Effects of transdermal flunixin meglumine on experimentally induced lameness in adult dairy cattle

Lameness is a common animal health condition with significant production and welfare implications. The transdermal formulation of flunixin meglumine is the only approved drug for pain control in cattle in the United States. Thirty adult dairy cows were enrolled in a study to determine the effect of transdermal flunixin on cattle with induced lameness. Cows were allocated to 1 of 3 treatment groups, with 10 cows per group: lameness and flunixin (L+F), lameness and placebo (L+P), or sham induction and placebo (S+P). An arthritis-synovitis was induced in the distal interphalangeal joint of the left hind lateral digit, using 20 mg of amphotericin B, 6 h before the application of treatment. Cows enrolled into the sham induction group had 4 mL of isotonic saline injected into the joint. Cows were dosed with transdermal flunixin at 3.33 mg/kg (1 mL/15 kg), or a placebo at 1 mL/15 kg, every 24 h for 3 d. The first treatment of flunixin or placebo was considered the start of the study, identified as time 0 h. Data were collected from all cows for 120 h following the initial treatment application. Outcome measures included plasma cortisol; substance P; visual lameness assessment; mechanical nociception threshold (MNT), presented as difference between left and right feet; infrared thermography (IRT), presented as difference between left and right feet; and gait analysis using a pressure mat. Cortisol concentrations were lower for the L+F group starting at 1.5 h after drug administration. Substance P levels showed no evidence for treatment differences among groups. Differences between the left hind MNT and right hind MNT were detected, with S+P having the lowest difference at ?0.04 kilograms-force (kgf; 95% CI: ?1.86 to 1.78 kgf), and L+P having the highest at ?2.96 kgf (95% CI: 1.55 to 4.36 kgf). The L+F group was intermediate at ?2.08 kgf (95% CI: 0.89 to 3.27 kgf). Similarly, when the difference between the maximum temperatures of the coronary band were examined via IRT, the L+P group had the highest difference at 1.64°C (95% CI: 1.02 to 2.26°C), with the L+F and S+P groups measuring 0.57°C (95% CI: 0.06 to 1.08°C) and 0.53°C (95% CI: ?0.2 to 1.25°C) respectively. We found no evidence for differences among treatment groups when analyzing force, contact pressure, step impulse, or stride length. Based on differences in MNT, IRT, and cortisol, transdermal flunixin is an effective analgesic agent for induced lameness. Multiple doses of transdermal flunixin may be required to be clinically effective, based on MNT and IRT data. Further investigation of transdermal flunixin and its analgesic effects is warranted in naturally occurring lameness.     

Short communication: Behavioral evaluation of the analgesic effect of flunixin meglumine in lame dairy cows

The objective of this study was to evaluate the effect of flunixin meglumine treatment on lameness pain in dairy cows. Twenty-four lactating Holstein cows were enrolled in the study based on visual observation of abnormal locomotion. The primary measurement endpoint was weight-shifting between the rear limbs. Weight-shifting was calculated as the standard deviation of the weight borne on the rear limbs over a 15 min period; this value correlates directly with lameness pain in dairy cows. After collecting baseline weight-bearing data, we randomly assigned cows to 1 of 2 treatment groups: 2.2 mg/kg body weight flunixin meglumine (2 mL/45 kg) or an equivalent volume of isotonic sterile saline solution. Weight-bearing data were collected from each cow at 2, 6, 12, and 24 h after a single intravenous drug treatment. Mean locomotion scores over the 2 d before treatment were 2.38/5 in the flunixin-treated group and 2.43/5 in the saline-treated control group; these values were not significantly different. Weight-shifting values were also not significantly different on either pretreatment day. Cows treated with flunixin meglumine showed significantly less weight-shifting between the rear limbs at 6, 12, and 24 h after treatment compared with saline-treated controls, providing evidence that flunixin meglumine alleviates lameness-associated pain.     

Effect of bupivacaine liposome suspension administered as a cornual nerve block on indicators of pain and distress during and after cautery dehorning in dairy calves

