and 12 (P = 0.10) hours after intramammary infusion. Cows challenge exposed with LPS and treated with flunixin meglumine had rumen sounds that were twofold more frequent than did cows challenge exposed with LPS and treated with PBS solution during those time periods.

Serum cortisol concentration—Cows receiving the LPS infusion had significantly (P = 0.01) elevated serum cortisol concentrations at 3, 6, and 12 hours after intramammary infusion, compared with concentrations for cows infused with PBS solution (Figure 2). Mean ± SEM cortisol concentrations of cows challenge exposed with LPS increased quickly and peaked at 26.8 ± 3.3 μg/dL, compared with cortisol concentrations in cows infused with PBS solution, which had a mean of 10.9 ± 2.9 μg/dL at 3 hours after intramammary infusion. Samples obtained 24 hours after intramammary infusion did not differ significantly between groups infused with LPS or PBS solution in regard to cortisol concentrations. No significant effects of flunixin meglumine treatment or the interaction between LPS infusion and flunixin meglumine treatment were found.

DMI—The DMI was not significantly affected by LPS infusion or flunixin meglumine treatment administered after challenge exposure (Figure 3). There was not a significant effect of the interaction between LPS infusion and flunixin meglumine treatment.

Figure 3—
Figure 3—

Mean ± SEM DMI (A) and milk production (B) of cows (n = 5 cows/treatment group) after intramammary infusion of LPS followed 4 hours later by IV injection of PBS solution (diagonal-striped bars), intramammary infusion of LPS followed 4 hours later by IV injection of flunixin meglumine (white bars), intramammary infusion of PBS solution followed 4 hours later by IV injection of PBS solution (cross-hatched bars), or intramammary infusion of PBS solution followed 4 hours later by IV injection of flunixin meglumine (black bars). Values for DMI and milk production are expressed as the percentage of the daily mean value after intramammary infusion divided by the mean value for the 7 days before intramammary infusion. Time of intramammary infusion was designated as time 0.

Citation: American Journal of Veterinary Research 72, 5; 10.2460/ajvr.72.5.620

Milk production—Milk production for cows infused with LPS decreased significantly 1 and 2 days after challenge exposure, compared with milk production for cows infused with PBS solution (Figure 3). The LPS infusion caused a 25% decrease in milk production 1 day after challenge exposure and a 7% decrease 2 days after challenge exposure, compared with the mean milk production for the 7 days before challenge exposure. No significant effects of flunixin meglumine treatment or the interaction between LPS infusion and flunixin meglumine treatment were found.

Amount of time spent eating—The amount of time spent eating differed between cows infused with LPS and cows infused with PBS solution (Figure 4). Cows challenge exposed with LPS spent significantly less time eating at 0 to 3 hours (P < 0.001), 3 to 6 hours (P = 0.01), and 6 to 9 hours (P = 0.004) after intramammary infusion than did cows infused with PBS solution. Cows that received flunixin meglumine treatment spent significantly more time eating at 9 to 12 hours after intramammary infusion than did cows that received treatment with PBS solution. A nonsignificant interaction was detected between LPS infusion and flunixin meglumine treatment at 3 to 6 hours (P = 0.15) and 6 to 9 hours (P = 0.11) after intramammary infusion. Cows infused with LPS and treated with flunixin meglumine spent more time eating during those periods than did cows infused with LPS and treated with PBS solution.

Figure 4—
Figure 4—

Mean ± SEM amount of time spent eating (A), amount of time spent chewing cud (B), and combined amount of time spent eating and chewing cud (C) for cows (n = 5 cows/treatment group) after intramammary infusion of LPS followed 4 hours later by IV injection of PBS solution (diagonal-striped bars), intramammary infusion of LPS followed 4 hours later by IV injection of flunixin meglumine (white bars), intramammary infusion of PBS solution followed 4 hours later by IV injection of PBS solution (cross-hatched bars), or intramammary infusion of PBS solution followed 4 hours later by IV injection of flunixin meglumine (black bars). Time of intramammary infusion was designated as time 0.

