• View in gallery

    Mean ± SEM plasma meloxicam concentration on days 0 and 3 for single-source healthy yearling crossbred beef steers that received meloxicam (1 mg/kg, PO) and then were (black bars; n = 20) or were not (white bars; 10) transported 1,300 km overnight (approx 15 hours) from the Mississippi State University Brown Loam Branch Experiment Station to the KSU LARC. Meloxicam was administered during the afternoon on day −1. The steers that were not transported after meloxicam administration had been moved from the experiment station to the KSU LARC 17 days earlier and allowed to acclimate to their new environment. Blood samples were collected from all steers immediately before treatment administration on day −1 and after the transported group arrived at the feedlot on days 0 and 3. *Within a day, value for the nontransported group differs significantly (P < 0.01) from that for the transported group.

  • View in gallery

    Mean ± SEM plasma cortisol concentration on study days −1, 0, and 3 for the steers of Figure 1 as well as control steers in both the transported (n = 20) and nontransported (10) groups that received a lactose placebo (1 bolus/steer, PO) on the afternoon of day −1. Plasma cortisol concentration was not significantly associated with transport status, treatment (meloxicam or placebo), or the interaction of transport status and treatment; therefore, the data from all 4 treatment groups were combined for reporting purposes. a,bMeans with different lowercase letters differ significantly (P < 0.01). See Figure 1 for remainder of key.

  • 1. Salak-Johnson JL, McGlone JJ. Making sense of apparently conflicting data: stress and immunity in swine and cattle. J Anim Sci 2007; 85(suppl 13):E81E88.

    • Search Google Scholar
    • Export Citation
  • 2. Sheridan JF, Dobbs C, Brown D, et al. Psychoneuroimmunology: stress effects on pathogenesis and immunity during infection. Clin Microbiol Rev 1994; 7:200212.

    • Search Google Scholar
    • Export Citation
  • 3. Fike K, Spire MF. Transportation of cattle. Vet Clin North Am Food Anim Pract 2006; 22:305320.

  • 4. Cernicchiaro N, White BJ, Renter DG, et al. Associations between the distance traveled from sale barns to commercial feedlots in the United States and overall performance, risk of respiratory disease, and cumulative mortality in feeder cattle during 1997 to 2009. J Anim Sci 2012; 90:19291939.

    • Search Google Scholar
    • Export Citation
  • 5. Sanderson MW, Dargatz DA, Wagner BA. Risk factors for initial respiratory disease in United States' feedlots based on producer-collected daily morbidity counts. Can Vet J 2008; 49:373378.

    • Search Google Scholar
    • Export Citation
  • 6. Van Engen NK, Platt R, Roth JA, et al. Impact of oral meloxicam and long-distance transport on cell-mediated and humoral immune responses in feedlot steers receiving modified live BVDV booster vaccination on arrival. Vet Immunol Immunopathol 2016; 175:4250.

    • Search Google Scholar
    • Export Citation
  • 7. Buckham Sporer KR, Burton JL, Earley B, et al. Transportation stress in young bulls alters expression of neutrophil genes important for the regulation of apoptosis, tissue remodeling, margination, and anti-bacterial function. Vet Immunol Immunopathol 2007; 118:1929.

    • Search Google Scholar
    • Export Citation
  • 8. Earley B, Murray M, Prendiville DJ, et al. The effect of transport by road and sea on physiology, immunity and behaviour of beef cattle. Res Vet Sci 2012; 92:531541.

    • Search Google Scholar
    • Export Citation
  • 9. Odore R, D'Angelo A, Badino P, et al. Road transportation affects blood hormone levels and lymphocyte glucocorticoid and beta-adrenergic receptor concentrations in calves. Vet J 2004; 168:297303.

    • Search Google Scholar
    • Export Citation
  • 10. Swanson JC, Morrow-Tesch J. Cattle transport: historical, research, and future perspectives. J Anim Sci 2001; 79(suppl E):E109E109.

    • Search Google Scholar
    • Export Citation
  • 11. Sweiger SH, Nichols MD. Control methods for bovine respiratory disease in stocker cattle. Vet Clin North Am Food Anim Pract 2010; 26:261271.

    • Search Google Scholar
    • Export Citation
  • 12. Stock ML, Coetzee JF. Clinical pharmacology of analgesic drugs in cattle. Vet Clin North Am Food Anim Pract 2015; 31:113138.

  • 13. Mosher RA, Coetzee JF, Cull CA, et al. Pharmacokinetics of oral meloxicam in ruminant and preruminant calves. J Vet Pharmacol Ther 2012; 35:373381.

    • Search Google Scholar
    • Export Citation
  • 14. Coetzee JF, KuKanich B, Mosher R, et al. Pharmacokinetics of intravenous and oral meloxicam in ruminant calves. Vet Ther 2009; 10:E1E8.

    • Search Google Scholar
    • Export Citation
  • 15. Perino LJ, Apley MD. Clinical trial design in feedlots. Vet Clin North Am Food Anim Pract 1998; 14:343365.

  • 16. Reuter RR, Carroll JA, Hulbert LE, et al. Technical note: development of a self-contained, indwelling rectal temperature probe for cattle research. J Anim Sci 2010; 88:32913295.

    • Search Google Scholar
    • Export Citation
  • 17. Burdick NC, Carroll JA, Dailey JW, et al. Development of a self-contained, indwelling vaginal temperature probe for use in cattle research. J Therm Biol 2012; 37:339343.

    • Search Google Scholar
    • Export Citation
  • 18. Van Engen NK, Stock ML, Engelken T, et al. Impact of oral meloxicam on circulating physiological biomarkers of stress and inflammation in beef steers after long-distance transportation. J Anim Sci 2014; 92:498510.

    • Search Google Scholar
    • Export Citation
  • 19. Bannikov GA, Hinds CA, Rajala-Schultz PJ, et al. Serum haptoglobin-matrix metalloproteinase 9 (Hp-MMP 9) complex as a biomarker of systemic inflammation in cattle. Vet Immunol Immunopathol 2011; 139:4149.

    • Search Google Scholar
    • Export Citation
  • 20. Hinds CA, Niehaus AJ, Premanandan C, et al. Characterization of the contributions of Hp-MMP 9 to the serum acute phase protein response of lipopolysaccharide challenged calves. BMC Vet Res 2014; 10:261.

    • Search Google Scholar
    • Export Citation
  • 21. Tris-buffered saline (TBS) 1X. Cold Spring Harb Protoc 2009. doi: 10.1101/pdb.rec11830.

  • 22. Nightingale CR, Sellers MD, Ballou MA. Elevated plasma haptoglobin concentrations following parturition are associated with elevated leukocyte responses and decreased subsequent reproductive efficiency in multiparous Holstein dairy cows. Vet Immunol Immunopathol 2015; 164:1623.

    • Search Google Scholar
    • Export Citation
  • 23. Ballou MA, Hanson DL, Cobb CJ, et al. Plane of nutrition influences the performance, innate leukocyte responses, and resistance to an oral Salmonella enterica serotype Typhimurium challenge in Jersey calves. J Dairy Sci 2015; 98:19721982.

    • Search Google Scholar
    • Export Citation
  • 24. DeDonder KD, Apley MD, Li M, et al. Pharmacokinetics and pharmacodynamics of gamithromycin in pulmonary epithelial lining fluid in naturally occurring bovine respiratory disease in multisource commingled feedlot cattle. J Vet Pharmacol Ther 2016; 39:157166.

    • Search Google Scholar
    • Export Citation
  • 25. Capik SF, White BJ, Lubbers BV, et al. Comparison of the diagnostic performance of bacterial culture of nasopharyngeal swab and bronchoalveolar lavage fluid samples obtained from calves with bovine respiratory disease. Am J Vet Res 2017; 78:350358.

    • Search Google Scholar
    • Export Citation
  • 26. Senthilkumaran C, Clark ME, Abdelaziz K, et al. Increased annexin A1 and A2 levels in bronchoalveolar lavage fluid are associated with resistance to respiratory disease in beef calves. Vet Res 2013; 44:24.

    • Search Google Scholar
    • Export Citation
  • 27. Dohoo I, Martin W, Stryhn H. Repeated measures data. In: Dohoo I, Martin W, Stryhn H, eds. Veterinary epidemiologic research. 2nd ed. Charlottetown, PEI, Canada: VER Inc, 2009;614.

    • Search Google Scholar
    • Export Citation
  • 28. Cole NA, Camp TH, Rowe LD Jr, et al. Effect of transport on feeder calves. Am J Vet Res 1988; 49:178183.

  • 29. Pettiford SG, Ferguson DM, Lea JM, et al. Effect of loading practices and 6-hour road transport on the physiological responses of yearling cattle. Aust J Exp Agric 2008; 48:10281033.

    • Search Google Scholar
    • Export Citation
  • 30. Warriss PD, Brown SN, Knowles TG, et al. Effects on cattle of transport by road for up to 15 hours. Vet Rec 1995; 136:319323.

  • 31. Morris DD. Alterations in the erythron and alterations in the leukogram. In: Smith BP, ed. Large animal internal medicine. 4th ed. St Louis: Mosby, 2009;401, 406.

    • Search Google Scholar
    • Export Citation
  • 32. DeVane CL. Substance P: a new era, a new role. Pharmacotherapy 2001; 21:10611069.