Dehorning is performed on a high percentage of dairies worldwide. Concern about the negative effect of dehorning on animal welfare has contributed to the development of new guidelines that require the use of pain management at the time of disbudding in the United States. However, livestock producers are limited in how to address this requirement due to a lack of (1) approved analgesic drugs, (2) analgesic options that control pain for an extended duration, and (3) analgesic formulations that are practical for producers to administer. The objective of this study was to evaluate the effectiveness of bupivacaine liposome suspension, a novel, long-acting, local anesthetic formulation administered as a nerve block at dehorning, compared with current industry standard analgesic approaches using lidocaine nerve blocks alone or in combination with the nonsteroidal anti-inflammatory drug meloxicam. Fifty male Holstein calves, 10 to 14 wk of age, were enrolled and randomly assigned to 1 of 5 treatment groups before cautery dehorning as follows: (1) bupivacaine liposome suspension block, oral placebo (BUP); (2) lidocaine block, oral placebo (LID); (3) lidocaine block, oral meloxicam (1 mg/kg of body weight; LID + MEL); (4) saline block, oral placebo (CON); and (5) saline block, oral placebo, sham dehorn (SHAM). Biomarkers were collected from 0 to 120 h postdehorning and included infrared thermography, mechanical nociceptive threshold (MNT), pressure mat gait analysis, chute defense and behavior scoring, and blood sampling for serum cortisol and prostaglandin E₂ metabolites. Responses were analyzed using repeated measures with calf nested in treatment designated as a random effect, and treatment, time, and their interaction designated as fixed effects. At 2 h postdehorning, the BUP group had a higher MNT compared with the CON group. Furthermore, at 24 h postdehorning, the BUP group had a higher MNT compared with the LID group. Gait distance differed significantly between treatment groups; the CON, LID, and LID + MEL groups had an increased gait distance relative to the SHAM group. The CON group exhibited a higher chute defense behavior score during the dehorning procedure compared with all other treatments. Furthermore, the CON group exhibited more ear flicks than the BUP and LID + MEL groups postdehorning. At 4 h and 24 h after dehorning, the LID + MEL group had a lower average prostaglandin E₂ metabolites concentration compared with all other treatment groups. These data showed that administration of bupivacaine liposome suspension as a cornual nerve block at the time of dehorning was as effective at controlling pain as a multimodal approach of lidocaine and meloxicam.     

The effect of transdermal flunixin meglumine on blood cortisol levels in dairy calves after cautery disbudding with local anesthesia

The objective of this study was to evaluate the effect of the nonsteroidal anti-inflammatory drug transdermal flunixin meglumine (Finadyne Transdermal) on plasma cortisol, average daily weight gain, and standing and lying behavior of calves, when given at the time of disbudding combined with local anesthesia. A sedative was not used to minimize pharmacological interactions. Seventy-one female Holstein Friesian calves aged 13 ± 2 d, with an average weight of 48.9 ± 4.26 kg were enrolled in the study. All calves were randomly assigned to one of 3 treatment groups: (1) control group (CON, n = 27), (2) 1-flunixin group (1-FLU, n = 26) with a single administration of transdermal flunixin meglumine at disbudding, and (3) 2-flunixin group (2-FLU, n = 24) with 2 administrations of transdermal flunixin meglumine, the first treatment at disbudding and the second 6 h after disbudding. Although the CON group received a placebo, 1-FLU and 2-FLU received flunixin meglumine transdermally. To account for plasma cortisol changes due to manipulation and handling of the calves, a sham disbudding procedure was performed one week before disbudding took place. Sham disbudding was conducted by using a cold cautery dehorner applied to each horn bud for 10 s. Disbudding was performed in a similar way by using a hot cautery dehorner. Plasma samples were collected to measure the stress biomarker cortisol at 7 different time points. Body weights were measured 4 times in 2 wk. Standing and lying behavior was assessed via 3-dimensional accelerometer. During sham disbudding and disbudding mean plasma cortisol concentrations were 6.09 ± 2.5 ng/mL and 5.16 ± 2.8 ng/mL, respectively. Treatment tended to have an effect on plasma cortisol concentrations during sham disbudding and had an effect on plasma cortisol concentrations during disbudding. Plasma cortisol concentrations were affected by treatment 2 h after disbudding in comparison to CON group. Furthermore, there was a significant effect on plasma cortisol concentrations 6 h after disbudding in contrast to CON. A return to baseline plasma cortisol levels (initial concentrations) was not achieved in CON during disbudding. There was no statistical difference between average daily weight gain and the treatment procedure. Total lying time was not affected by treatment after disbudding. In conclusion, transdermal flunixin meglumine given at the time of disbudding combined with local anesthesia decreased concentrations of the stress biomarker cortisol, but a second dose 6 h after disbudding had no further effect on plasma cortisol levels.     

Short communication: Effects of meloxicam administration on protein metabolism and growth performance in transported Jersey calves

Our objective was to investigate the effects of administering the nonsteroidal anti-inflammatory drug meloxicam (MEL) before transport on various indicators of protein metabolism and growth performance over the first 96 h after transport in Jersey calves. Calves (age ± SD; 2 ± 1 d) sourced from a commercial farm were randomly administered, at 1 mg/kg of body weight, either meloxicam (MEL; n = 11) or a whey protein placebo (CON; n = 10) orally before transport to a calf facility (669 km; 8.5-h road trip). Calves were weighed and rectal temperature was recorded before departure (0 h), on arrival (8.5 h), and 96 h after arrival. Blood was collected at the same time as calves were weighed, and samples were analyzed for total protein (0-h sample), cortisol (0- and 8.5-h samples), haptoglobin (0- and 96-h samples), and amino acids, 3-methylhistidine, and urea-N (96 h). Milk replacer (MR) intake was recorded on arrival and over the next 4 d. Serum total protein concentration did not differ for CON and MEL calves. Plasma cortisol concentration was similar across treatments at 0 h; however, it was lower for CON than for MEL calves at 8.5 h. Although serum haptoglobin concentration tended to be greater for CON than MEL calves 96 h after transport, 3-methylhistidine and plasma urea-N concentrations did not differ across treatments. Plasma Asp, Asn, Glu, Lys, Met, Ser, and Trp were greater and plasma Arg, Gly, Pro, and Thr concentrations tended to be greater at 96 h after arrival for MEL compared with CON calves. Intake of MR and average daily gain were higher in MEL than in CON calves. In summary, although it had no effect on 3-methylhistidine or urea-N concentrations, administration of MEL before transport tended to reduce haptoglobin concentration, altered the amino acid profile, and was beneficial in preventing a decrease in MR intake and average daily gain in Jersey calves.     

