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Determination of milk concentrations and pharmacokinetics of salicylic acid following acetylsalicylic acid (aspirin) administration in postpartum dairy cows

Open AccessPublished:October 04, 2022DOI:https://doi.org/10.3168/jds.2021-21507

      ABSTRACT

      The objectives of this descriptive study were to (1) describe the pharmacokinetics of salicylic acid (SA) in the milk and plasma of postpartum dairy cattle following oral administration of acetylsalicylic acid (ASA; aspirin), (2) to estimate a recommended milk withdrawal period for dairy cattle treated with ASA, and (3) to determine the effect of ASA administration on plasma prostaglandin E2 metabolite (PGEM) concentrations. Primiparous (n = 3) and multiparous (n = 7) postpartum Holstein dairy cows received 2 oral treatments with ASA at 200 mg/kg of body weight, 24 h apart. Concentrations of SA in plasma and milk from 0 h through 120 h after ASA administration were analyzed using ultra performance liquid chromatography triple quadrupole mass spectrometry and a milk withdrawal period was estimated using the United States Food and Drug Administration Milk Discard App in R. Two withdrawal periods were estimated: (1) a whole-herd treatment scenario with no dilution factor and (2) an individual animal treatment scenario with a bulk tank factor included in analysis. Plasma PGEM concentrations in samples from 0 h to 24 h after ASA administration were determined using a commercially available competitive ELISA. Milk SA concentrations were undetected in all cows by 48 h after the last ASA treatment. Secondary peaks were observed in plasma at 58 and 82 h after the last treatment and in milk at 87 h after the last treatment. In the absence of a tolerance for SA in milk, the estimated milk withdrawal periods were (1) 156 h for the whole-herd treatment scenario and (2) 120 h for the individual animal treatment scenario. Plasma PGEM concentrations were reduced compared with baseline for up to 12 h after ASA administration, with the greatest reduction observed at 2 h. Results from this study suggest that the current milk withhold recommendation for dairy cattle administered ASA may need revision to 120 h (5 d) and that ASA administration may mitigate postpartum inflammation through reduction in prostaglandin production for up to 12 h after treatment. Pharmacokinetic and milk withdrawal data from this study will inform future recommendations for extra-label use of aspirin in postpartum dairy cows. Further research is required to determine the basis for the secondary SA peaks and to elucidate the long-term effects of ASA administration on dairy cow health.