  • 33. Buckham Sporer KR, Weber PS, Burton JL, et al. Transportation of young beef bulls alters circulating physiological parameters that may be effective biomarkers of stress. J Anim Sci 2008; 86:13251334.

    • Search Google Scholar
    • Export Citation
  • 34. Crookshank HR, Elissalde MH, White RG, et al. Effect of transportation and handling of calves upon blood serum composition. J Anim Sci 1979; 48:430435.

    • Search Google Scholar
    • Export Citation
  • 35. Earley B, O'Riordan EG. Effects of transporting bulls at different space allowances on physiological, haematological and immunological responses to a 12-h journey by road. Ir J Agric Food Res 2006; 45:3950.

    • Search Google Scholar
    • Export Citation
  • 36. Burdick NC, Carroll JA, Hulbert LE, et al. Relationships between temperament and transportation with rectal temperature and serum concentrations of cortisol and epinephrine in bulls. Livest Sci 2010; 129:166172.

    • Search Google Scholar
    • Export Citation
  • 37. Burdick NC, Carroll JA, Randel RD, et al. Influence of temperament and transportation on physiological and endocrinological parameters in bulls. Livest Sci 2011; 139:213221.

    • Search Google Scholar
    • Export Citation
  • 38. Mitchell G, Hattingh J, Ganhao M. Stress in cattle assessed after handling, after transport and after slaughter. Vet Rec 1988; 123:201205.

    • Search Google Scholar
    • Export Citation
  • 39. Thun R, Eggenberger E, Zerobin K, et al. Twenty-four-hour secretory pattern of cortisol in the bull: evidence of episodic secretion and circadian rhythm. Endocrinology 1981; 109:22082212.

    • Search Google Scholar
    • Export Citation
  • 40. Earley B, Fisher AD, O'Riordan EG. Effects of pre-transport fasting on the physiological responses of young cattle to 8-hour road transport. Ir J Agric Food Res 2006; 45:5160.

    • Search Google Scholar
    • Export Citation
  • 41. Jacobsen S, Andersen PH, Toelboell T, et al. Dose dependency and individual variability of the lipopolysaccharide-induced bovine acute phase protein response. J Dairy Sci 2004; 87:33303339.

    • Search Google Scholar
    • Export Citation
  • 42. Lomborg SR, Nielsen LR, Heegaard PM, et al. Acute phase proteins in cattle after exposure to complex stress. Vet Res Commun 2008; 32:575582.

    • Search Google Scholar
    • Export Citation
  • 43. Coetzee JF, Edwards LN, Mosher RA, et al. Effect of oral meloxicam on health and performance of beef steers relative to bulls castrated on arrival at the feedlot. J Anim Sci 2012; 90:10261039.

    • Search Google Scholar
    • Export Citation

Advertisement

Effect of oral administration of meloxicam prior to transport on inflammatory mediators and leukoctye function of cattle at feedlot arrival

View More View Less
  • 1 Department of Diagnostic Medicine/Pathobiology, Kansas State University, Manhattan, KS 66506.
  • | 2 Department of Clinical Sciences, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506.
  • | 3 Department of Clinical Sciences, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506.
  • | 4 Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA 50010.
  • | 5 Department of Diagnostic Medicine/Pathobiology, Kansas State University, Manhattan, KS 66506.
  • | 6 Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA 50010.
  • | 7 Department of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210.
  • | 8 Department of Animal and Food Sciences, College of Agricultural Sciences and Natural Resources, Texas Tech University, Lubbock, TX 79409.
  • | 9 Department of Animal Sciences and Industry, College of Agriculture, Kansas State University, Manhattan, KS 66506.
  • | 10 Mississippi Agricultural and Forestry Experiment Station, Department of Animal and Dairy Sciences, College of Agricultural and Life Sciences, Mississippi State University, Raymond, MS 39154.
  • | 11 Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, ON N1G 2W1, Canada.
  • | 12 Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, ON N1G 2W1, Canada.
  • | 13 Livestock Issues Research Unit, USDA Agricultural Research Service, 1604 E FM 1294, Lubbock, TX 79403.
  • | 14 Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA 50010.

Abstract

OBJECTIVE To investigate the effects of meloxicam administration before long-distance transport on inflammatory mediators and leukocyte function of cattle at feedlot arrival.

ANIMALS 60 healthy yearling beef steers.

PROCEDURES Single-source steers were assigned to a transported (n = 40) or nontransported (20) group. Then, half of the steers within each group were assigned to receive meloxicam (1 mg/kg, PO) or a lactose placebo (1 bolus/steer, PO). All steers were transported approximately 1,300 km overnight to a feedlot; however, the nontransported group was moved before treatment (meloxicam or placebo) administration and allowed a 17-day acclimation period, whereas the transported group was moved immediately after treatment administration on day −1. Blood samples for measurement of inflammatory mediators and leukocyte function were collected from all steers on days −1, 0, and 3.

RESULTS For steers that received meloxicam, mean plasma meloxicam concentration for the transported group was significantly greater than that for the nontransported group on day 0. For steers that received the placebo, mean haptoglobin-matrix metalloproteinase-9 complex for the transported group was significantly greater than that for the nontransported group on day 0. Mean haptoglobin concentration, neutrophil L-selectin intensity, and polymorphonuclear leukocyte count for the transported group were significantly greater than those for the nontransported group. Mean substance P concentration for nontransported steers that received meloxicam was significantly lower than that for the other 3 treatment groups.

CONCLUSIONS AND CLINICAL RELEVANCE Results indicated meloxicam administration to healthy steers immediately before long-distance transport did not significantly mitigate the effects of transport-induced stress on leukocyte function or inflammatory markers.

Abstract

OBJECTIVE To investigate the effects of meloxicam administration before long-distance transport on inflammatory mediators and leukocyte function of cattle at feedlot arrival.

ANIMALS 60 healthy yearling beef steers.

PROCEDURES Single-source steers were assigned to a transported (n = 40) or nontransported (20) group. Then, half of the steers within each group were assigned to receive meloxicam (1 mg/kg, PO) or a lactose placebo (1 bolus/steer, PO). All steers were transported approximately 1,300 km overnight to a feedlot; however, the nontransported group was moved before treatment (meloxicam or placebo) administration and allowed a 17-day acclimation period, whereas the transported group was moved immediately after treatment administration on day −1. Blood samples for measurement of inflammatory mediators and leukocyte function were collected from all steers on days −1, 0, and 3.

RESULTS For steers that received meloxicam, mean plasma meloxicam concentration for the transported group was significantly greater than that for the nontransported group on day 0. For steers that received the placebo, mean haptoglobin-matrix metalloproteinase-9 complex for the transported group was significantly greater than that for the nontransported group on day 0. Mean haptoglobin concentration, neutrophil L-selectin intensity, and polymorphonuclear leukocyte count for the transported group were significantly greater than those for the nontransported group. Mean substance P concentration for nontransported steers that received meloxicam was significantly lower than that for the other 3 treatment groups.

CONCLUSIONS AND CLINICAL RELEVANCE Results indicated meloxicam administration to healthy steers immediately before long-distance transport did not significantly mitigate the effects of transport-induced stress on leukocyte function or inflammatory markers.

Stress is a complex physiologic response to either a single stimulus or multiple stimuli that, depending on severity and duration, have both positive and negative effects on various physiologic variables in cattle.1,2 Although stress can have negative effects on the immune system, the mere presence of stress, whether physiologic or psychological, does not always lead to clinical disease.2

Within the beef production system, cattle are exposed to various stressors throughout their lives, including weaning, handling for basic processing or other procedures, commingling at auction markets or other facilities, and transport between locations in the production system.3 Cattle are frequently transported multiple times over various distances within their lifetimes.3 Long-distance transport is considered a significant source of stress for cattle and is associated with an increase in the risk of BRD morbidity.4,5 Results of multiple studies6–8 indicate that long-distance transport induces changes in various measures of inflammation and immunity along with appreciable differences in weight, hydration status, and other alterations in normal physiologic status. Although most studies indicate that long-distance transport has an overall negative effect on cattle, results for some variables are inconsistent, and considerable individual variability within certain variables has been reported,9 most likely because transport is stressful on multiple levels for cattle and involves more than the distance traveled and time spent in the truck. Handling and restraint prior to or immediately following transport, weather conditions during transit, temperament, and other variables may all affect the overall stress experienced by an individual animal.10

Bovine respiratory disease remains a substantial problem for cattle despite many advances in treatment and prevention.11 Administration of antimicrobials to cattle that are at high risk for or affected by BRD is a common practice. Adjunct treatments to decrease morbidity and mortality rates associated with BRD are being investigated in an effort to decrease antimicrobial use in cattle. Meloxicam, an NSAID that preferentially inhibits cyclooxygenase-2 and thereby the inflammatory response, is one such potential adjunct treatment.12,13 Although meloxicam has not been approved by the FDA for use in cattle in the United States, it can be legally administered in an extralabel manner under AMDUCA. In 1 study,14 meloxicam (1 mg/kg, PO) administered to ruminating calves had high bioavailability and a mean plasma half-life of approximately 28 hours, which suggests that meloxicaminduced analgesia may last for several days following oral administration of a single dose of the drug. Given that stress and transport can affect certain measures of inflammation and leukocyte responses in cattle, we hypothesized that administration of a single dose of meloxicam (1 mg/kg, PO) to cattle prior to shipping will mitigate some of the negative effects of long-distance transport. Therefore, the objective of the study reported here was to investigate the effects of meloxicam administration prior to shipping on inflammatory mediators and leukocyte function of cattle during and after long-distance transport.