The effects of periparturient administration of flunixin meglumine on the health and production of dairy cattle

Research on the assessment and management of pain in cows following difficult or assisted calving is still limited, especially on the effects of analgesics intended to mitigate this pain. The purpose of this study was to assess the effects of flunixin meglumine on the health and production of Holstein cows after calving. In total, 34 flunixin-treated and 38 placebo-treated animals were enrolled in a precalving treatment trial. A total of 633 animals given flunixin and 632 animals administered a placebo were enrolled in a postcalving treatment trial. In both cases, animals were randomly assigned to treatment, and researchers were blind to treatment condition until after analysis. A total of 1,265 animal records were analyzed for milk production for the first 14 d in milk and health outcomes for the first 30 d in milk. Animals treated with flunixin meglumine before calving had a significantly increased risk of stillbirth. Animals treated immediately after calving had increased odds of having a retained placenta and, in turn, increased risk of a high temperature, decreased milk production, and an increased risk of developing metritis. The administration of flunixin meglumine within 24 h of parturition is not recommended in dairy cattle.     

The binding of orally dosed hydrophobic active pharmaceutical ingredients to casein micelles in milk

Casein proteins (α_S1-, α_S2-, β- and κ-casein) account for 80% of the total protein content in bovine milk and form casein micelles (average diameter = 130 nm, approximately 10¹⁵ micelles/mL). The affinity of native casein micelles with the 3 hydrophobic active pharmaceutical ingredients (API), meloxicam [351.4 g/mol; log P = 3.43; acid dissociation constant (pK_a) = 4.08], flunixin (296.2 g/mol; log P = 4.1; pK_a = 5.82), and thiabendazole (201.2 g/mol; log P = 2.92; pK_a = 4.64), was evaluated in bovine milk collected from dosed Holstein cows. Native casein micelles were separated from raw bovine milk by mild techniques such as ultracentrifugation, diafiltration, isoelectric point precipitation (pH 4.6), and size exclusion chromatography. Acetonitrile extraction of hydrophobic API was then done, followed by quantification using HPLC-UV. For the API or metabolites meloxicam, 5-hyroxy flunixin and 5-hydroxy thiabendazole, 31 ± 3.90, 31 ± 1.3, and 28 ± 0.5% of the content in milk was associated with casein micelles, respectively. Less than ～5.0% of the recovered hydrophobic API were found in the milk fat fraction, and the remaining ～65% were associated with the whey/serum fraction. A separate in vitro study showed that 66 ± 6.4% of meloxicam, 29 ± 0.58% of flunixin, 34 ± 0.21% of the metabolite 5-hyroxy flunixin, 50 ± 4.5% of thiabendazole, and 33 ± 3.8% of metabolite 5-hydroxy thiabendazole was found partitioned into casein micelles. Our study supports the hypothesis that casein micelles are native carriers for hydrophobic compounds in bovine milk.     

Lying behavior as an indicator of lameness in dairy cows

Lameness is widely recognized as one of the most serious welfare and production concerns in the dairy industry. Our objectives were to evaluate the associations between lying behavior and lameness, and to determine whether lying behavior can be used as a diagnostic tool for lameness. Electronic data loggers recorded lying behavior of 1,319 cows from 28 farms at 1-min intervals for 5 d. These cows were gait scored according to a 5-point Numerical Rating System (NRS), and categorized as NRS ≤2, NRS = 3, or NRS = 4; no cow was scored as NRS = 5. Lameness was dichotomized twice: LAME (NRS ≥3) and SEVLAME (NRS = 4). Data were divided into 2 groups: 11 farms using deep-bedded stalls (DB) and 17 farms using mattress stalls (MAT). Differences in the daily lying time (h/d), frequency of lying bouts (n/d), duration of lying bouts (min/bout), and the standard deviation of bout duration (min/bout) between LAME or SEVLAME cows and those that were not were tested using mixed models. Receiver operating characteristic curves were constructed to identify behavioral thresholds to distinguish SEVLAME cows from the rest. Odds ratios for SEVLAME were estimated using logistic regression. Overall, 28.5% of cows were LAME including 7.3% that were SEVLAME. The prevalence of SEVLAME was higher on MAT farms than on DB farms (9.3 ± 1.3 vs. 4.4 ± 1.2%, respectively). SEVLAME cows on DB farms spent 12.8 [confidence interval (CI): 12.0 to 13.7] h/d lying down compared with 11.2 (CI: 10.7 to 11.8) h/d for cows that were not SEVLAME. These cows had longer duration of lying bouts [95.3 (CI: 84.6 to 107.3) vs. 80.3 (CI: 74.9 to 86.1) min/bout] and greater SD of bout duration [44.4 (CI: 41.1 to 48.0) vs. 50.7 (CI: 44.1 to 58.3) min/bout]. There were no behavioral differences among lameness categories on MAT farms. Within DB farms, cows with lying times >14.5 h/d had 16.2 (5.8 to 45.2) times higher odds of being SEVLAME. Cows with average lying bouts >90 min/bout were at 3.0 (1.2 to 7.4) times higher odds of being SEVLAME, and cows with average SD of bout duration >55 min/bout were at 4.1 (1.7 to 9.9) times higher odds of being SEVLAME. These results show that high lying times, long lying bouts, and variability in the duration of lying bouts were associated with lameness, and that stall surface influenced the behavioral responses of lame cows.     