Citation: American Journal of Veterinary Research 72, 5; 10.2460/ajvr.72.5.620

Cumulative amount of time spent eating during the first 12 hours after intramammary infusion differed between the LPS- and PBS solution—infused cows (Figure 5). Cows challenge exposed with LPS spent significantly less time eating during the first 12 hours after intramammary infusion than did cows infused with PBS solution. Flunixin meglumine treatment did not significantly affect cumulative amount of time spent eating during the first 12 hours after intramammary infusion. During the first 12 hours after intramammary infusion, a significant (P = 0.01) interaction between LPS infusion and flunixin meglumine treatment was detected. Cows infused with LPS and treated with flunixin meglumine spent more time eating during the first 12 hours after intramammary infusion than did cows infused with LPS and treated with PBS solution.

Figure 5—
Figure 5—

Mean ± SEM cumulative amount of time spent eating (A), cumulative amount of time spent chewing cud (B), and combined cumulative amount of time spent eating and chewing cud (C) for cows (n = 5 cows/treatment group) after intramammary infusion of LPS followed 4 hours later by IV injection of PBS solution (diagonal-striped bars), intramammary infusion of LPS followed 4 hours later by IV injection of flunixin meglumine (white bars), intramammary infusion of PBS solution followed 4 hours later by IV injection of PBS solution (cross-hatched bars), or intramammary infusion of PBS solution followed 4 hours later by IV injection of flunixin meglumine (black bars). Time of intramammary infusion was designated as time 0.

Citation: American Journal of Veterinary Research 72, 5; 10.2460/ajvr.72.5.620

Time spent chewing cud—The amount of time spent chewing cud by cows infused with LPS was significantly (P < 0.001) less at 3 to 6 hours and 6 to 9 hours after intramammary infusion than that spent by cows infused with PBS solution (Figure 4). In fact, cows challenge exposed with PBS solution spent approximately twice as much time chewing cud during these 2 time periods as did cows challenge exposed with LPS. Type of IV treatment also affected the amount of time spent chewing cud. Cows treated with flunixin meglumine spent a significantly greater amount of time chewing cud at 3 to 6 hours and 6 to 9 hours after intramammary infusion than did cows treated with PBS solution. At 6 to 9 hours after intramammary infusion, there was a nonsignificant (P = 0.13) interaction between LPS infusion and flunixin meglumine treatment. Cows challenge exposed with LPS followed by treatment with flunixin meglumine spent approximately twofold more time chewing cud than did cows challenge exposed with LPS followed by treatment with PBS solution.

Cows infused with LPS spent significantly less cumulative time chewing cud during the first 12 hours after intramammary infusion, compared with that for cows infused with PBS solution (Figure 5). Cows receiving flunixin meglumine treatment spent a significantly higher amount of time chewing cud during the first 9 hours after intramammary infusion, compared with that for cows receiving treatment with PBS solution. We did not detect a significant interaction between LPS infusion and flunixin meglumine treatment on cumulative amount of time spent chewing cud.

Combined amount of time spent eating and chewing cud—Combined amount of time spent eating and chewing cud for cows infused with LPS was significantly less at 0 to 3 hours (P < 0.05), 3 to 6 hours (P < 0.001), and 6 to 9 hours (P < 0.001), compared with the amount of time for cows infused with PBS solution (Figure 4). Treatment with flunixin meglumine or PBS solution did not significantly affect the combined amount of time spent eating and chewing cud. A significant interaction between LPS infusion and flunixin meglumine treatment was detected at 3 to 6 hours (P < 0.001) and 6 to 9 hours (P < 0.001) after intramammary infusion. Cows infused with LPS and treated with flunixin meglumine spent twice the amount of time eating and chewing cud, compared with the amount of time for cows infused with LPS and treated with PBS solution.

Cows challenge exposed with LPS spent significantly less cumulative amount of time eating and chewing cud during the first 3, 6, 9, and 12 hours after intramammary infusion, compared with results for cows infused with PBS solution (Figure 5). Cumulative amount of time spent eating and chewing cud did not differ significantly between cows treated with flunixin meglumine or PBS solution. Cows infused with LPS and treated with flunixin meglumine spent significantly more time eating and chewing cud, compared with that for cows infused with LPS and treated with PBS solution, during the first 9 and 12 hours after intramammary infusion.

Amount of time spent lying—The amount of time spent lying within stratified time periods (Figure 6) and the cumulative amount of time spent lying were not significantly affected by LPS infusion or flunixin meglumine treatment. No significant interaction was detected between LPS infusion and flunixin meglumine treatment.