Materials and Methods

Animals

All study procedures were reviewed and approved by the KSU (protocol No. 3335) and Iowa State University (protocol No. 5-12-7355-B) Institutional Animal Care and Use Committees. Sixty crossbreed beef steers (mean ± SD weight, 309.5 ± 5.3 kg) from the Mississippi State University Brown Loam Branch Experiment Station were used for the study. The steers were born on the research station during the spring of 2013 and ranged in age from 15.2 to 17.8 months (mean ± SD age, 16.7 ± 0.08 months) at the start of the study. The steers were castrated within approximately 24 hours after birth and had received an 8-way clostridial vaccine,a 5-way modified-live virus respiratory vaccine,b Mannheimia haemolytica toxoid vaccine,c and pour-on anthelminticd 28 days before weaning and again at weaning. The steers were weaned in accordance with protocols established by Mississippi State University personnel approximately 8 months prior to study initiation. All steers were visually confirmed to be healthy by trained observers (NVE and SFC) prior to study initiation.

Study design

Prior to study initiation, the 60 steers selected for the study were randomly assigned by means of a random number generator to a transported (n = 40) or nontransported control (20) group. Then, within each group, half of the steers were randomly assigned by means of a random number generator to receive meloxicame (1 mg/kg, PO) or a lactose placebo (1 bolus/steer, PO). Thus, the study had an unbalanced 2 × 2 factorial design, with transport status (transported or nontransported) and treatment (meloxicam or placebo) as the 2 factors.

To ensure personnel administering the boluses were unaware of (blinded to) the treatment assigned to each steer, the appropriate amount of meloxicam along with sufficient lactose powder to hide the tablets was placed in bolus capsules identical to the capsules that contained only lactose powder. The bolus prepared for each steer was labeled with the steer's identification number to ensure it was administered to the correct steer.

Study timeline

Treatment administration was designated as day −1. Two days prior to scheduled transport, steers were removed from grass pasture, gathered in a pen near the working facility at the experiment station, and provided ad libitum access to grass hay and water. On day −18, steers in the nontransported group were transported approximately 1,300 km overnight (15 hours) from the Brown Loam Branch Experiment Station to the KSU LARC, where they were allowed to acclimate to the facility and recover from transport from day −17 to 0. At approximately the same time on day −1, the transported and nontransported steers received the assigned treatments at their respective locations. The steers in the transported group were then transported overnight to the KSU LARC.

Once all the study cattle were at the KSU LARC, steers within each of the 4 treatment groups were randomly assigned by means of a random number generator to 1 of 4 open-air dirt pens (15 steers/pen) such that the distribution of steers from each group was fairly equal among the 4 pens. Steers were provided grass hay and water ad libitum, were fed a grain ration that was appropriate for their age and weight twice daily, and had access to a 3-sided shelter for the duration of the study.

Monitoring

For 28 days after arrival at the KSU LARC, all steers were observed by the same veterinarian (SFC) for signs of BRD or other health issues twice daily and assigned a CIS by use of a previously described modified 4-point scale,15 where 1 = clinically normal; 2 = moderately depressed with or without a cough or other signs of BRD; 3 = severely depressed with or without labored breathing, a cough, or other signs of BRD; and 4 = recumbent and moribund. Following the morning observation, steers that were assigned a CIS ≥ 2 the evening before or that morning were removed from their assigned pens for a physical examination. Bovine respiratory disease was defined as a CIS ≥ 2 and rectal temperature ≥ 40°C. Steers with BRD or other health issues were treated by the attending veterinarian in accordance with current standard beef industry protocols. Steers that were treated with antimicrobials remained in the study, but steers that required treatment with any type of NSAID were excluded from the study from that point forward and removed from the study population.

On day −2, the steers in the transported group were instrumented with a rectal temperature probef as described16 with a minor modification. Specifically, a smaller temperature sensor (length, 25.4 mm; diameter, 8.3 mm; and weight, 3.3 g) was used; therefore, the probe container that attached to the end of the plastic cross-linked polyethylene tubing was also smaller.17 The probes were monitored twice daily for proper placement and signs of swelling or discomfort and removed when necessary. Each probe recorded the rectal temperature of the steer at 5-minute intervals before, during, and after transport and was removed on day 3.

Each steer in the transported group also had a digital thermographic image of the medial canthus of the left eye obtained on days −1 (immediately prior to transport and treatment), 0, and 3. Each image was translated to temperature readings by internally calibrated software within the camera.g

Blood sample collection

Each steer in the transported and nontransported groups was restrained in a manual squeeze chute, and blood (40 mL) was obtained by jugular venipuncture on days −1 (immediately prior to treatment), 0, and 3 for measurement of various markers of inflammation and immune function. Blood was collected into 5 types of blood collection tubes. Blood collected in tubes containing lithium heparin was used to harvest plasma for measurement of cortisol and meloxicam concentrations, whereas blood collected in tubes containing sodium heparin was used to harvest plasma for measurement of haptoglobin concentration, neutrophil L-selectin intensity, and neutrophil phagocytic and oxidative burst activity. Blood collected in serum-separator tubes was used to harvest serum for determination of Hp-MMP-9 concentrations, and blood collected in EDTA-containing tubes was used for hematologic analysis. Blood for measurement of substance P concentration was first collected into a sterile tube without any additives, then 6 mL of blood was immediately removed from the tube with a needle and syringe and carefully added to a previously prepared blood collection tube containing 300 μL of EDTA and 0.9 mg of benzamidine. Blood samples in sodium heparin tubes were stored at room temperature (approx 22 °C) and processed within 24 hours after collection. Blood samples in EDTA-containing tubes were stored on ice for transport to the laboratory and processed within several hours after collection.

On day −1, the 40 steers in the transported group at the Brown Loam Branch Experiment Station were separated into 2 groups that were approximately equal in size, and it took approximately 60 minutes to collect samples from all steers in each group. The 20 steers in the nontransported group at the KSU LARC were likewise allocated into 2 equally sized groups for sample collection on day −1, and it took between 30 and 40 minutes to collect samples from all steers in each group. On days 0 and 3, blood samples from all 60 steers were collected over approximately 6 hours. The steers were managed such that they were restrained in the handling facility (tub, alley, and squeeze chute) for < 30 minutes to minimize the effect of handling stress on the various blood variables measured.

Blood sample analysis

All laboratory personnel were blinded to the transport status and treatment assigned to each steer during sample analysis. Because of laboratory constraints, blood samples for measurement of hematologic variables and neutrophil activity could not be evaluated within 24 hours after collection on day −1; therefore, those variables were analyzed only on days 0 and 3.

Plasma meloxicam concentration—Plasma meloxicam concentrations were determined by use of high-performance liquid chromatographyh with mass spectrometry detectioni as described.18 For each steer, all samples were analyzed in the same batch, or run, to limit variability. The standard curve applied to each run was linear for meloxicam concentrations ranging from 1 to 20,000 ng/mL. For all runs, the correlation coefficient was > 0.97, and all measured standard curve samples were within 25% of the designated values, with most samples within < 15%. The meloxicam assay for bovine plasma had a mean ± SD accuracy of 93.3 ± 7.5% of the actual concentration, and the coefficient of variation was 3.8% as determined for 4 sets of replicates with meloxicam concentrations of 100 and 1,000 ng/mL.

Plasma cortisol concentration—Plasma cortisol concentrations were determined by use of a commercially available radioimmunoassayj as described.18 The optimal detection range for the assay was cortisol concentrations between 3 and 500 ng/mL. The interassay and intra-assay variabilities for the radioimmunoassay were calculated with a commercial programk and were 11.1% and 12.6%, respectively.

Plasma substance P concentration—Plasma substance P concentrations were determined by use of a double-antibody radioimmunoassay with primary antibodies against substance Pl and 125I-substance Pm as described.18 Protease inhibitors used in the assay included EDTA (13mM) and benzamidine (1mM). The detection range for substance P was 10 to 160 pg/mL, with a coefficient of variation of 9.59% for intra-assay variability and 16.28% for interassay variability.

Serum Hp-MMP-9 concentration—An ELISA was performed as described19,20 to determine serum Hp-MMP-9 concentrations. The capture antibody used was a monoclonal anti-bovine MMP-9 (clone 10.1 [native bovine neutrophil MMP-9 antigen]), and wells were blocked by the addition of 300 μL of TBS-blocking buffer.n All plates were prepared at the same time to minimize plate-to-plate variation.

The serum Hp-MMP-9 standards prepared for use in the ELISA were from a cow that was ill and had serum Hp-MMP-9 concentrations that ranged from 9 to 913 ng/mL as quantified in another study.19 Serum samples from that cow were stored in frozen aliquots. The serum aliquots used for the ELISA were thawed on ice and sonicated for 1 minute 3 times, vortexed, and serially diluted so that standards had an Hp-MMP-9 concentration of 228, 114, 57, 28, 14, 7, and 3.5 ng/mL. Blank wells contained all reagents, along with serum from a healthy steer, diluted 1:10 in TBS solution (pH, 7.5) to which 0.05% Tween 20 (TBS + Tween 20 buffer) was added.21 Affinity chromatography and an ELISA indicated that the serum Hp-MMP-9 concentration for that healthy steer was below the limit of detection for Hp-MMP-9 (3.5 ng/mL; unpublished data).