      Key words

      INTRODUCTION

      Administration of nonsteroidal anti-inflammatory drugs (NSAID) at calving may reduce inflammation in the transition period and the associated negative effect on milk production (
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      Anti-inflammatory salicylate treatment alters the metabolic adaptations to lactation in dairy cattle.
      ). Previous work has shown that periparturient administration of sodium salicylate (SS), acetylsalicylic acid (ASA), meloxicam, ketoprofen, or carprofen increases milk yield (
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      Sodium salicylate treatment in early lactation increases whole-lactation milk and milk fat yield in mature dairy cows.
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      Short communication: Effects of analgesic use postcalving on cow welfare and production.
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      Hot topic: Early postpartum treatment of commercial dairy cows with non-steroidal anti-inflammatory drugs increases whole-lactation milk yield.
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      Administration of acetylsalicylic acid after parturition in lactating dairy cows under certified organic management: Part I. Milk yield, milk components, activity patterns, fertility, and health.
      ). Reported benefits of NSAID administration include decreased SCC, decreased culling rates, increased activity, and increased feeding behavior (
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      Effect of meloxicam administration after calving on milk production, acute phase proteins, and behavior in dairy cows.
      ;
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      • Schubert H.
      • Broom D.M.
      Short communication: Effects of analgesic use postcalving on cow welfare and production.
      ;
      • Shock D.A.
      • Renaud D.L.
      • Roche S.M.
      • Poliquin R.
      • Thomson R.
      • Olson M.E.
      Evaluating the impact of meloxicam oral suspension administered at parturition on subsequent production, health, and culling in dairy cows: A randomized clinical field trial.
      ).
      The use of the NSAID, SS, and ASA (aspirin) has also been described in postpartum dairy cows (
      • Farney J.K.
      • Mamedova L.K.
      • Coetzee J.F.
      • Minton J.E.
      • Hollis L.C.
      • Bradford B.J.
      Sodium salicylate treatment in early lactation increases whole-lactation milk and milk fat yield in mature dairy cows.
      ;
      • Carpenter A.J.
      • Ylioja C.M.
      • Vargas C.F.
      • Mamedova L.K.
      • Mendonça L.G.
      • Coetzee J.F.
      • Hollis L.C.
      • Gehring R.
      • Bradford B.J.
      Hot topic: Early postpartum treatment of commercial dairy cows with non-steroidal anti-inflammatory drugs increases whole-lactation milk yield.
      ;
      • Barragan A.A.
      • Bauman L.M.
      • da Costa L.
      • Velez J.
      • Gonzalez J.D.R.
      • Schuenemann G.M.
      • Menichetti B.
      • Piñeiro J.
      • Bas S.
      Administration of acetylsalicylic acid after parturition in lactating dairy cows under certified organic management: Part I. Milk yield, milk components, activity patterns, fertility, and health.
      ). Acetylsalicylic acid and its active metabolite, salicylic acid (SA), inhibit cyclooxygenase-1 and -2, reduce tumor necrosis factor-α (TNF-α) mRNA transcription, and prevent nuclear factor kappa B activation by binding to the protein inhibitor of nuclear factor kappa B kinase subunit beta (
      • Mitchell J.A.
      • Akarasereenont P.
      • Thiemermann C.
      • Flower R.J.
      • Vane J.R.
      Selectivity of nonsteroidal antiinflammatory drugs as inhibitors of constitutive and inducible cyclooxygenase.
      ;
      • Yin M.J.
      • Yamamoto Y.
      • Gaynor R.
      The anti-inflammatory agents aspirin and salicylate inhibit the activity of IκB kinase-β.
      ;
      • Myers M.J.
      • Scott M.L.
      • Deaver C.M.
      • Farrell D.E.
      • Yancy H.F.
      Biomarkers of inflammation in cattle determining the effectiveness of anti-inflammatory drugs.