Figure 6—
Figure 6—

Mean ± SEM amount of time spent lying (A) and cumulative amount of time spent lying (B) for cows (n = 5 cows/treatment group) after intramammary infusion of LPS followed 4 hours later by IV injection of PBS solution (diagonal-striped bars), intramammary infusion of LPS followed 4 hours later by IV injection of flunixin meglumine (white bars), intramammary infusion of PBS solution followed 4 hours later by IV injection of PBS solution (cross-hatched bars), or intramammary infusion of PBS solution followed 4 hours later by IV injection of flunixin meglumine (black bars). Time of intramammary infusion was designated as time 0.

Citation: American Journal of Veterinary Research 72, 5; 10.2460/ajvr.72.5.620

Correlations among behavioral and physiologic responses—Rectal temperature was significantly negatively correlated with the amount of time spent chewing cud (r = −0.49), amount of time spent eating (r = −0.35), combined amount of time spent eating and chewing cud (r = −0.59), and amount of time spent lying (r = −0.23) during the first 12 hours after LPS challenge exposure. Serum cortisol concentration was also significantly negatively associated with amount of time spent chewing cud (r = −0.41), combined amount of time spent eating and chewing cud (r = −0.32), and amount of time spent lying (r = −0.21) during the first 12 hours after LPS challenge exposure. Amyloid A concentration and SCC in milk from infused mammary glands were not significantly correlated with behavioral responses during the 12 hours after intramammary infusion.

Discussion

Lipopolysaccharide-induced acute clinical mastitis caused changes in both behavioral and physiologic variables in lactating dairy cows. Values for the behavioral variables of the amount of time spent chewing cud and amount of time spent eating, measured separately or in combination, were lower in cows receiving LPS, compared with values in cows receiving PBS solution. Eating and chewing cud were mutually exclusive events as timed during observation; however, the inter-dependency of the actions led to the analyses of these variables separately and in combination, as reported elsewhere.15 Regardless of whether tested separately or in combination, the reduced amount of time spent eating and chewing cud during the first 12 hours after LPS challenge exposure corresponded with the peak in systemic clinical signs at 3 to 9 hours after intramammary infusion. In addition, the behavioral variables of the amount of time spent eating and chewing cud were negatively correlated with the systemic response variables of rectal temperature and blood cortisol concentrations during the 12 hours after intramammary infusion. Changes in the amount of time spent eating and chewing cud appeared to be limited to the first 12 hours after intramammary infusion. The cumulative amount of time spent eating and chewing cud for 24 hours after intramammary infusion did not differ between cows with acute LPS-induced mastitis and control cows receiving PBS solution as an intramammary infusion.

Treatment with flunixin meglumine reduced the negative effects of LPS-induced clinical mastitis with regard to the amount of time spent eating and chewing cud. In fact, treatment with flunixin meglumine increased the amount of time spent eating and chewing cud in cows infused with LPS or PBS solution during 5 to 9 hours after IV treatment (ie, 9 to 13 hours after intramammary infusion). Results of the present study suggested that treatment with flunixin meglumine had a stimulatory effect on eating behavior and rumen activity, which are typically reduced as a result of acute mastitis. In contrast, investigators in another study16 reported that DMI was reduced in postparturient cows treated with flunixin meglumine. A possible explanation for this discrepancy was the difference in metabolic status of periparturient cows16 and cows in the present study that were between 65 and 110 days in lactation.

Cumulative amount of time spent lying did not differ between intramammary infusion groups, and flunixin meglumine treatment did not affect the amount of time cows spent lying in stalls. These data differ from those of a Finnish study7 in which cows with mastitis spent more time resting from 0 to 2 hours after challenge exposure and less time resting from 3 to 11 hours after challenge exposure, compared with the amount of time spent lying before challenge exposure. The interval between milking and feeding and intramammary infusion may have influenced the amount of time spent lying in the present study. Both LPS- and PBS solution—infused cows spent a considerably greater amount of time lying in stalls during the period from 12 to 24 hours after intramammary infusion, compared with the amount of time spent lying during the first 12 hours after intramammary infusion (the period when acute signs of mastitis were evident in LPS-infused cows). Despite the lack of differences in the amount of time spent lying between LPS- and PBS solution—infused cows, the amount of time spent lying was negatively correlated with rectal temperature and blood cortisol concentrations during the first 12 hours after intramammary infusion.