Measuring lameness prevalence: Effects of case definition and assessment frequency

Lameness assessments are commonly conducted at a single point in time, but such assessments are subject to multiple sources of error. We conducted a longitudinal study, assessing the gait of 282 lactating dairy cows weekly during the first 12 wk of lactation, with the aim of assessing how lameness prevalence changed in relation to case definition and assessment frequency. Gait was scored using a 5-point scale where scores of 1 and 2 were considered sound, 3 was clinically lame, and 4 and 5 were severely lame. We created 5 lameness definitions using increasingly stringent thresholds based upon the number of consecutive events of locomotion score ≥3. In LAME1, a cow was considered lame when locomotion score was ≥3 at any scoring event, in LAME2, LAME3, LAME4, and LAME5, a cow was considered lame when locomotion score was 3 or higher during 2, 3, 4, and 5 consecutive scoring events, respectively. We also assessed the effect of assessment frequency on measures of prevalence and incidence using weekly assessment (ASSM1), 1 assessment every 2 wk (ASSM2), 1 assessment every 3 wk (ASSM3), and 1 assessment every 4 wk (ASSM4). Using LAME1, 69.2% of cows were considered lame at some point during the trial, with an average point prevalence of 31.8% (SD: 2.8) and average incidence rate of 10.9 cases/100 cow weeks (SD: 3.7). Lameness prevalence decreased to 28.0% when using LAME5. Survival analysis was used to assess the effects of parity, using these different case definitions. Parity is a known risk for lameness, such that case definitions and prevalence estimates should be stratified by parity to inform management decisions. Using the LAME3 criterion, primiparous cows had the highest chance of reaching 12 wk without a lameness event, and fourth and higher parities had the lowest. Weighted linear and quadratic kappa values were used to assess agreement between different assessment frequencies and lameness definitions; we found substantial to excellent agreement between ASSM1 and ASSM2 using LAME1, LAME2, and LAME3 definitions. Agreement was fair to substantial between ASSM1 and ASSM3 and low to fair between ASSM1 and ASSM4. Likewise, the agreement between LAME1 and LAME2 was fair in primiparous cows, substantial in second and third parity cows, and poor to fair in fourth and greater parity cows. We conclude that lameness prevalence estimates are dependent upon case definition and that the use of more stringent case definitions results in fewer cows classified as lame. These results suggest that routine locomotion assessments be conducted at least every 2 wk, and that cows should be defined as lame on the basis of 2 consecutive assessments.     

Effect of analgesia during hoof trimming on gait, weight distribution, and activity of dairy cattle

Sixty-six lactating cows were either injected with flunixin meglumine (2.2 mg/kg of BW) immediately before hoof trimming (n = 28), injected with a saline solution immediately before hoof trimming (n = 28), or injected with a saline solution immediately before sham hoof trimming (control; n = 10). Gait scores, time spent lying down, frequency of steps, and how cows distributed their weight among their legs when standing before, during, and after injections were measured to assess whether automated measures of activity and weight distribution can detect lameness and the effects of pain mitigation during hoof trimming. The overall gait score was positively correlated with the variability of the weight applied the rear legs (r = 0.32) and negatively correlated with the rear leg weight ratio (LWR; r = −0.52) and the frequency of steps (r = −0.43). The rear LWR was the best predictor of cows being lame (NRS >3), accounting for 27% of the variation in the likelihood of a cow being lame and 11% of the variation in the likelihood of a cow having an infectious hoof lesion. For each 5% increase in the rear LWR, the likelihood of being lame decreased by 30% (odds ratio = 0.71; 95% confidence interval = 0.56, 0.90) and the likelihood of being afflicted with an infectious hoof disease decreased by 20% (odds ratio = 0.81; 95% confidence interval = 0.67, 0.98). Neither hoof trimming nor a combination of hoof trimming and analgesia significantly affected gait score or any measure of weight distribution. Daily lying time increased during the 2 d following hoof trimming independently of the flunixin meglumine injection. However, this increase was not sustained for longer than 2 d when cows were injected with flunixin meglumine. Measures of weight shifting between legs while cows are standing and measures of activity show great potential as automated methods of detecting lameness and may also provide a tool for future evaluation of lameness therapies, such as hoof trimming and pain mitigation.     