      ). Acetylsalicylic acid is not currently approved in the United States for use in lactating dairy cattle (
      • Smith G.W.
      • Davis J.L.
      • Tell L.A.
      • Webb A.I.
      • Riviere J.E.
      Extralabel use of nonsteroidal anti-inflammatory drugs in cattle.
      ). Many authors have reported increased milk yields after treating postpartum, multiparous cows with ASA, similar to the effects seen with other NSAID (
      • Trevisi E.
      • Bertoni G.
      Attenuation with acetylsalicylate treatments of inflammatory conditions in periparturient dairy cows.
      ;
      • Farney J.K.
      • Mamedova L.K.
      • Coetzee J.F.
      • Minton J.E.
      • Hollis L.C.
      • Bradford B.J.
      Sodium salicylate treatment in early lactation increases whole-lactation milk and milk fat yield in mature dairy cows.
      ;
      • Barragan A.A.
      • Bauman L.M.
      • da Costa L.
      • Velez J.
      • Gonzalez J.D.R.
      • Schuenemann G.M.
      • Menichetti B.
      • Piñeiro J.
      • Bas S.
      Administration of acetylsalicylic acid after parturition in lactating dairy cows under certified organic management: Part I. Milk yield, milk components, activity patterns, fertility, and health.
      ). A recent study found that cows treated with ASA had a significantly lower incidence of metritis and a tendency toward lower rates of endometritis (
      • Barragan A.A.
      • Bas S.
      • Hovingh E.
      • Byler L.
      Effects of postpartum acetylsalicylic acid on uterine diseases and reproductive performance in dairy cattle.
      ). The authors also reported a tendency in the ASA group to faster conception rates, a lower incidence of pregnancy losses, and increased conception rates following pregnancy loss compared with untreated cattle.
      Although the potential benefits of ASA administration are attractive to producers, milk residues need to be investigated. Currently, no published pharmacokinetic (PK) data are on SA in milk. Though PK data are available for serum salicylate concentrations (
      • Gingerich D.A.
      • Baggot J.D.
      • Yeary R.A.
      Pharmacokinetics and dosage of aspirin in cattle.
      ;
      • Anderson J.G.
      • Oehme F.W.
      • Moore W.E.
      The effect of oral salicylate (aspirin) on hematologic and biochemical profiles of dairy cattle.
      ;
      • Coetzee J.F.
      • Gehring R.
      • Bettenhausen A.C.
      • Lubbers B.V.
      • Toerber S.E.
      • Thomson D.U.
      • Kukanich B.
      • Apley M.D.
      Attenuation of acute plasma cortisol response in calves following intravenous sodium salicylate administration prior to castration.
      ;
      • Kotschwar J.L.
      • Coetzee J.F.
      • Anderson D.E.
      • Gehring R.
      • KuKanich B.
      • Apley M.D.
      Analgesic efficacy of sodium salicylate in an amphotericin B induced bovine synovitis-arthritis model.
      ;
      • Baldridge S.L.
      • Coetzee J.F.
      • Dritz S.S.
      • Reinbold J.B.
      • Gehring R.
      • Havel J.
      • Kukanich B.
      Pharmacokinetics and physiologic effects of intramuscularly administered xylazine hydrochloride-ketamine hydrochloride-butorphanol tartrate alone or in combination with orally administered sodium salicylate on biomarkers of pain in Holstein calves following castration and dehorning.
      ;
      • Bergamasco L.
      • Coetzee J.F.
      • Gehring R.
      • Murray L.
      • Song T.
      • Mosher R.A.
      Effect of intravenous sodium salicylate administration prior to castration on plasma cortisol and electroencephalography parameters in calves.
      ), the treatment regimens used were unlike those described in more recent studies. Some of these earlier studies investigated the use of SS through either a single intravenous administration at 50 mg/kg of BW (
      • Coetzee J.F.
      • Gehring R.
      • Bettenhausen A.C.
      • Lubbers B.V.
      • Toerber S.E.
      • Thomson D.U.
      • Kukanich B.
      • Apley M.D.
      Attenuation of acute plasma cortisol response in calves following intravenous sodium salicylate administration prior to castration.
      ;
      • Kotschwar J.L.
      • Coetzee J.F.