Results of the study reported here are in agreement with those of other reports14,17 in which intramammary challenge exposure with LPS caused increased rectal temperature, increased serum cortisol concentrations, and decreased rumen activity. In the present study, changes in each of these variables peaked rapidly during the first 12 hours after challenge exposure and returned to values comparable to those of PBS solution—infused (control) cows by 24 hours after intramammary infusion. Investigators in one of the aforementioned studies14 reported that cows with endotoxin-induced mastitis developed fever and had reduced rumen activity within the first 24 hours after challenge exposure. In the other aforementioned study,17 intramammary infusion of dairy cows with LPS caused mastitis and a 5- to 10-fold increase in serum cortisol concentrations after challenge exposure.

Treatment with flunixin meglumine 4 hours after intramammary infusion mitigated the systemic responses of increased rectal temperature and decreased rumen activity in the cows of the present study. Flunixin meglumine is an antipyretic drug18 capable of reducing the febrile response to LPS. Results of the present study are in agreement with those of another study,11 which indicates that cattle challenge exposed with LPS and treated with flunixin meglumine have greater frequency of rumen sounds, compared with results for untreated cattle challenge exposed with LPS. The administration of flunixin meglumine to cows with mastitis experimentally induced by the use of E coli reduces the deleterious effects of the disease on rumen activity.19 The effects of flunixin meglumine on rumen contractions led authors in 1 study10 to speculate that the decrease in rumen motility during E coli—induced mastitis is at least partly attributable to a mechanism involving prostaglandin. Serum cortisol concentration was not responsive to flunixin meglumine treatment in the present study. The lack of response to flunixin meglumine after LPS challenge exposure may have been attributable to the timing of the treatment. Flunixin meglumine was administered 4 hours after intramammary infusion of LPS, and the peak cortisol response in cows was detected 3 hours after intramammary infusion of LPS.

Milk production was significantly reduced after LPS infusion. Reduction in milk production following intramammary infusion of LPS in the study reported here is consistent with results in other reports.20,21 The decrease in milk production during LPS release into the mammary gland appears to be mediated locally via secretory tissue and systemically via a reduction in appetite.22 Treatment with flunixin meglumine was not a factor for milk production or DMI. In contrast to results of another study21 of experimentally induced mastitis, LPS infusion did not significantly alter DMI in the present study. In that study,21 investigators reported that cows receiving LPS had decreased feed intake on the day of the challenge exposure and that feed intake was still 19% lower than in the control cows the day after challenge exposure. Time of feeding on the day of challenge exposure may explain discrepancies on DMI in cows challenge exposed with LPS between the present study and that other study.21 Cows in the present study were fed 2.5 hours before challenge exposure, whereas cows in that other study21 were fed at the time of challenge exposure. Feeding activity peaks immediately after feed delivery.23

Intramammary infusion of LPS caused local changes in the mammary gland. The SCC and milk amyloid A concentration increased within hours after the intramammary infusion and remained elevated at 24 hours after challenge exposure. These results support those in another report24 in that LPS infusion caused a rapid influx of neutrophils into the mammary gland and a subsequent increase in milk amyloid A concentration. In contrast to systemic signs of clinical mastitis, these local factors of inflammation (ie, SCC and milk amyloid A concentration) were not altered by treatment with flunixin meglumine. The initial influx of somatic cells into challenge-exposed mammary glands preceded treatment with flunixin meglumine; therefore, a change in SCCs was not expected because of flunixin meglumine. Flunixin meglumine treatment of clinical mastitis failed to influence local mammary gland responses in other studies.3,25 Local mammary gland responses following intramammary challenge exposure were not correlated with behavioral variables in the study reported here.

The method used in the present study to monitor the behavior of cows following LPS challenge exposure was chosen to test whether there were concurrent changes in behavioral and physiologic variables during acute mastitis. The behavioral traits of the amount of time spent eating and chewing cud were measurable variables altered by acute mastitis, and administration of flunixin meglumine mitigated the adverse effects of acute mastitis on both behavioral and systemic physiologic variables. The correlations among behavioral and physiologic variables suggest that observation of behavior is an ancillary noninvasive method for monitoring the well-being and recovery of dairy cows with acute LPS-induced mastitis.