Serum samples from the study steers were diluted 1:10 with TBS + Tween 20 buffer prior to ELISA analysis. Diluted standards and study serum samples were placed in duplicate wells (100 μL/well) of a 96-well plate (16 standards and 40 serum samples/plate). The plate was incubated on a plate shaker at room temperature for 2 hours, after which it was washed 4 times with TBS (50mM Tris and 150mM NaCl; pH, 7.5) containing 0.05% Tween 20. Then, 100 μL of prediluted rabbit anti-bovine haptoglobin-horseradish peroxidase conjugateo was added to each well to bind to haptoglobin that was bound to MMP-9 in the samples. The plate was incubated on a plate shaker at room temperature for 1 hour, after which it was washed 5 times with TBS + Tween 20 buffer. To each well, 100 μL of 3,3′,5,5′-tetramethylbenzidine substratep was added, and the plate was incubated at room temperature for 20 minutes to allow color development. Then, 100 μL of 0.1 N hydrochloric acid was added to each well to stop the enzymatic reaction. Standard and sample absorbance was determined at 450 nm by use of a microplate reader. All serum samples with an absorbance greater than that of the highest standard (n = 56 samples) were diluted to 1:50 with TBS + Tween 20 buffer and reassayed. For each serum sample, the Hp-MMP-9 concentration was determined by use of the intercept and slope calculated from linear regression of the known standard concentration versus absorbance value, which was then corrected for the sample dilution (10- or 50-fold).

Plasma haptoglobin concentration, neutrophil L-selectin intensity, neutrophil phagocytic and oxidative burst activity, and hematologic values—Plasma haptoglobin concentrations, neutrophil phagocytic and oxidative burst activity, neutrophil L-selectin intensity, and hematologic values were determined as described.22,23

BALF sample collection and annexin A1 analysis

Bronchoalveolar lavage fluid samples were obtained only from the steers in the transported group on days 0 and 3. Collection of BALF samples was performed as described24,25 with some modifications. Briefly, 200 mL of sterile saline (0.9% NaCl) solution was introduced into a bronchus and immediately aspirated in 2 separate aliquots of 120 mL and 80 mL. The aspirated fluid was filtered through sterile gauze into 50-mL conical tubes, chilled on ice, and then centrifuged for 10 minutes at 500 × g. The resulting supernatant was decanted from each tube and stored frozen at −80°C until analysis.

The annexin A1 concentration was measured in the BALF samples as described26 with some modifications. Briefly, the BALF samples were concentrated by centrifugal filtration with a 3-kDa molecular weight–limit deviceq and purifiedr for measurement of protein concentrations.s

Two samples of concentrated BALF that had intermediate annexin A1 concentrations were used as standards and loaded on each gel to facilitate comparison of test sample results between gels and blots. Samples containing 10 μg of protein were loaded on SDS polyacrylamide gelst and separated at 200 V for 1 hour. Proteins were visualized by densitometry,u and the total protein in each lane was quantified by densitometry.v Proteins were transferred to a membranew that was used for western blot analysis. The membrane was blocked with 5% bovine serum albumin for 1 hour and then incubated with rabbit anti-human annexin A1 antibodyx overnight (approx 18 hours) at 4°C, washed, incubated with peroxidase-conjugated goat anti-rabbit immunoglobuliny as a secondary antibody for 30 minutes, and washed for 1 hour. Proteins on the membrane were visualized by chemiluminescence.z Bands with a size (37 kDa) consistent with annexin A126 were quantified by densitometry.u Results were reported as the density of the band on the western blot membrane adjusted for the background density of the blot normalized to the corresponding adjusted total protein value obtained by densitometric analysis of the SDS polyacrylamide gels. Quantitative data were confirmed by visual inspection of the blots and gels. Laboratory personnel were blinded to the treatment assigned to each steer during analysis of BALF samples.

Statistical analysis

For each steer in the transported group, rectal temperature data were aggregated, and the mean was calculated for every 6-hour period beginning immediately after treatment administration and continuing through day 2 (2 days after arrival at the KSU LARC). Additionally, the digital thermographic images were evaluated to determine the maximum temperature around the medial canthus of the left eye on days −1, 0, and 3, and those data were used for statistical analyses.

Generalized linear mixed modelsaa were used to evaluate the effect of transport status (transported or nontransported) and treatment (meloxicam or placebo) on all outcomes of interest except Hp-MMP-9 concentration. Continuous outcomes of interest included rectal temperature, ocular temperature, plasma cortisol concentration, plasma meloxicam concentration, neutrophil L-selectin intensity, RBC count, MCHC, and hemoglobin concentration. Continuous outcomes of interest that required a natural logarithmic transformation to normalize the data distribution prior to analysis included plasma substance P concentration, serum haptoglobin concentration, oxidative burst intensity, phagocytosis intensity, WBC count, lymphocyte count, polymorphonuclear leukocyte count, and monocyte count. A Gaussian distribution, an identity link, and residual pseudo-likelihood estimation were specified for the models for continuous outcomes. Results for outcomes that required a natural logarithmic transformation to normalize the data distribution were back-transformed and reported as the means and corresponding 95% CIs. Generalized linear mixed models were also used to model categorical outcomes. Mean cell volume was modeled as a dichotomous outcome (normal [40 to 65 fL] or low [< 40 fL]) on the basis of the reference range established for that variable at the laboratory where the samples were analyzed. The model specified for mean cell volume included a binary distribution with a logit link and residual pseudo-likelihood estimation. Models for the percentage of phagocytosing and oxidative neutrophils and Hct were fit with a β distribution.

Each model included a random intercept for pen (4-level categorical variable) and a random covariance structure for unequally spaced repeated measures within steers when necessary. When the outcome variable of interest was measured only on days 0 and 3, a simple random intercept for steer was included in the model.27 Given the 2 × 2 factorial design of the study, all models included main (fixed) effects for transport status and treatment as well as study day (a 2- or 3-level categorical variable depending on the outcome being modeled) and the 2-way interaction between transport status and treatment when data were available for all 4 treatment groups. For outcomes measured for the transported group only (rectal temperature, ocular temperature, and BALF annexin A1 concentration), models included fixed effects for treatment, study day or time period, and the interaction between treatment and study day or time period. Additionally, the annexin A1 concentration data underwent a natural logarithmic transformation prior to analysis, and the model included a random intercept for blot number. Unless otherwise noted, model estimates were reported as model-adjusted mean ± SEM. An analysis of Studentized (continuous outcomes) or Pearson (dichotomized or categorical outcomes) residual plots was performed for all models, and residuals with an absolute value ≥ 3 were further evaluated as potential outliers or influential observations. Fixed effects with values of P ≤ 0.05 were considered significant. A more stringent value of P ≤ 0.01 was considered significant for effects with > 1 pairwise comparison to reduce the likelihood of type I errors.

Residuals for the Hp-MMP-9 concentration model were not normally distributed or homoscedastic despite several attempts to transform and normalize the data distribution. Therefore, a Kruskal-Wallis test was used to compare Hp-MMP-9 concentration among the 4 treatment groups within the 2 × 2 factorial design, followed by the Steel-Dwass methodbb for multiple comparisons (α = 0.01) between days.

Results

Steers

All steers in the transported and nontransported groups were assigned a CIS of 1 (clinically normal) during the first 3 days after arrival at the KSU LARC. Several steers were assigned a CIS of 2 at various times during the 28 days after treatment (meloxicam or placebo) administration, but none had a rectal temperature ≥ 40°C and therefore did not meet the criteria for diagnosis of BRD. One steer in the transported group that received meloxicam and 1 steer in the nontransported group that received the placebo had signs of lameness during days 0 to 3, but did not require NSAID treatment and were retained in the study and all analyses.

Rectal and ocular temperatures

The rectal temperature probes placed in 5 steers of the transported group (meloxicam, n = 3; placebo, 2) malfunctioned and did not provide any data for analysis. Several additional probes malfunctioned or had to be removed at various times between placement and day 3. One probe was removed on arrival at the KSU LARC because of swelling (placebo). One probe fell out on arrival at the KSU LARC (meloxicam), and another fell out on day 1 (meloxicam). One probe was found partially displaced from the rectum of a steer on day 2 (meloxicam), and 1 probe fell out during the last 6 hours of the data collection period (meloxicam). Analysis of the available data indicated that rectal temperature was not significantly associated with the interaction between time and treatment but was significantly (P < 0.01) associated with time (aggregated into 6-hour intervals). The mean rectal temperature did not differ significantly (P = 0.83) between transported steers that were treated with meloxicam and those treated with the placebo.

Ocular temperature was not significantly associated with treatment or the interaction between treatment and study day. However, the mean ± SEM ocular temperature on day 0 (39.4 ± 0.08°C) was significantly (P < 0.01) greater than that on days −1 (38.9 ± 0.08°C) and 3 (38.8 ± 0.11°C).