      • Anderson D.E.
      • Gehring R.
      • KuKanich B.
      • Apley M.D.
      Analgesic efficacy of sodium salicylate in an amphotericin B induced bovine synovitis-arthritis model.
      ;
      • Bergamasco L.
      • Coetzee J.F.
      • Gehring R.
      • Murray L.
      • Song T.
      • Mosher R.A.
      Effect of intravenous sodium salicylate administration prior to castration on plasma cortisol and electroencephalography parameters in calves.
      ) or oral administration through drinking water for 5 d at doses ranging from 13.6–152 mg/kg of BW (
      • Baldridge S.L.
      • Coetzee J.F.
      • Dritz S.S.
      • Reinbold J.B.
      • Gehring R.
      • Havel J.
      • Kukanich B.
      Pharmacokinetics and physiologic effects of intramuscularly administered xylazine hydrochloride-ketamine hydrochloride-butorphanol tartrate alone or in combination with orally administered sodium salicylate on biomarkers of pain in Holstein calves following castration and dehorning.
      ). Two studies investigated oral administration of ASA, either at 50 mg/kg of BW for one treatment (
      • Coetzee J.F.
      • Gehring R.
      • Bettenhausen A.C.
      • Lubbers B.V.
      • Toerber S.E.
      • Thomson D.U.
      • Kukanich B.
      • Apley M.D.
      Attenuation of acute plasma cortisol response in calves following intravenous sodium salicylate administration prior to castration.
      ) or at 100 mg/kg of BW per treatment every 12 h for 5 d (
      • Gingerich D.A.
      • Baggot J.D.
      • Yeary R.A.
      Pharmacokinetics and dosage of aspirin in cattle.
      ). More recently,
      • Barragan A.A.
      • Bauman L.M.
      • da Costa L.
      • Velez J.
      • Gonzalez J.D.R.
      • Schuenemann G.M.
      • Menichetti B.
      • Piñeiro J.
      • Bas S.
      Administration of acetylsalicylic acid after parturition in lactating dairy cows under certified organic management: Part I. Milk yield, milk components, activity patterns, fertility, and health.
      ,
      • Barragan A.A.
      • Bauman L.M.
      • Schuenemann G.M.
      • Velez J.
      • Lakritz J.
      • Coetzee J.F.
      • Gonzalez J.D.R.
      • Piñeiro J.M.
      • Menichetti B.
      • Bas S.
      Administration of acetylsalicylic acid after parturition in lactating dairy cows under certified organic management: Part II. Biomarkers of nociception, inflammation, and stress.
      ,
      • Barragan A.A.
      • Bas S.
      • Hovingh E.
      • Byler L.
      Effects of postpartum acetylsalicylic acid on uterine diseases and reproductive performance in dairy cattle.
      ) reported that a short course of ASA treatment—200 mg/kg every 24 h for 2 d—has positive effects on milk production and cow metabolic and reproductive well-being. This revised treatment protocol may be more practical and, thus, more likely to be adopted by researchers and producers for use in postpartum cows. In addition to the updated dosing regimen, the improved quality and sensitivity of analytical equipment make PK data gathered today more accurate. Drug concentration data in plasma and milk are needed to establish the pharmacokinetic profile of SA, to recommend milk withhold times, and to inform future approval decisions and regulations regarding milk withholding periods for lactating dairy cattle. Supplying producers with appropriate, well-founded data on milk residues is critical to maintaining consumer trust and food supply integrity. Minimal pharmacodynamic data are available for dairy cattle following oral administration of ASA. Determining the short- and long-term effects of ASA on dairy cow health would provide producers with information necessary to form optimal postpartum cow health management programs.
      The objectives of this study were to (1) describe the pharmacokinetics of SA in the milk and plasma of postpartum dairy cattle following oral administration of aspirin, (2) to establish a recommended milk withdrawal interval (WDI) for dairy cattle treated with ASA at 200 mg/kg twice, 24 h apart, and (3) to determine the effect of ASA administration on plasma concentrations of PGE2 metabolites (PGEM) compared with baseline PGEM values.