ABBREVIATIONS

DMI

Dry-matter intake

LPS

Lipopolysaccharide

SCC

Somatic cell count

a.

Jorgensen Laboratories Inc, Loveland, Colo.

b.

Sigma Chemical Co, St Louis, Mo.

c.

Gelman Laboratory, Ann Arbor, Mich.

d.

Banamine, Schering-Plough Animal Health Corp, Union, NJ.

e.

Mast ID RANGE, Tridelta Development, Maynooth, Ireland.

f.

ACTIVE Cortisol EIA, Diagnostic Systems Laboratories Inc, Webster, Tex.

g.

Geovision GV-1000, USA Vision Systems Inc, Irvine, Calif.

h.

Maxi Day/Night Cam security camera, Swann Corp Inc, Santa Fe Springs, Calif.

i.

PROC MIXED, SAS, version 9, SAS Institute Inc, Cary, NC.

References

  • 1.

    Jain NC, Schalm OW, Lasmanis J. Neutrophil kinetics in endotoxin-induced mastitis. Am J Vet Res 1978; 39: 16621667.

  • 2.

    Barrett JJ, Hogan JS, Weiss WP, et al. Concentrations of alpha-tocopherol after intramammary infusion of Escherichia coli or lipopolysaccharide. J Dairy Sci 1997; 80: 28262832.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 3.

    Anderson KL, Smith AR, Shanks RD, et al. Endotoxin-induced bovine mastitis: immunoglobulins, phagocytosis, and effect of flunixin meglumine. Am J Vet Res 1986; 47: 24052410.

    • Search Google Scholar
    • Export Citation
  • 4.

    Jackson JA, Shuster DE, Silvia WJ, et al. Physiological responses to intramammary or intravenous treatment with endotoxin in lactating cows. J Dairy Sci 1990; 73: 627632.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 5.

    Bannerman DD, Paape MJ, Hare WR, et al. Increased levels of LPS-binding protein in bovine blood and milk following bacterial lipopolysaccharide challenge. J Dairy Sci 2003; 86: 31283137.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 6.

    Wagner SA, Jones DE, Apley MD. Effect of endotoxic mastitis on epithelial cell numbers in the milk of dairy cows. Am J Vet Res 2009; 70: 796799.

  • 7.

    Hänninen L, Kaihilahti J, Taponen S, et al. Does behaviour predict acute endotoxin mastitis in dairy cows? in Proceedings. 41st Int Cong Int Soc Appl Ethol 2007; 508.

    • Search Google Scholar
    • Export Citation
  • 8.

    von Keyserlingk MAG, Rushen J, Passille AM, et al. The welfare of dairy cattle—key concepts and the role of science. J Dairy Sci 2009; 92: 41014111.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 9.

    Anderson KL, Smith AR, Shanks RD, et al. Efficacy of flunixin meglumine for the treatment of endotoxin-induced bovine mastitis. Am J Vet Res 1986; 47: 13661372.

    • Search Google Scholar
    • Export Citation
  • 10.

    Lohuis JACM, Van Leeuwen W, Verheijden JHM, et al. Effect of steroidal anti-inflammatory drugs on Escherichia coli endotoxin-induced mastitis in the cow. J Dairy Sci 1989; 72: 241249.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11.

    Wagner SA, Apley MD. Effects of two anti-inflammatory drugs on physiologic variables and milk production in cows with endotoxin-induced mastitis. Am J Vet Res 2004; 65: 6468.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 12.

    National Mastitis Council. Microbiological procedures for the diagnosis of bovine udder infection and determination of milk quality. 4th ed. Fort Atkinson, Wis: National Mastitis Council and WD Hoard and Sons Co, 2004; 18.

    • Search Google Scholar
    • Export Citation
  • 13.

    Hogan JS, Weiss WP, Smith KL, et al. Effects of an Escherichia coli J5 vaccine on mild clinical coliform mastitis. J Dairy Sci 1995; 78: 285290.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14.

    Perkins KH, VandeHaar MJ, Burton JL, et al. Clinical responses to intramammary endotoxin infusion in dairy cows subjected to feed restriction. J Dairy Sci 2002; 85: 17241731.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 15.