液质联用测定肉制品中氟尼辛葡甲胺前处理方法的优化

以液相色谱-四级杆串联质谱法(LC-MS/MS为检测手段,通过考察回收率大小测定肉制品中氟尼辛葡甲胺的残留量,并对其的前处理条件进行了优化,最终确定了肉制品中氟尼辛葡甲胺前处理方法:采用乙腈-乙酸乙酯(11V/V提取,PCX固相萃取小柱净化,用正己烷-乙酸乙酯(91V/V淋洗,甲醇和氨化甲醇(595V/V洗脱,再用体积浓度0.1%甲酸(含0.5 mmol/L乙酸铵)定容,待测。方法回收率为85.0%110.0%相对标准偏差(RSD6.0%。     

Effect of nonsteroidal anti-inflammatory drugs on the inflammatory response of bovine endometrial epithelial cells in vitro

Chronic postpartum uterine infection detrimentally affects subsequent fertility. Nonsteroidal anti-inflammatory drugs (NSAID) are used to alleviate pain and treat inflammatory conditions in transition dairy cows with varying success. To screen the efficacy of NSAID in the absence of animal experiments, we have established an in vitro model to study uterine inflammation. Inflammation was induced in cultured bovine endometrial epithelial cells by challenging cells with an inflammation cocktail: lipopolysaccharide and proinflammatory cytokines, interleukin-1β (IL1β) and tumor necrosis factor α (TNFα). Release of the inflammation markers, serum amyloid A (SAA) and α-1-acid glycoprotein (αAGP), was measured by ELISA. Concentration of these markers was used to indicate the effectiveness in dampening inflammation of 5 NSAID: meloxicam, flunixin meglumine, aspirin, ketoprofen, and tolfenamic acid. Three NSAID, meloxicam, flunixin meglumine, and tolfenamic acid, were successful at dampening the release of SAA and αAGP into cell-culture supernatant, and the corresponding treated cells were selected for down-stream mRNA expression analysis. Expression of 192 genes involved in regulation of inflammatory pathways were investigated using Nanostring. Of the genes investigated, 81 were above the mRNA expression-analysis threshold criteria and were included in expression analysis. All SAA genes investigated (SAA2, SAA3, M-SAA3.2) were upregulated in response to the inflammation cocktail, relative to mRNA expression in control cells; however, AGP mRNA expression was below the expression analysis threshold and was, therefore, excluded from analysis. Treatment with NSAID downregulated genes involved in regulating chemokine signaling (e.g., CXCL2, CXCR4, CXCL5, and CXCL16) and genes that regulate the eicosanoid pathway (e.g., LTA4H, PTGS2, PLA2G4A, and PTGDS). Of the 5 NSAID investigated, meloxicam, flunixin meglumine, and tolfenamic acid are recommended for further investigation into treatment of postpartum uterine inflammation. The results from this study confirm the immunomodulatory properties of the endometrial epithelium in response to inflammatory stimuli and suggest that NSAID may be beneficial in alleviating uterine inflammation.     

Plasma pharmacokinetics and milk residues of flunixin and 5-hydroxy flunixin following different routes of administration in dairy cattle

The objective of this study was to determine if the plasma pharmacokinetics and milk elimination of flunixin (FLU) and 5-hydroxy flunixin (5OH) differ following intramuscular and subcutaneous injection of FLU compared with intravenous injection. Twelve lactating Holstein cows were used in a randomized crossover design study. Cows were organized into 2 groups based on milk production (<20 or >30 kg of milk/d). All cattle were administered 2 doses of 1.1 mg of FLU/kg at 12-h intervals by intravenous, intramuscular, and subcutaneous injections. The washout period between routes of administration was 7 d. Blood samples were collected from the jugular vein before FLU administration and at various time points up to 36 h after the first dose of FLU. Composite milk samples were collected before FLU administration and twice daily for 5 d after the first dose of FLU. Samples were analyzed by ultra-HPLC with mass spectrometric detection. For FLU plasma samples, a difference in terminal half-life was observed among routes of administration. Harmonic mean terminal half-lives for FLU were 3.42, 4.48, and 5.39 h for intravenous, intramuscular, and subcutaneous injection, respectively. The mean bioavailability following intramuscular and subcutaneous dosing was 84.5 and 104.2%, respectively. The decrease in 5OH milk concentration versus time after last dose was analyzed with the nonlinear mixed effects modeling approach and indicated that both the route of administration and rate of milk production were significant covariates. The number of milk samples greater than the tolerance limit for each route of administration was also compared at each time point for statistical significance. Forty-eight hours after the first dose, 5OH milk concentrations were undetectable in all intravenously injected cows; however, one intramuscularly injected and one subcutaneously injected cow had measurable concentrations. These cows had 5OH concentrations above the tolerance limit at the 36-h withdrawal time. The high number of FLU residues identified in cull dairy cows by the United States Department of Agriculture Food Safety Inspection Service is likely related to administration of the drug by an unapproved route. Cattle that received FLU by the approved (intravenous) route consistently eliminated the drug before the approved withdrawal times; however, residues can persist beyond these approved times following intramuscular or subcutaneous administration. Cows producing less than 20 kg of milk/d had altered FLU milk clearance, which may also contribute to violative FLU residues.     