Plasma meloxicam, cortisol, and substance P concentrations

Meloxicam was not detected in the plasma of any of the steers prior to treatment administration on day −1 or in any of the steers treated with the placebo on days 0 and 3. The mean plasma meloxicam concentration for the transported steers treated with meloxicam was significantly greater than that for the nontransported steers treated with meloxicam on day 0, but did not differ significantly on day 3 (Figure 1).

Figure 1—
Figure 1—

Mean ± SEM plasma meloxicam concentration on days 0 and 3 for single-source healthy yearling crossbred beef steers that received meloxicam (1 mg/kg, PO) and then were (black bars; n = 20) or were not (white bars; 10) transported 1,300 km overnight (approx 15 hours) from the Mississippi State University Brown Loam Branch Experiment Station to the KSU LARC. Meloxicam was administered during the afternoon on day −1. The steers that were not transported after meloxicam administration had been moved from the experiment station to the KSU LARC 17 days earlier and allowed to acclimate to their new environment. Blood samples were collected from all steers immediately before treatment administration on day −1 and after the transported group arrived at the feedlot on days 0 and 3. *Within a day, value for the nontransported group differs significantly (P < 0.01) from that for the transported group.

Citation: American Journal of Veterinary Research 78, 12; 10.2460/ajvr.78.12.1426

Plasma cortisol concentration was not significantly associated with transport status, treatment, or the interaction between transport status and treatment. However, it was significantly associated with study day, with the mean plasma cortisol concentration significantly greater on day 3 than on day −1 (Figure 2).

Figure 2—
Figure 2—

Mean ± SEM plasma cortisol concentration on study days −1, 0, and 3 for the steers of Figure 1 as well as control steers in both the transported (n = 20) and nontransported (10) groups that received a lactose placebo (1 bolus/steer, PO) on the afternoon of day −1. Plasma cortisol concentration was not significantly associated with transport status, treatment (meloxicam or placebo), or the interaction of transport status and treatment; therefore, the data from all 4 treatment groups were combined for reporting purposes. a,bMeans with different lowercase letters differ significantly (P < 0.01). See Figure 1 for remainder of key.

Citation: American Journal of Veterinary Research 78, 12; 10.2460/ajvr.78.12.1426

Plasma substance P concentration was not significantly associated with study day but was significantly (P < 0.01) associated with the interaction between transport status and treatment. The mean ± SEM plasma substance P concentration did not differ significantly among the transported steers that were treated with meloxicam (77.8 ± 1.05 pg/mL), transported steers treated with the placebo (72.1 ± 1.05 pg/mL), and nontransported steers treated with the placebo (73.8 ± 1.08 pg/mL), but the mean ± SEM plasma substance P concentration for the nontransported steers treated with meloxicam (53.4 ± 1.08 pg/mL) was significantly less than that for each of the other 3 groups.

Serum Hp-MMP-9 concentration

On day −1, the mean serum Hp-MMP-9 concentration did not differ significantly (P = 0.57) among the 4 treatment groups. On day 0, the mean ± SEM serum Hp-MMP-9 concentration for the transported steers treated with the placebo (377.8 ± 140.65 ng/mL) was significantly (P < 0.01) greater than that for the nontransported steers treated with the placebo (0 ± 0 ng/mL). On day 3, although there was an overall significant (P < 0.01) difference in the mean serum Hp-MMP-9 concentrations among the 4 treatment groups, none of the pairwise comparisons were significant.

Hematologic analysis

Hematologic results for all 4 treatment groups were summarized (Table 1). None of the hematologic variables were significantly associated with the interaction between transport status and treatment. Blood hemoglobin concentration, mean cell volume, lymphocyte count, and monocyte count were not significantly associated with transport status, treatment, study day, or any 2-way interactions evaluated. The main effects for transport status and treatment were not significantly associated with RBC count, Hct, or MCHC. The overall mean ± SEM RBC count on day 0 (7.76 × 106 RBCs/μL ± 0.14 × 106 RBCs/μL) was significantly (P < 0.01) greater than that on day 3 (7.54 × 106 RBCs/μL ± 0.14 × 106 RBCs/μL). Similarly, the mean ± SEM Hct on day 0 (30.2 ± 0.46%) was significantly (P < 0.01) greater than that on day 3 (29.4 ± 0.46%), whereas the mean ± SEM MCHC on day 0 (35.6 ± 0.22 g/dL) was significantly (P < 0.01) less than that on day 3 (36.6 ± 0.22 g/dL). Neither transport status nor treatment was significantly associated with WBC count, but the overall mean WBC count on day 0 (11,485 WBCs/μL; 95% CI, 10,824 to 12,187 WBCs/μL) was significantly (P = 0.03) greater than that on day 3 (10,895 WBCs/μL; 95% CI, 10,271 to 11,558 WBCs/μL). Transport status and study day but not treatment were significantly associated with the polymorphonuclear leukocyte count. The mean polymorphonuclear leukocyte count for the transported group (3,800 cells/μL; 95% CI, 3,406 to 4,239 cells/μL) was significantly (P < 0.01) greater than that for the nontransported group (2,697 cells/μL; 95% CI, 2,310 to 3,149 cells/μL), and the overall mean polymorphonuclear leukocyte count on day 0 (3,744 cells/μL; 95% CI, 3,348 to 4,186 cells/μL) was significantly (P < 0.01) greater than that on day 3 (2,737 cells/μL; 95% CI, 2,450 to 3,058 cells/μL).

Table 1—

Descriptive hematologic results for 60 crossbred beef steers that were (n = 40) or were not (20) transported 1,300 km overnight (approx 15 hours) after receiving meloxicam (1 mg/kg, PO) or a lactose placebo (1 bolus/steer, PO) on day −1.

  Nontransported groupTransported group
  Meloxicam (n = 10)Placebo (n = 10)Meloxicam (n = 20)Placebo (n = 20)
VariableDayMean ± SDMedian (range)Mean ± SDMedian (range)Mean ± SDMedian (range)Mean ± SDMedian (range)
RBC07.75 ± 1.397.83 (5.72–10.83)7.77 ± 1.057.47 (6.31–9.79)7.73 ± 0.997.77 (6.00–9.57)7.86 ± 0.817.69 (6.36–9.46)
(× 106 cells/μL)37.52 ± 1.257.41 (6.08 −10.29)7.69 ± 0.807.57 (6.67–8.97)7.44 ± 1.117.37 (5.27–9.53)7.58 ± 0.857.38 (5.94–9.40)
Hemoglobin (g/dL)010.47 ± 1.3910.25 (8.20–13.70)10.94 ± 1.2510.95 (8.70–13.10)10.64 ± 1.1010.65 (8.30–12.50)10.85 ± 1.0310.95 (9.10–12.50)
 310.65 ± 1.3710.25 (8.90–13.90)11.19 ± 0.9211.20 (9.80–12.50)10.54 ± 1.3310.55 (7.20–13.10)10.70 ± 1.0010.80 (8.80–12.90)
Hct (%)029.42 ± 4.5229.60 (22.20–38.10)31.00 ± 3.6130.30 (27.10–37.60)29.76 ± 3.0130.25 (22.60–35.10)30.56 ± 3.0530.65 (25.70–35.40)
 328.96 ± 3.6928.15 (22.60–35.50)30.72 ± 2.2730.10 (27.30–34.30)28.74 ± 3.5728.60 (18.90–34.00)29.48 ± 3.2429.55 (24.00–38.80)
MCV (fL)038.21 ± 2.9037.80 (34.20–42.70)40.05 ± 2.0540.25 (36.70–42.90)38.73 ± 2.9637.75 (35.10–45.20)39.00 ± 2.9439.65 (34.30–44.30)
 338.82 ± 3.5137.80 (34.20–43.60)40.11 ± 2.0040.30 (37.40–43.00)38.83 ± 3.0238.25 (35.50–45.40)39.02 ± 3.1539.50 (34.20–45.20)
MCHC (g/dL)035.79 ± 2.9435.45 (33.10–43.00)35.32 ± 1.6235.25 (32.10–37.40)35.78 ± 1.2935.80 (33.50–37.70)35.54 ± 1.7235.40 (31.90–38.90)
 336.84 ± 1.6236.55 (34.30–39.40)36.43 ± 1.3336.70 (34.10–38.20)36.71 ± 1.5537.00 (34.10–39.20)36.34 ± 1.2936.30 (33.20–38.90)
WBC count010.91 ± 1.1811.18 (8.44–12.18)10.72 ± 2.1410.37 (8.83–16.46)12.19 ± 2.8512.00 (8.56–16.74)12.68 ± 2.3612.36 (8.92–18.48)
(× 103 cells/μL)311.28 ± 2.0311.06 (8.00–14.47)10.40 ± 2.0410.00 (8.73–15.90)11.56 ± 3.2712.40 (7.09–20.90)11.74 ± 2.9411.02 (7.74–17.86)
Polymorphonuclear03.34 ± 0.723.34 (2.55–4.65)2.95 ± 0.912.90 (1.72–4.89)4.52 ± 1.944.34 (1.92–8.73)5.18 ± 1.965.20 (2.02–10.18)
leukocyte count (× 103 cells/μL)32.83 ± 0.863.24 (0.71–3.47)2.32 ± 0.922.49 (1.06–4.03)3.50 ± 1.633.21 (1.32–7.99)3.60 ± 1.933.01 (1.64–9.40)
Lymphocyte count05.60 ± 1.225.88 (3.27–7.14)5.87 ± 1.225.69 (4.35–8.51)5.76 ± 1.125.69 (4.24–8.35)5.85 ± 1.195.89 (3.71–8.18)
(× 103 cells/μL)36.13 ± 1.755.71 (4.04–8.91)5.94 ± 1.425.63 (4.62–9.38)5.91 ± 1.335.48 (4.00–8.60)6.09 ± 1.365.90 (3.71–8.69)
Monocyte count01.47 ± 0.251.47 (1.15–2.10)1.63 ± 0.381.53 (1.36–2.65)1.68 ± 0.651.47 (0.91–2.97)1.48 ± 0.431.56 (0.78–2.32)
(× 103 cells/μL)31.59 ± 0.271.60 (1.16–2.11)1.67 ± 0.261.59 (1.34–2.05)1.51 ± 0.641.36 (0.66–3.04)1.53 ± 0.511.35 (0.85–2.90)