    Allen DM, Grant RJ. Interactions between forage and wet corn gluten feed as sources of fiber in diets for lactating cows. J Dairy Sci 2000; 73: 322331.

    • Search Google Scholar
    • Export Citation
  • 16.

    Shwartz G, Hill K, VanBaale M, et al. Effects of flunixin meglumine on pyrexia and bioenergetic variables in postparturient dairy cows. J Dairy Sci 2008; 92: 19631970.

    • Search Google Scholar
    • Export Citation
  • 17.

    Shuster DE, Harmon RJ. High cortisol concentrations and mediation of the hypogalactia during endotoxin-induced mastitis. J Dairy Sci 1992; 75: 739746.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 18.

    Smith GW, Davis JL, Tell LA, et al. Extralabel use of nonsteroidal anti-inflammatory drugs in cattle. J Am Vet Med Assoc 2008; 232: 697701.

  • 19.

    Lohuis JACM, Van Leeuwen W, Verheijden JHM, et al. Flunixin meglumine and flurbiprofen in cows with experimental Escherichia coli mastitis. Vet Rec 1989; 124: 305308.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 20.

    Lehtolainen T, Suominen S, Kutila T. Effect of intramammary Escherichia coli endotoxin in early- vs. late-lactating dairy cows. J Dairy Sci 2003; 86: 23272333.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 21.

    Waldron MR, Kulick AE, Bell AW, et al. Acute experiment mastitis is not causal toward the development of energy-related metabolic disorders in early postpartum dairy cows. J Dairy Sci 2006; 89: 596610.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 22.

    Hogan J, Smith KL. Coliform mastitis. Vet Res 2003; 34: 507519.

  • 23.

    Mäntysaari P, Khalili H, Sariola J. Effect of feeding frequency of a total mixed ration on the performance of high-yielding dairy cows. J Dairy Sci 2006; 89: 43124320.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 24.

    Boosman R, Niewold TA, Mutsaers CW, et al. Serum amyloid A concentrations in cows given endotoxin as an acute-phase stimulant. Am J Vet Res 1989; 50: 16901694.

    • Search Google Scholar
    • Export Citation
  • 25.

    Dascanio JJ, Mechor G, Gröhn Y, et al. Effect of phenylbutazone and flunixin meglumine on acute toxic mastitis in dairy cows. Am J Vet Res 1995; 56: 12131218.

    • Search Google Scholar
    • Export Citation
  • Figure 1—

    Mean ± SEM SCC (A), clinical mastitis score (B), and milk amyloid A concentration (C) in mammary glands of cows (n = 5 cows/treatment group) after intramammary infusion of 25 μg of LPS followed 4 hours later by IV injection of PBS solution (1 mL/45 kg; black circles [A and C] and diagonal-striped bars [B]), intramammary infusion of 25 μg of LPS followed 4 hours later by IV injection of flunixin meglumine (50 mg/45 kg; white circles [A and C] and white bars [B]), intramammary infusion of 10 mL of PBS solution followed 4 hours later by IV injection of 10 mL of PBS solution (1 mL/45 kg; inverted black triangles [A and C] and cross-hatched bars [B]), or intramammary infusion of 10mL of PBS solution followed 4 hours later by IV injection of flunixin meglumine (50 mg/45 kg; white triangles [A and C] and black bars [B]). Time of intramammary infusion was designated as time 0. Clinical mastitis score was measured on a 5-point scale (1 = clinically normal mammary gland and clinically normal milk, 2 = clinically normal mammary gland but small flakes in milk, 3 = clinically normal mammary gland but abnormal milk, 4 = swollen mammary gland and abnormal milk, and 5 = systemic signs of infection, swollen mammary gland, and abnormal milk).

  • Figure 2—

    Mean ± SEM rectal temperature (A), frequency of rumen contractions (B), and serum cortisol concentration (C) of cows (n = 5 cows/treatment group) that received intramammary infusion of LPS followed 4 hours later by IV injection of PBS solution (black circles), intramammary infusion of LPS followed 4 hours later by IV injection of flunixin meglumine (white circles), intramammary infusion of PBS solution followed 4 hours later by IV injection of PBS solution (inverted black triangles), or intramammary infusion of PBS solution followed 4 hours later by IV injection of flunixin meglumine (white triangles). Time of intramammary infusion was designated as time 0.