Meloxicam affects the inflammatory responses of bovine mammary epithelial cells

Nonsteroidal anti-inflammatory drugs are used as supportive therapy with antimicrobial treatments for mastitis in cows to alleviate pain of the inflamed mammary gland. They act mainly by inhibition of cyclooxygenases. Meloxicam (MEL) is a drug designed for cyclooxygenase-2 selectivity, which is upregulated upon inflammation, acting as a key enzyme for the conversion of arachidonic acid to prostaglandins. Although some studies in dairy cows showed positive results in recovery from mastitis when MEL was added to the treatments, direct effects of MEL on the immune system of mastitic cows are unknown. The aim of this study was to investigate effects of MEL on the immune response of bovine mammary epithelial cells (MEC) with or without simultaneous immune stimulation by pathogen-associated molecular patterns of common mastitis pathogens. Mammary epithelial cells from 4 cows were isolated and cultured. To evaluate dose effects of MEL, MEC were challenged with or without 0.2 µg/mL lipopolysaccharide (LPS; serotype O26:B6 from Escherichia coli) with addition of increasing concentrations of MEL (0, 0.25, 0.5, 1.0, 1.5, or 2.0 mg/mL). The addition of MEL prevented the increase of mRNA expression of key inflammatory factors in LPS-challenged MEC in a dose-dependent manner. To investigate the effects of MEL on pathogen-specific immune responses of MEC, treatments included challenges with LPS from E. coli and lipoteichoic acid from Staphylococcus aureus with or without 1.5 mg/mL MEL for 3, 6, and 24 h. Meloxicam prevented the increase of mRNA abundance of key inflammatory mediators in response to LPS and lipoteichoic acid, such as tumor necrosis factor, serum amyloid A, inducible nitric oxide synthase, and the chemokines IL-8 and CXC chemokine ligands 3 and 5. The prostaglandin E₂ synthesis in challenged and nonchallenged cells was reduced by MEL within 24 h. Furthermore, MEL reduced the viability and consequently the total RNA yield of the cells. However, mRNA abundance of apoptosis-related enzymes was not affected by any treatment. Meloxicam had clear dose-dependent effects on the immune response of MEC to pathogen-associated molecular patterns of common mastitis pathogens by preventing increased expression of important factors involved in inflammation. This nonsteroidal anti-inflammatory drug also has detrimental effects on cell viability. How these effects would influence the elimination of pathogens from an infected mammary gland during mastitis therapy with meloxicam needs to be further investigated.     

The pharmacokinetics of transdermal flunixin in lactating dairy goats

Flunixin is a nonsteroidal anti-inflammatory drug approved for use in cattle to manage pyrexia associated with bovine respiratory disease, mastitis, and endotoxemia. In the United States, no nonsteroidal anti-inflammatory drugs are approved for use in goats, but analgesics are needed for management of painful conditions to improve animal welfare. The objective of this study was to evaluate the pharmacokinetics of transdermal flunixin in dairy goats to determine a milk withdrawal interval (WDI) to avoid violative residue contamination in the food supply. Six adult lactating dairy goats received 3.3 mg/kg of transdermal flunixin before milk, interstitial fluid (ISF), and blood samples were collected at various time points for 360 h. The samples were analyzed using tandem mass spectrometry to detect flunixin as well as the flunixin marker metabolite, 5-hydroxyflunixin followed by a pharmacokinetic WDI calculation using the US Food and Drug Administration tolerance limit method to propose safe residue levels in goat milk. The mean flunixin apparent plasma half-life was 21.63 h. The apparent milk half-life for 5-hydroxyflunixin was 17.52 h. Our findings provide a milk WDI of 60 h using the US Food and Drug Administration tolerance of 0.002 µg/mL (established for bovine milk) and a more conservative WDI of 96 h using a limit of quantification of 0.001 µg/mL following the extralabel use of transdermal flunixin in dairy goats.     

Effects of a combination butaphosphan and cyanocobalamin product and insulin on ketosis resolution and milk production