      MATERIALS AND METHODS

      The Institutional Animal Care and Use Committee at Kansas State University approved the methods outlined below (protocol number 4432).

      Animals, Housing, and Treatments

      In this descriptive study, postpartum Holstein cattle (Bos taurus; n = 10; 3 primiparous, 7 multiparous) of an average weight of 662 kg (554–859 kg) from the Kansas State University Dairy Teaching and Research Center were enrolled between May 2021 until July 2021, within 3 to 27 h after calving. The Dairy Teaching and Research Center houses approximately 300 cows. A common, straw-bedded pen was used to house cows that were within approximately 30 d of calving. To be eligible for enrollment, cows were required to be free of illness; have a lameness score of ≤2 out of 5 using a visual lameness scoring system (
      • Sprecher D.J.
      • Hostetler D.E.
      • Kaneene J.B.
      A lameness scoring system that uses posture and gait to predict dairy cattle reproductive performance.
      ); have a dystocia score of 1 out of 5 (
      • Barragan A.A.
      • Hovingh E.
      • Bas S.
      • Lakritz J.
      • Byler L.
      • Ludwikowski A.
      • Takitch S.
      • Zug J.
      • Hann S.
      Effects of postpartum acetylsalicylic acid on metabolic status, health, and production in lactating dairy cattle.
      ); and have no history of receiving ASA or other NSAID in the past 30 d. If any cows developed lameness or illness or required other medications during the study period, they were unenrolled. Throughout the enrollment period for each cow, body temperature was measured at least once per day using rectal thermometry and urine ketones were analyzed once per day with ketone reagent strips supplied by the Kansas State Dairy Teaching and Research Center. Farm personnel administered a single bolus of BoviKalc (Boehringer Ingelheim Vetmedica) to cows at the time of calving. Eligible cows were enrolled following the morning milking. Before enrollment, cows were weighed. Following enrollment, cows were moved to an open-sided, freestall barn bedded with sand, along with other postpartum cows. Cows were milked at 0700, 1700, and 2200 h and fed TMR once daily in the morning. The diet was formulated to meet or exceed nutrient requirements for high-producing lactating dairy cattle (
      • NRC
      Nutrient Requirements of Dairy Cattle.
      ). Cows were provided water ad libitum.
      Cattle received oral ASA treatments at enrollment and 24 h later (following the 0700 h milking). A bolus gun was used to administer 3 to 5 boluses of 480 grain (31,104 mg/bolus) ASA (MWI Animal Health) to achieve the target dose of 200 mg/kg of BW. Milk and whole blood were obtained at each time point for SA concentration determination.
      Time points for milk collection were before ASA administration (0 h) and 10, 15, 24, 34, 39, 48, 58, 63, 72, 82, 87, 96, 106, 111, and 120 h after ASA administration. Each milk collection time point occurred when cows were scheduled to enter the milking parlor. Cows were milked into floor pails so the entire milking was obtained. Following milking, the milk was well stirred and a milk sample was collected in sample vials (Thermo Fisher Scientific). The milk samples were immediately placed on ice and transported to the laboratory for storage at −20°C until analysis.
      Time points for blood collection were before ASA administration and 2, 6, 10, 24, 34, 48, 58, 72, 82, 96, 106, and 120 h after ASA administration. At each collection time point, cows were restrained in a chute or head-lock, and 6 mL of whole blood was obtained by jugular or coccygeal venipuncture using a 1.5 in 18- or 20-gauge needle. Blood samples were collected using sterile 6-mL lithium heparin-coated tubes (Vacuette, Greiner Bio-One). Blood was immediately placed on ice, transported to the laboratory, and centrifuged (4,000 × g for 10 min at 4°C). Plasma was harvested and stored at −80°C until analysis.