  • Figure 3—

    Mean ± SEM DMI (A) and milk production (B) of cows (n = 5 cows/treatment group) after intramammary infusion of LPS followed 4 hours later by IV injection of PBS solution (diagonal-striped bars), intramammary infusion of LPS followed 4 hours later by IV injection of flunixin meglumine (white bars), intramammary infusion of PBS solution followed 4 hours later by IV injection of PBS solution (cross-hatched bars), or intramammary infusion of PBS solution followed 4 hours later by IV injection of flunixin meglumine (black bars). Values for DMI and milk production are expressed as the percentage of the daily mean value after intramammary infusion divided by the mean value for the 7 days before intramammary infusion. Time of intramammary infusion was designated as time 0.

  • Figure 4—

    Mean ± SEM amount of time spent eating (A), amount of time spent chewing cud (B), and combined amount of time spent eating and chewing cud (C) for cows (n = 5 cows/treatment group) after intramammary infusion of LPS followed 4 hours later by IV injection of PBS solution (diagonal-striped bars), intramammary infusion of LPS followed 4 hours later by IV injection of flunixin meglumine (white bars), intramammary infusion of PBS solution followed 4 hours later by IV injection of PBS solution (cross-hatched bars), or intramammary infusion of PBS solution followed 4 hours later by IV injection of flunixin meglumine (black bars). Time of intramammary infusion was designated as time 0.

  • Figure 5—

    Mean ± SEM cumulative amount of time spent eating (A), cumulative amount of time spent chewing cud (B), and combined cumulative amount of time spent eating and chewing cud (C) for cows (n = 5 cows/treatment group) after intramammary infusion of LPS followed 4 hours later by IV injection of PBS solution (diagonal-striped bars), intramammary infusion of LPS followed 4 hours later by IV injection of flunixin meglumine (white bars), intramammary infusion of PBS solution followed 4 hours later by IV injection of PBS solution (cross-hatched bars), or intramammary infusion of PBS solution followed 4 hours later by IV injection of flunixin meglumine (black bars). Time of intramammary infusion was designated as time 0.

  • Figure 6—

    Mean ± SEM amount of time spent lying (A) and cumulative amount of time spent lying (B) for cows (n = 5 cows/treatment group) after intramammary infusion of LPS followed 4 hours later by IV injection of PBS solution (diagonal-striped bars), intramammary infusion of LPS followed 4 hours later by IV injection of flunixin meglumine (white bars), intramammary infusion of PBS solution followed 4 hours later by IV injection of PBS solution (cross-hatched bars), or intramammary infusion of PBS solution followed 4 hours later by IV injection of flunixin meglumine (black bars). Time of intramammary infusion was designated as time 0.

  • 1.

    Jain NC, Schalm OW, Lasmanis J. Neutrophil kinetics in endotoxin-induced mastitis. Am J Vet Res 1978; 39: 16621667.

  • 2.

    Barrett JJ, Hogan JS, Weiss WP, et al. Concentrations of alpha-tocopherol after intramammary infusion of Escherichia coli or lipopolysaccharide. J Dairy Sci 1997; 80: 28262832.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 3.

    Anderson KL, Smith AR, Shanks RD, et al. Endotoxin-induced bovine mastitis: immunoglobulins, phagocytosis, and effect of flunixin meglumine. Am J Vet Res 1986; 47: 24052410.

    • Search Google Scholar
    • Export Citation
  • 4.

    Jackson JA, Shuster DE, Silvia WJ, et al. Physiological responses to intramammary or intravenous treatment with endotoxin in lactating cows. J Dairy Sci 1990; 73: 627632.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 5.

    Bannerman DD, Paape MJ, Hare WR, et al. Increased levels of LPS-binding protein in bovine blood and milk following bacterial lipopolysaccharide challenge. J Dairy Sci 2003; 86: 31283137.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 6.

    Wagner SA, Jones DE, Apley MD. Effect of endotoxic mastitis on epithelial cell numbers in the milk of dairy cows. Am J Vet Res 2009; 70: 796799.

  • 7.