Prior to study initiation, the 60 steers selected for the study were randomly assigned to a transported (n = 40) or nontransported control (20) group. Then, within each group, half of the steers were randomly assigned to receive meloxicam or a lactose placebo (day −1). Thus, the study had an unbalanced 2 × 2 factorial design with transport status (transported or nontransported) and treatment (meloxicam or placebo) as the 2 factors. Blood samples were collected from all steers for hematologic analysis after feedlot arrival on day 0 (1 day after treatment administration) and on day 3 (4 days after treatment administration).

Plasma haptoglobin concentration

Plasma haptoglobin concentration was not significantly (P = 0.46) associated with the interaction between transport status and treatment or the main effect of treatment but was significantly (P < 0.01) associated with transport status and study day. The mean plasma haptoglobin concentration for the steers in the transported group (59.4 μg/mL; 95% CI, 41.9 to 84.1 μg/mL) was greater than that for steers in the nontransported group (25.9 μg/mL; 95% CI, 15.7 to 42.6 μg/mL). The mean plasma haptoglobin concentration on day 0 (26.7 μg/mL; 95% CI, 18.2 to 39.1 μg/mL) was significantly (P = 0.01) less than that on day 3 (47.1 μg/mL; 95% CI, 31.2 to 71.1 μg/mL), but the mean plasma haptoglobin concentration on day −1 (47.9 μg/mL; 95% CI, 31.4 to 73.1 μg/mL) did not differ significantly from that on either day 0 or 3.

Neutrophil L-selectin intensity and neutrophil activity

Neutrophil L-selectin intensity was not significantly associated with treatment or the interaction between transport status and treatment but was significantly associated with transport status and study day. The mean ± SEM neutrophil L-selectin intensity for the transported group (58.6 ± 5.68 GMFI) was significantly (P < 0.01) greater than that for the nontransported group (48.0 ± 6.13 GMFI). Additionally, the mean ± SEM neutrophil L-selectin intensity on day 0 (47.2 ± 6.24 GMFI) was significantly (P < 0.01) less than that on day 3 (59.4 ± 5.70 GMFI).

Neutrophil activity was not significantly associated with the interaction between transport status and treatment or the main effects for transport status and treatment but was significantly associated with study day. Mean phagocytosis intensity on day 0 (87.6 GMFI; 95% CI, 77.5 to 99.0 GMFI) was significantly (P < 0.01) less than that on day 3 (98.9 GMFI; 95% CI, 87.4 to 111.8 GMFI), whereas the mean percentage of phagocytosing and oxidative burst neutrophils on day 0 (65.5 ± 2.23%) was significantly (P < 0.01) greater than that on day 3 (55.7 ± 1.59%). The intensity of neutrophil oxidative burst activity was not significantly associated with any of the independent variables assessed.

BALF annexin A1 concentration

The BALF annexin A1 concentration was not significantly associated with the main effects of transport status, treatment, or study day or the interaction between transport status and treatment.

Discussion

In the present study, the effect of long-distance transport on cattle was evaluated in the absence of other stressors commonly associated with transport such as weaning, commingling, and time spent at a livestock auction market. Although the steers of the present study were weaned 8 months prior to initiation of the study and obtained from a single source, they were still deprived of food and water during transport. Both the transported and nontransported steers were transported 1,300 km overnight from the Mississippi State University Brown Loam Branch Experiment Station to the KSU LARC; however, the steers in the nontransported group were allowed to acclimate to the KSU LARC for 17 days prior to treatment (meloxicam or lactose placebo) administration. Both groups of steers were handled at the same time points immediately before and after treatment administration. The adverse effects of food and water deprivation during transport of cattle have been described,28 and some researchers29,30 consider stress associated with handling to have a greater effect on cattle than the actual long-distance transport event itself. Nevertheless, none of the steers of this study developed BRD during the observation period despite being exposed to multiple stressors. Thus, the results of this study provided information regarding the effect of transportation on healthy cattle.

In cattle, the maximum plasma concentration of meloxicam is achieved approximately 12 hours after oral administration, and the mean half-life of the drug is approximately 28 hours.14 For the transported steers that received meloxicam in the present study, the mean plasma meloxicam concentration 1 day after drug administration (day 0) was similar to that reported for transported cattle of another study18 but significantly greater than that for the nontransported steers that received meloxicam. In the present study, the transported and nontransported steers received the same dose of meloxicam at approximately the same time. It is possible that the transported steers became dehydrated during transport, which led to hemoconcentration and an elevated plasma meloxicam concentration. However, on day 0, the Hct was within the reference range31 (as calculated from RBC and mean corpuscular volume reference values) for all study steers and the RBC count was within the reference range31 (5.0 × 106 RBCs/μL to 10 × 106 RBCs/μL) for all but 2 steers in the nontransported group that received meloxicam, which suggested that the steers in the transported group were not appreciably dehydrated. Alternatively, a transport-induced decrease in rumen fill may have altered the rate of passage and absorption of meloxicam in the gastrointestinal tract because of less drug binding to forages in the rumen.13

The only variable assessed in the present study that was significantly associated with treatment was plasma substance P concentration. Substance P is a neuroactive peptide that is involved in pain and stress responses.32 In the present study, the mean plasma substance P concentration for the nontransported steers that received meloxicam was significantly lower than that for the other 3 treatment groups. The reason that the mean plasma substance P concentration for the transported steers that received meloxicam was not similarly decreased and in fact did not differ from that of the control steers (transported and nontransported) that received the placebo is unclear. It is possible that the handling events on days −2 and −1 caused some level of pain or stress for the steers of all 4 treatment groups, which was mitigated by meloxicam administration in the nontransported steers but not the transported steers because of the additional stress and discomfort caused by long-distance transport and placement of the rectal temperature probes. The reason the mean plasma substance P concentrations did not differ between both transported groups and the nontransported control steers that received the placebo may have been because the steers of this study were older and had less total stress associated with transport (ie, weaning or commingling); therefore, the long-distance transport and associated handling and sampling events that were supposed to induce inflammation, discomfort, and stress were not sufficient to cause an increase in substance P concentration. However, that does not explain why the mean plasma substance P concentration for the nontransported steers that were treated with meloxicam was significantly lower than that for the other 3 groups. Although meloxicam administration did not affect plasma substance P concentration in the transported steers of this study, it may decrease the plasma substance P concentrations for younger, recently weaned, or extensively commingled cattle that are concurrently exposed to multiple stressors and warrants further research.

The effect of transport on plasma cortisol concentrations of cattle is unclear. Results of some studies indicate that plasma cortisol concentration increases following transport6,9,33 or transport in conjunction with weaning,34 whereas results of other studies indicate that transport is associated with a decrease28 or no change35,36 in plasma cortisol concentrations of cattle. Responses to stress differ; therefore, it is likely that the relationship between stress and cortisol concentration also differs depending on the magnitude, type, duration, or total amount of stress to which cattle are exposed.10 Separation of the effect of stress induced by transport from the effect of stress induced by unloading, handling, and acquisition of blood samples is challenging, and only a few studies34,37,38 have attempted to do so. Although attempts were made to minimize the stress associated with handling and sample acquisition for the steers of the present study, it is likely that the stress associated with those procedures contributed to the lack of a significant association between transport status and plasma cortisol concentration in this study. The mean plasma cortisol concentration for the study population on day −1 was significantly less than that on day 3. The time of day during which the steers were sampled might have contributed to that difference. Blood samples were collected late in the afternoon on day −1 in contrast to early in the morning on days 0 and 3. However, mean plasma cortisol concentrations were fairly high on all 3 days, which indicated that all steers were exposed to some form of stress regardless of treatment group and made it difficult to identify circadian rhythms in cortisol concentrations.39

In addition to the conflicting reports regarding the effect of stress on cortisol concentrations of cattle, there is also considerable controversy regarding the effect of glucocorticoids on the immune function of cattle. Some forms of stress potentially improve some aspects of the immune response,1 whereas acute or prolonged stress generally has a negative effect on immune function.35,40 The relationship between stress and immune system function is complex and complicated by factors such as genetics, portion of the immune system examined, and type and chronicity of the stressors.1,28 Although long-distance transport is generally considered a substantially stressful event for cattle, the leukograms for the transported steers of the present study were not characteristic of a classic stress response (eg, leukocytosis with lymphopenia and mild neutrophilia). In fact, the only part of the leukogram that was significantly associated with transport status was the polymorphonuclear leukocyte count, and although the mean polymorphonuclear leukocyte count for the transported steers was significantly greater than that for the nontransported steers, it was still within the reference range.31 Also, although the mean total WBC and polymorphonuclear leukocyte counts for all steers on day 0 were significantly greater than those on day 3, they were within the respective reference ranges31 on both days. Moreover, even though the mean RBC count and Hct for all steers increased significantly and the mean MCHC decreased significantly between days 0 and 3 of the present study, all those variables remained within the respective reference ranges31 except for the Hct for 1 steer and RBC counts of 2 steers. The significant differences observed between sampling times for those hematologic variables made sense physiologically; however, the lack of baseline values immediately prior to treatment administration on day −1 and the fact that all those variables remained within the respective reference ranges made it difficult to interpret the clinical relevance of the differences observed. As previously mentioned, it is possible that the long-distance transport event was not stressful enough to induce a classic stress leukogram in the steers of this study, and a different response might be observed in other types of cattle.