The objective of this study was to determine the effects of butaphosphan-cyanocobalamin (B+C), glargine insulin, and propylene glycol on resolution of ketosis and average daily milk yield after treatment. Cows from 16 herds in Ontario, Canada, and 1 herd in Michigan were tested at weekly intervals between 3 and 16 DIM. Ketosis was defined as blood β-hydroxybutyrate (BHB) ≥1.2 mmol/L. All ketotic cows were given a baseline treatment of 3 d of 300 g of propylene glycol orally. Animals were then randomly assigned to treatment with 3 doses of either 25 mL of B+C or 25 mL of saline placebo and 1 dose of either 2 mL (200 IU) of glargine insulin or 2 mL of saline placebo in a 2 × 2 factorial arrangement. Outcomes of interest on all farms were ketosis cure (blood BHB <1.2 mmol/L 1 wk postenrollment), maintenance of ketosis cure (blood BHB <1.2 mmol/L 1 and 2 wk postenrollment), and blood BHB concentrations at 1 and 2 wk postenrollment. Milk weights were collected daily in 1 large freestall herd. Repeated measures ANOVA was used to evaluate blood BHB concentrations 2 wk after treatment and milk production for 30 d after treatment. Poisson regression was used to examine the effect of treatment on cure and maintenance of cure. Due to a regulatory delay causing temporary unavailability of B+C in Canada, data were analyzed in 2 sets of models: one for insulin and the corresponding placebo (n = 620) and one for the full trial (n = 380). Animals with blood glucose concentrations ≤2.2 mmol/L at the time of ketosis diagnosis were 2.1 times more likely (95% CI = 1.2 to 3.7) to be cured if treated with B+C. Animals in lactation 3 or higher that had blood glucose concentrations <2.2 mmol/L at enrollment produced 4.2 kg/d (95% CI = 1.4 to 7.1) more milk if treated with insulin versus placebo and 2.8 kg/d (95% CI = 0.9 to 4.7) more milk if treated with B+C versus placebo. Animals in lactation 3 or higher with blood glucose ≥2.2 mmol/L that were treated with insulin produced 2.3 kg/d (95% CI = 0.3 to 4.4) less milk than untreated controls. No interaction was observed between treatments. This evidence suggests that B+C and insulin may be beneficial for ketosis treatment in animals with blood glucose <2.2 mmol/L at ketosis diagnosis. It also suggests that blood glucose concentration may be an important predictor of success of ketosis treatment.     

The impact of meloxicam on postsurgical stress associated with cautery dehorning

The objectives were to determine the duration of the stress response associated with cautery dehorning and to assess the effectiveness of the nonsteroidal anti-inflammatory drug meloxicam (Metacam, 20 mg/mL solution for injection) for reducing that response. Sixty Holstein heifer calves were blocked by age and randomly assigned to receive an i.m. injection of meloxicam or a placebo (0.5 mg/kg). All calves were given a lidocaine cornual nerve block delivered 5 mL per side 10 min before dehorning. To establish baseline values, calves were sham dehorned 24 h before actual dehorning. Blood samples were taken via indwelling jugular catheters at 0, 0.5, 1, 1.5, 2, 4, 6, and 24 h after the procedure. Heart and respiratory rates were also taken at these times. Data were analyzed using PROC MIXED in SAS. Analysis of covariance was employed to assess the difference between sham and dehorning at each time period. Dehorning was associated with elevated serum cortisol (d −1: 33.9 ± 1.26; d 0: 46.2 ± 2.33 nmol/L) and heart rate (d −1: 108 ± 1.8; d 0: 109.4 ± 2.4 beats per minute) in both groups for 24 h, and elevated respiratory rate (sham: 42.2 ± 1.95 vs. dehorning: 45.1 ± 2.19 respirations per minute) in both groups for 6 h. A treatment × time interaction was found for cortisol, with meloxicam calves having lower serum cortisol than controls until 6 h after dehorning (meloxicam: 49.7 ± 4.37 vs. control: 63.0 ± 6.94 nmol/L). There was no difference between the treatment groups at 24 h (meloxicam: 35.2 ± 2.74 and control: 34.8 ± 3.64 nmol/L of cortisol). Overall, the changes in heart rates (increase meloxicam: 3.74 ± 0.96 vs. control: 4.70 ± 1.87) and respiratory rates (increase meloxicam: 2 ± 0.1 vs. control: 4 ± 0.2) were greater in the control group compared with the meloxicam group. These results indicate that meloxicam reduced the physiological stress response to dehorning.     

Analgesic efficacy of sodium salicylate in an amphotericin B-induced bovine synovitis-arthritis model

This study examined the efficacy of sodium salicylate for providing analgesia in an amphotericin B-induced bovine synovitis-arthritis model using 10 male Holstein calves, 4 to 6 mo old and weighing approximately 250 kg. The study used a repeated measures partial crossover design with 2 phases, consisting of 3 treatment periods within each phase. Calves were blocked by body weight and randomly assigned to the sodium salicylate (50 mg/kg i.v.) or placebo group for phase 1. In period 1, lameness induction was simulated with a needle prick of the coronary band, followed by drug or placebo administration. At predetermined time points, serial blood samples for cortisol and salicylate concentrations, electrodermal activity measurements, heart rates, and pressure mat data were collected. Visual lameness scores were recorded by an observer blinded to treatments. In period 2, lameness was induced with injection of amphotericin B into the distal interphalangeal joint, followed by drug or placebo administration, with sample collection as described previously. In period 3, the drug or placebo was administered to the respective calves with sample collection. After a 10-d washout period, phase 2 was conducted with treatments crossed over between groups. Cortisol and salicylate samples were analyzed by competitive chemiluminescent immunoassay and fluorescence polarization immunoassay, respectively. The pharmacokinetic data were analyzed using compartmental analysis. Mean intravenous salicylate apparent volume of distribution was 0.2 ± 0.005 L/kg, total body clearance was 4.3 ± 0.2 mL/min·kg, and elimination half-life was 36.9 ± 1.2 min. The repeated measures data were analyzed based on a univariate split-plot approach with a random effects-mixed model. Differences in stance phase duration and serum cortisol concentration values were seen both between periods and between treatment group × periods; differences in heart rate, contact surface area, and contact pressure values were seen between periods, suggesting that our lameness model was effective. No differences were seen between treatment groups. When analyzed by visual lameness score, differences were seen in heart rate, contact surface area, contact pressure, and cortisol concentrations. Area under the time-effect curves, determined by using the trapezoidal rule, had results similar to the repeated measures data, except for a difference in period for electrodermal activity. This amphotericin B-induced synovitis-arthritis model is a useful tool for studying changes associated with lameness in cattle. Sodium salicylate was not effective in providing analgesia after lameness.     