    Hänninen L, Kaihilahti J, Taponen S, et al. Does behaviour predict acute endotoxin mastitis in dairy cows? in Proceedings. 41st Int Cong Int Soc Appl Ethol 2007; 508.

    • Search Google Scholar
    • Export Citation
  • 8.

    von Keyserlingk MAG, Rushen J, Passille AM, et al. The welfare of dairy cattle—key concepts and the role of science. J Dairy Sci 2009; 92: 41014111.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 9.

    Anderson KL, Smith AR, Shanks RD, et al. Efficacy of flunixin meglumine for the treatment of endotoxin-induced bovine mastitis. Am J Vet Res 1986; 47: 13661372.

    • Search Google Scholar
    • Export Citation
  • 10.

    Lohuis JACM, Van Leeuwen W, Verheijden JHM, et al. Effect of steroidal anti-inflammatory drugs on Escherichia coli endotoxin-induced mastitis in the cow. J Dairy Sci 1989; 72: 241249.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11.

    Wagner SA, Apley MD. Effects of two anti-inflammatory drugs on physiologic variables and milk production in cows with endotoxin-induced mastitis. Am J Vet Res 2004; 65: 6468.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 12.

    National Mastitis Council. Microbiological procedures for the diagnosis of bovine udder infection and determination of milk quality. 4th ed. Fort Atkinson, Wis: National Mastitis Council and WD Hoard and Sons Co, 2004; 18.

    • Search Google Scholar
    • Export Citation
  • 13.

    Hogan JS, Weiss WP, Smith KL, et al. Effects of an Escherichia coli J5 vaccine on mild clinical coliform mastitis. J Dairy Sci 1995; 78: 285290.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14.

    Perkins KH, VandeHaar MJ, Burton JL, et al. Clinical responses to intramammary endotoxin infusion in dairy cows subjected to feed restriction. J Dairy Sci 2002; 85: 17241731.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 15.

    Allen DM, Grant RJ. Interactions between forage and wet corn gluten feed as sources of fiber in diets for lactating cows. J Dairy Sci 2000; 73: 322331.

    • Search Google Scholar
    • Export Citation
  • 16.

    Shwartz G, Hill K, VanBaale M, et al. Effects of flunixin meglumine on pyrexia and bioenergetic variables in postparturient dairy cows. J Dairy Sci 2008; 92: 19631970.

    • Search Google Scholar
    • Export Citation
  • 17.

    Shuster DE, Harmon RJ. High cortisol concentrations and mediation of the hypogalactia during endotoxin-induced mastitis. J Dairy Sci 1992; 75: 739746.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 18.

    Smith GW, Davis JL, Tell LA, et al. Extralabel use of nonsteroidal anti-inflammatory drugs in cattle. J Am Vet Med Assoc 2008; 232: 697701.

  • 19.

    Lohuis JACM, Van Leeuwen W, Verheijden JHM, et al. Flunixin meglumine and flurbiprofen in cows with experimental Escherichia coli mastitis. Vet Rec 1989; 124: 305308.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 20.

    Lehtolainen T, Suominen S, Kutila T. Effect of intramammary Escherichia coli endotoxin in early- vs. late-lactating dairy cows. J Dairy Sci 2003; 86: 23272333.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 21.

    Waldron MR, Kulick AE, Bell AW, et al. Acute experiment mastitis is not causal toward the development of energy-related metabolic disorders in early postpartum dairy cows. J Dairy Sci 2006; 89: 596610.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 22.

    Hogan J, Smith KL. Coliform mastitis. Vet Res 2003; 34: 507519.

  • 23.

    Mäntysaari P, Khalili H, Sariola J. Effect of feeding frequency of a total mixed ration on the performance of high-yielding dairy cows. J Dairy Sci 2006; 89: 43124320.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 24.

    Boosman R, Niewold TA, Mutsaers CW, et al. Serum amyloid A concentrations in cows given endotoxin as an acute-phase stimulant. Am J Vet Res 1989; 50: 16901694.

    • Search Google Scholar
    • Export Citation
  • 25.

    Dascanio JJ, Mechor G, Gröhn Y, et al. Effect of phenylbutazone and flunixin meglumine on acute toxic mastitis in dairy cows. Am J Vet Res 1995; 56: 12131218.

    • Search Google Scholar
    • Export Citation

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