Results of the present study indicated that the mean plasma haptoglobin concentration of the transported yearling steers was significantly greater than that for the nontransported yearling steers, which was in contrast to results of another study35 in which the mean plasma haptoglobin concentration did not differ significantly between cattle that were and were not transported. Nonetheless, similar to the present study, the mean plasma haptoglobin concentration did not differ significantly between pretransport and posttransport blood samples in that study.35 In the present study, the mean plasma haptoglobin concentration on day 3 was significantly greater than that on day 0. This delayed small increase in plasma haptoglobin concentration may have been caused by the fact that haptoglobin is a late acute-phase protein41 and is unlikely to be detectably increased within 24 hours after a stress event. However, in another study,33 the mean plasma haptoglobin concentration for calves was significantly decreased at 4.5 and 9.75 hours after initiation of a 9-hour transport period, compared with that immediately before transport (baseline), but did not differ significantly from the baseline concentration thereafter. Additionally, the acute-phase protein response to stress42 and inflammatory stimuli such as lipopolysaccharide41 varies considerably among individual animals. It is possible that transport does not induce a consistent amount of inflammation in cattle or that varying factors between studies such as duration of transport, road conditions, and environmental conditions confound the effects of transport and other stressful events on plasma haptoglobin concentrations.

Transported steers, even those at a fairly low risk for BRD such as the steers of the present study, were expected to have greater cortisol and inflammatory marker concentrations or activities, decreased leukocyte function, and a stress leukogram, compared with results for nontransported steers. Although comparing results among studies that evaluated the effect of transport stress on cattle is complicated by variability among study populations, transport events, and timing of sample acquisition, the results observed in the present study were surprising and hinted at the complexity of what constitutes a stressful event in different groups of cattle. In this study, even though efforts were made to handle the steers of all 4 treatment groups similarly and minimize the amount of time each steer was handled, substantial time was required to collect samples on days −1, 0, and 3 and may have introduced unintended variability in the results, which limited the ability to detect significant differences. It is also possible that samples need to be collected more frequently to detect physiologic changes associated with long-distance transport in apparently healthy steers that are at low risk for disease.7,37

Results of the present study indicated that administration of a single dose (1 mg/kg, PO) of meloxicam to previously weaned healthy yearling steers from a single source immediately prior to long-distance transport did not mitigate the effects of transport-induced stress on inflammatory mediators and leukocyte function. However, meloxicam may have a beneficial effect in other populations of calves that are at high risk of disease (eg, recently weaned and commingled from multiple sources; high-risk cattle) or exposed to additional stressors or inflammatory events in conjunction with long-distance transport.43 Further research is necessary to evaluate the effect of meloxicam in high-risk cattle that undergo long-distance transport and investigate the complex interplay of different types and sources of stress on disease development in cattle.

Acknowledgments

Supported by the USDA National Institute of Food and Agriculture (Agriculture and Food Research Initiative Competitive Grant No. 2013-67015-21332). The funding source did not have any involvement in the study design, data analysis and interpretation, or writing and publication of the manuscript.

The authors declare that there were no conflicts of interest.

The authors thank Drs. Mathew Stock, Kaitlynn Abell, and Nicole Sanchez for technical assistance.

Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA. The USDA is an equal opportunity provider and employer.

ABBREVIATIONS

BALF

Bronchoalveolar lavage fluid

BRD

Bovine respiratory disease

CI

Confidence interval

CIS

Clinical illness score

GMFI

Geometric mean fluorescence intensity

Hp-MMP-9

Haptoglobin-matrix metalloproteinase-9 complex

KSU

Kansas State University

LARC

Large Animal Research Center

MMP-9

Metalloproteinase-9

MCHC

Mean corpuscular hemoglobin concentration

TBS

Tris-buffered saline

Footnotes

a.

Ultrabac 8, Zoetis, Florham Park, NJ.

b.

Pyramid 5, Boehringer Ingelheim, St Joseph, Mo.

c.

Presponse SQ, Boehringer Ingelheim, St Joseph, Mo.

d.

Eprinex, Merial Inc, Duluth, Ga.

e.

Meloxicam (NDC code 65862-098-05), Aurobindo Pharma USA Inc, Dayton, NJ.

f.

Starr-Oddi DST micro-T, MeterMall USA, Marysville, Ohio.

g.

Thermacam Research Pro 2.8 SR-1, FLIR Systems, Nashua, NH.

h.

Surveyor MS Pump and Autosampler, Thermo Scientific, San Jose, Calif.

i.

TSQ Quantum Discovery MAX, Thermo Scientific, San Jose, Calif.

j.

MP Biomedical LLC, Santa Ana, Calif.

k.

Excel, version 2013, Microsoft Inc, Redmond, Wash.

l.

1:20,000, product No. H-061-05, lot No. 1286-1, Phoenix Pharmaceuticals, Burlingame, Calif.

m.

20,000 counts/min, PerkinElmer Inc, Waltham, Mass.

n.

SuperBlock T20 (TBS), Thermo Scientific, Pierce, Rockford, Ill.

o.

RHPT-10A 1:5,000 dilution, Immunology Consultants Laboratory, Portland, Ore.

p.

50-76-11, Kirkegaard & Perry Laboratories, Gaithersburg, Md.

q.

Amicon Ultra-15, Millipore, Billerica, Mass.

r.

Ready Prep 2-D Cleanup Kit, Bio-Rad Laboratories Inc, Hercules, Calif.

s.

Nanodrop 2000C, Thermo Scientific, Rockford, Ill.

t.

TGX Stain-Free Fast Cast Acrylamide solutions, Bio-Rad Laboratories Inc, Hercules, Calif.

u.

ChemiDoc, Bio-Rad Laboratories Ltd, Mississauga, ON, Canada.

v.

Image Lab software, Bio-Rad Laboratories, Hercules, Calif.

w.

TransBlot Turbo PVDF Membrane, Bio-Rad Laboratories Inc, Mississauga, ON, Canada.

x.

1:375; H00000301-D01P, Novus Biologicals, Oakville, ON, Canada.

y.

1:3000; DakoCytomation, Glostrup, Denmark.

z.

Clarity ECL western blotting substrate, Bio-Rad Laboratories Inc, Mississauga, ON, Canada.

aa.

PROC GLIMMIX, SAS, version 9.4, SAS Institute Inc, Cary, NC.

bb.

JMP 12, SAS Institute Inc, Cary, NC.

References

  • 1. Salak-Johnson JL, McGlone JJ. Making sense of apparently conflicting data: stress and immunity in swine and cattle. J Anim Sci 2007; 85(suppl 13):E81E88.

    • Search Google Scholar
    • Export Citation
  • 2. Sheridan JF, Dobbs C, Brown D, et al. Psychoneuroimmunology: stress effects on pathogenesis and immunity during infection. Clin Microbiol Rev 1994; 7:200212.

    • Search Google Scholar
    • Export Citation
  • 3. Fike K, Spire MF. Transportation of cattle. Vet Clin North Am Food Anim Pract 2006; 22:305320.

  • 4. Cernicchiaro N, White BJ, Renter DG, et al. Associations between the distance traveled from sale barns to commercial feedlots in the United States and overall performance, risk of respiratory disease, and cumulative mortality in feeder cattle during 1997 to 2009. J Anim Sci 2012; 90:19291939.

    • Search Google Scholar
    • Export Citation
  • 5. Sanderson MW, Dargatz DA, Wagner BA. Risk factors for initial respiratory disease in United States' feedlots based on producer-collected daily morbidity counts. Can Vet J 2008; 49:373378.

    • Search Google Scholar
    • Export Citation
  • 6. Van Engen NK, Platt R, Roth JA, et al. Impact of oral meloxicam and long-distance transport on cell-mediated and humoral immune responses in feedlot steers receiving modified live BVDV booster vaccination on arrival. Vet Immunol Immunopathol 2016; 175:4250.

    • Search Google Scholar
    • Export Citation
  • 7. Buckham Sporer KR, Burton JL, Earley B, et al. Transportation stress in young bulls alters expression of neutrophil genes important for the regulation of apoptosis, tissue remodeling, margination, and anti-bacterial function. Vet Immunol Immunopathol 2007; 118:1929.