The effect of meloxicam on behavior and pain sensitivity of dairy calves following cautery dehorning with a local anesthetic

Effects of a single injection of meloxicam on calf behavior, pain sensitivity, and feed and water intakes were examined following dehorning. Sixty Holstein heifer calves were blocked by age and randomly assigned to receive an i.m. injection of meloxicam (0.5 mg/kg) or a placebo. All calves were given a lidocaine cornual nerve block (5 mL per horn). Treatments and nerve blocks were administered 10 min before cautery dehorning. Continuous sampling of behavior was performed during five 1-h intervals using video recordings, and total daily activity was monitored using an accelerometer. A pain sensitivity test was administered with a pressure algometer, and feed and water intakes were recorded daily. Calves were sham-dehorned 24 h before actual dehorning to establish baseline values, and all variables were assessed at the same times following dehorning and sham dehorning for up to 48 h post-dehorning. Meloxicam-treated calves displayed less ear flicking during the 44 h following dehorning (increases of 4.29 ± 1.10 and 1.31 ± 0.66 ear flicks/h in the first 24 h, and increases of 3.27 ± 0.89 and 0.55 ± 0.50 ear flicks/h during the second 24 h, for control and meloxicam calves, respectively) and less head shaking during the first 9 h following dehorning (increase of 2.53 ± 0.54 and 0.85 ± 0.46 headshakes/h over baseline for control and meloxicam, respectively). Meloxicam-treated calves were less active than controls during the first 5 h following dehorning (activity 34.1 ± 3.2 and 30.6 ± 2.6 for control and meloxicam, respectively) and displayed less sensitivity to pressure algometry 4 h after dehorning (pressure tolerance of 1.62 ± 0.13 kg of force and 2.13 ± 0.15 kg of force for control and meloxicam calves, respectively). Changes in behavior suggest that meloxicam was effective for reducing post-surgical pain and distress associated with calf dehorning.     

The effect of timing of oral meloxicam administration on physiological responses in calves after cautery dehorning with local anesthesia

Dehorning is a painful husbandry procedure that is commonly performed in dairy calves. Parenteral meloxicam combined with local anesthesia mitigates the physiological and behavioral effects of dehorning in calves. The purpose of this study was to determine the influence of timing of oral meloxicam administration on physiological responses in calves after dehorning. Thirty Holstein bull calves, 8 to 10 wk of age (28–70 kg), were randomly assigned to 1 of 3 treatment groups: placebo-treated control group (n = 10), calves receiving meloxicam administered orally (1 mg/kg) in powdered milk replacer 12 h before cautery dehorning (MEL-PRE; n = 10), and calves receiving meloxicam administered as an oral bolus (1 mg/kg) at the time of dehorning (MEL-POST; n = 10). Following cautery dehorning, blood samples were collected to measure cortisol, substance P (SP), haptoglobin, ex vivo prostaglandin E₂ (PgE₂) production after lipopolysaccharide stimulation and meloxicam concentrations. Maximum ocular temperature and mechanical nociceptive threshold (MNT) were also assessed. Data were analyzed using noncompartmental pharmacokinetic analysis and repeated measures ANOVA models. Mean peak meloxicam concentrations were 3.61 ± 0 0.21 and 3.27 ± 0.14 μg/mL with average elimination half-lives of 38.62 ± 5.87 and 35.81 ± 6.26 h for MEL-PRE and MEL-POST, respectively. Serum cortisol concentrations were lower in meloxicam-treated calves compared with control calves at 4 h postdehorning. Substance P concentrations were significantly higher in control calves compared with meloxicam-treated calves at 120 h after dehorning. Prostaglandin E₂ concentrations were lower in meloxicam-treated calves compared with control calves. Mechanical nociceptive threshold was higher in control calves at 1 h after dehorning, but meloxicam-treated calves tended to have a higher MNT at 6 h after dehorning. No effect of timing of meloxicam administration on serum cortisol concentrations, SP concentrations, haptoglobin concentrations, maximum ocular temperature, or MNT was observed. However, PgE₂ concentrations in MEL-PRE calves were similar to control calves after 12 h postdehorning, whereas MEL-POST calves had lower PgE₂ concentrations for 3 d postdehorning. These findings support that meloxicam reduced cortisol, SP, and PgE₂ after dehorning, but only PgE₂ production was significantly affected by the timing of meloxicam administration.