    • Search Google Scholar
    • Export Citation
  • 8. Earley B, Murray M, Prendiville DJ, et al. The effect of transport by road and sea on physiology, immunity and behaviour of beef cattle. Res Vet Sci 2012; 92:531541.

    • Search Google Scholar
    • Export Citation
  • 9. Odore R, D'Angelo A, Badino P, et al. Road transportation affects blood hormone levels and lymphocyte glucocorticoid and beta-adrenergic receptor concentrations in calves. Vet J 2004; 168:297303.

    • Search Google Scholar
    • Export Citation
  • 10. Swanson JC, Morrow-Tesch J. Cattle transport: historical, research, and future perspectives. J Anim Sci 2001; 79(suppl E):E109E109.

    • Search Google Scholar
    • Export Citation
  • 11. Sweiger SH, Nichols MD. Control methods for bovine respiratory disease in stocker cattle. Vet Clin North Am Food Anim Pract 2010; 26:261271.

    • Search Google Scholar
    • Export Citation
  • 12. Stock ML, Coetzee JF. Clinical pharmacology of analgesic drugs in cattle. Vet Clin North Am Food Anim Pract 2015; 31:113138.

  • 13. Mosher RA, Coetzee JF, Cull CA, et al. Pharmacokinetics of oral meloxicam in ruminant and preruminant calves. J Vet Pharmacol Ther 2012; 35:373381.

    • Search Google Scholar
    • Export Citation
  • 14. Coetzee JF, KuKanich B, Mosher R, et al. Pharmacokinetics of intravenous and oral meloxicam in ruminant calves. Vet Ther 2009; 10:E1E8.

    • Search Google Scholar
    • Export Citation
  • 15. Perino LJ, Apley MD. Clinical trial design in feedlots. Vet Clin North Am Food Anim Pract 1998; 14:343365.

  • 16. Reuter RR, Carroll JA, Hulbert LE, et al. Technical note: development of a self-contained, indwelling rectal temperature probe for cattle research. J Anim Sci 2010; 88:32913295.

    • Search Google Scholar
    • Export Citation
  • 17. Burdick NC, Carroll JA, Dailey JW, et al. Development of a self-contained, indwelling vaginal temperature probe for use in cattle research. J Therm Biol 2012; 37:339343.

    • Search Google Scholar
    • Export Citation
  • 18. Van Engen NK, Stock ML, Engelken T, et al. Impact of oral meloxicam on circulating physiological biomarkers of stress and inflammation in beef steers after long-distance transportation. J Anim Sci 2014; 92:498510.

    • Search Google Scholar
    • Export Citation
  • 19. Bannikov GA, Hinds CA, Rajala-Schultz PJ, et al. Serum haptoglobin-matrix metalloproteinase 9 (Hp-MMP 9) complex as a biomarker of systemic inflammation in cattle. Vet Immunol Immunopathol 2011; 139:4149.

    • Search Google Scholar
    • Export Citation
  • 20. Hinds CA, Niehaus AJ, Premanandan C, et al. Characterization of the contributions of Hp-MMP 9 to the serum acute phase protein response of lipopolysaccharide challenged calves. BMC Vet Res 2014; 10:261.

    • Search Google Scholar
    • Export Citation
  • 21. Tris-buffered saline (TBS) 1X. Cold Spring Harb Protoc 2009. doi: 10.1101/pdb.rec11830.

  • 22. Nightingale CR, Sellers MD, Ballou MA. Elevated plasma haptoglobin concentrations following parturition are associated with elevated leukocyte responses and decreased subsequent reproductive efficiency in multiparous Holstein dairy cows. Vet Immunol Immunopathol 2015; 164:1623.

    • Search Google Scholar
    • Export Citation
  • 23. Ballou MA, Hanson DL, Cobb CJ, et al. Plane of nutrition influences the performance, innate leukocyte responses, and resistance to an oral Salmonella enterica serotype Typhimurium challenge in Jersey calves. J Dairy Sci 2015; 98:19721982.

    • Search Google Scholar
    • Export Citation
  • 24. DeDonder KD, Apley MD, Li M, et al. Pharmacokinetics and pharmacodynamics of gamithromycin in pulmonary epithelial lining fluid in naturally occurring bovine respiratory disease in multisource commingled feedlot cattle. J Vet Pharmacol Ther 2016; 39:157166.

    • Search Google Scholar
    • Export Citation
  • 25. Capik SF, White BJ, Lubbers BV, et al. Comparison of the diagnostic performance of bacterial culture of nasopharyngeal swab and bronchoalveolar lavage fluid samples obtained from calves with bovine respiratory disease. Am J Vet Res 2017; 78:350358.

    • Search Google Scholar
    • Export Citation
  • 26. Senthilkumaran C, Clark ME, Abdelaziz K, et al. Increased annexin A1 and A2 levels in bronchoalveolar lavage fluid are associated with resistance to respiratory disease in beef calves. Vet Res 2013; 44:24.

    • Search Google Scholar
    • Export Citation
  • 27. Dohoo I, Martin W, Stryhn H. Repeated measures data. In: Dohoo I, Martin W, Stryhn H, eds. Veterinary epidemiologic research. 2nd ed. Charlottetown, PEI, Canada: VER Inc, 2009;614.

    • Search Google Scholar
    • Export Citation
  • 28. Cole NA, Camp TH, Rowe LD Jr, et al. Effect of transport on feeder calves. Am J Vet Res 1988; 49:178183.

  • 29. Pettiford SG, Ferguson DM, Lea JM, et al. Effect of loading practices and 6-hour road transport on the physiological responses of yearling cattle. Aust J Exp Agric 2008; 48:10281033.

    • Search Google Scholar
    • Export Citation
  • 30. Warriss PD, Brown SN, Knowles TG, et al. Effects on cattle of transport by road for up to 15 hours. Vet Rec 1995; 136:319323.

  • 31. Morris DD. Alterations in the erythron and alterations in the leukogram. In: Smith BP, ed. Large animal internal medicine. 4th ed. St Louis: Mosby, 2009;401, 406.

    • Search Google Scholar
    • Export Citation
  • 32. DeVane CL. Substance P: a new era, a new role. Pharmacotherapy 2001; 21:10611069.

  • 33. Buckham Sporer KR, Weber PS, Burton JL, et al. Transportation of young beef bulls alters circulating physiological parameters that may be effective biomarkers of stress. J Anim Sci 2008; 86:13251334.

    • Search Google Scholar
    • Export Citation
  • 34. Crookshank HR, Elissalde MH, White RG, et al. Effect of transportation and handling of calves upon blood serum composition. J Anim Sci 1979; 48:430435.

    • Search Google Scholar
    • Export Citation
  • 35. Earley B, O'Riordan EG. Effects of transporting bulls at different space allowances on physiological, haematological and immunological responses to a 12-h journey by road. Ir J Agric Food Res 2006; 45:3950.

    • Search Google Scholar
    • Export Citation
  • 36. Burdick NC, Carroll JA, Hulbert LE, et al. Relationships between temperament and transportation with rectal temperature and serum concentrations of cortisol and epinephrine in bulls. Livest Sci 2010; 129:166172.

    • Search Google Scholar
    • Export Citation
  • 37. Burdick NC, Carroll JA, Randel RD, et al. Influence of temperament and transportation on physiological and endocrinological parameters in bulls. Livest Sci 2011; 139:213221.

    • Search Google Scholar
    • Export Citation
  • 38. Mitchell G, Hattingh J, Ganhao M. Stress in cattle assessed after handling, after transport and after slaughter. Vet Rec 1988; 123:201205.

    • Search Google Scholar
    • Export Citation
  • 39. Thun R, Eggenberger E, Zerobin K, et al. Twenty-four-hour secretory pattern of cortisol in the bull: evidence of episodic secretion and circadian rhythm. Endocrinology 1981; 109:22082212.

    • Search Google Scholar
    • Export Citation
  • 40. Earley B, Fisher AD, O'Riordan EG. Effects of pre-transport fasting on the physiological responses of young cattle to 8-hour road transport. Ir J Agric Food Res 2006; 45:5160.

    • Search Google Scholar
    • Export Citation
  • 41. Jacobsen S, Andersen PH, Toelboell T, et al. Dose dependency and individual variability of the lipopolysaccharide-induced bovine acute phase protein response. J Dairy Sci 2004; 87:33303339.

    • Search Google Scholar
    • Export Citation
  • 42. Lomborg SR, Nielsen LR, Heegaard PM, et al. Acute phase proteins in cattle after exposure to complex stress. Vet Res Commun 2008; 32:575582.

    • Search Google Scholar
    • Export Citation
  • 43. Coetzee JF, Edwards LN, Mosher RA, et al. Effect of oral meloxicam on health and performance of beef steers relative to bulls castrated on arrival at the feedlot. J Anim Sci 2012; 90:10261039.

    • Search Google Scholar
    • Export Citation

Contributor Notes

Dr. Capik's present address is Texas A&M AgriLife Research, Texas A&M University System, Amarillo, TX 79106; and the Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, 77843.

Ms. Jacob's present address is Faculty of Medicine, University of Toronto, Toronto, ON M55 1A8, Canada.

Dr. Coetzee's present address is Department of Anatomy and Physiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506.

Address correspondence to Dr. Capik (sarah.capik@ag.tamu.edu).