      Plasma and Milk Salicylic Acid Analysis

      Salicylic acid concentrations in milk and plasma samples were determined using ultra performance liquid chromatography triple quadrupole MS (UPLC-MS/MS). Salicylic acid (analytical standard) and salicylic acid-d4 (SA-d4; internal standard, Cerilliant) were purchased from Sigma-Aldrich Inc. Ultrapure water (18.2 MΩ-cm ) was obtained from an on-site system (Thermo Fisher Scientific). Optima LC/MS grade acetonitrile, ammonium formate, and ammonium hydroxide, and HPLC grade 85% phosphoric acid were obtained from Thermo Fisher Scientific. The LC/MS and HPLC grade methanol were obtained from Honeywell (Burdick and Jackson). Formic acid (MS standard) was obtained from Waters Corp. Blank filtered lithium heparin bovine plasma (negative control plasma; NCP) was obtained from Lampire Biological Laboratories Inc. Negative control milk was obtained from one of the animals enrolled in the study before ASA administration.
      Before sample preparation, plasma samples from 2, 6, 10, and 34 h were diluted 500-fold, and samples from 24 and 48 h were diluted 100-fold with NCP; the remaining plasma samples were prepared undiluted. The lipid layers from the milk samples in the collection vials were removed, and 1-mL aliquots from each milk sample were centrifuged at 1,500 × g at 4°C for 10 min. After centrifugation, the remaining lipid layers were removed and the milk samples were vortexed.
      Salicylic acid stock solution was prepared at 1,000 µg/mL in methanol and stored at −20°C. Salicylic acid-d4 100 µg/mL stock solution was stored in a glass vial (Waters Corp.) at −20°C. A 100 µg/mL working stock solution of SA was prepared daily by diluting the 1,000 µg/mL stock in 4% phosphoric acid. A 0.050 µg/mL working solution of SA-d4 was prepared daily by diluting the 100 µg/mL stock in 4% phosphoric acid. Working solutions for SA calibration standards (STC) and quality controls (QC) were prepared fresh daily in NCP or negative control milk. The STC were prepared at 8 concentrations (0.025, 0.050, 0.100, 0.250, 0.500, 1.000, 2.500, and 5.000 µg/mL SA) and QC were prepared at 3 concentrations (0.180, 1.800, and 3.600 µg/mL SA).
      For the STC, QC, negative controls, and internal standard controls, 100 µL of NCP or negative control milk was aliquoted. To this, for the STC and QC, 10 µL of the appropriate STC or QC stock and 190 µL of 0.05 µg/mL SA-d4 in 4% phosphoric acid were added. To the internal standard controls, 10 µL of 4% phosphoric acid and 190 µL of 0.05 µg/mL SA-d4 in 4% phosphoric acid were added. To the negative controls, 200 µL of 4% phosphoric acid was added. For the samples, 100 µL of plasma or milk was combined with 10 µL of 4% phosphoric acid and 190 µL of 0.05 µg/mL SA-d4 in 4% phosphoric acid. Samples, STC, QC, negative controls, and internal standard controls were vortexed and then centrifuged at 1,500 × g at 4°C for 10 min.
      Analytes were extracted via solid phase extraction using Oasis MAX 96-well µElution Plates (30µm; Waters Corp.) and a solid phase extraction positive pressure manifold (Positive Pressure-96 Processor, Waters Corp.). The solid phase extraction cartridges were conditioned with 300 µL methanol followed by 300 µL 18.2 MΩ-cm water. After 300 µL sample, control, STC, or QC solution was loaded into the appropriate wells, the cartridge was washed with 300 µL 5% ammonium hydroxide in 18.2 MΩ-cm water followed by 300 µL methanol. Eluate was collected in a clean collection plate (96-well, 2 mL; Waters Corp.) using 50 µL 2% formic acid in acetonitrile-methanol (60:40) and 50 µL 18.2 MΩ-cm water was added to all collection plate wells. The collection plates were covered with pre-slit silicone cap mats (Waters Corp., Milford, MA) and vortexed gently.
      Collection plates were loaded onto an ACQUITY H-Class PLUS UPLC system (Waters Corp.). Chromatographic separation was achieved using an ACQUITY UPLC HSS T3 C18 column (100 × 2.1 mm, 1.8 µm; Waters Corp.) kept at 40°C. The UPLC mobile phases consisted of 0.1% formic acid in 5 mM ammonium formate in water (mobile phase A) and 0.1% formic acid in 5 mM ammonium formate in acetonitrile-water (90:10; mobile phase B). A gradient program was used to achieve analyte separation. After sample injection (0 min), a combination of 99% mobile phase A and 1% mobile phase B was linearly changed to a combination of 0% mobile phase A and 100% mobile phase B until 1.49 min. Mobile phase B was linearly reversed to 1% at 2.0 min, and the original mobile phase mixture was held from 2.01 min to 3 min. The flow rate was 0.6 mL/min and the sample injection volume was 2 µL. The LC effluent was diverted to waste for the first 0.7 min and the last 1.0 min of each chromatographic run.
      The mass spectrometer was a Xevo TQ-S tandem mass spectrometer (MS/MS) equipped with a Z-spray electrospray ionization interface set in negative ion mode (Waters Corp.). Data were acquired and processed by MassLynx and TargetLynx software, respectively (Waters Corp.). The quantifying transition for SA was m/z 137.0636→92.8904 and the qualifying transition was m/z 137.0636→64.8659. The quantifying transition for SA-d4 was m/z 141.1081→96.9367. The cone voltage and collision energy for the SA quantifying and qualifying transitions were −42 V/−14 V and −42 V/−22 V, respectively. The parameters for SA-d4 were −44 V/−16 V. The dwell time for all compounds was 3 ms.
      The concentrations of the eluted standards were 0.0025, 0.005, 0.010, 0.025, 0.050, 0.100, 0.250, and 0.500 µg/mL. The concentrations of the eluted QC were 0.018, 0.180, and 0.360 µg/mL. The limit of detection (LOD) was 0.0025 µg/mL and the lower limit of quantification (LLOQ) was 0.0043 µg/mL. The standard curve was linear from 0.0025 to 0.500 µg/mL; the correlation coefficient was accepted if it was at least 0.975. The intra-day accuracy and precision were determined by analyzing replicates of SA at 3 different QC levels. Inter-day accuracy and precision calculated by analyzing the 3 levels of QC samples were determined to be 108.38 and 4.29%, respectively.

      Plasma Pharmacokinetic Analysis

      Pharmacokinetic analysis based on each cow's plasma concentration time curve was performed using PKSolver (
      • Zhang Y.
      • Huo M.
      • Zhou J.
      • Xie S.
      PKSolver: An add-in program for pharmacokinetic and pharmacodynamic data analysis in Microsoft Excel.
      ) in Excel (Microsoft Inc.). Using the semi-logarithmic plots of SA, noncompartmental analysis was performed with uniform weighting based on statistical moment theory. Average peak plasma SA concentration (Cmax) and time to peak concentration (Tmax) were determined. The log-linear portion of the terminal section of the log plasma concentration time curve was used to calculate the average terminal elimination rate constant for plasma (λz) using a linear regression technique. The average terminal half-life of plasma (T1/2λz) was determined using the equation T1/2λz = ln2/λz. The average area under the concentration time curve from 0 to 120 h (AUC0–120h) was calculated using the linear-log trapezoidal method. To account for total drug exposure, the average area under the plasma concentration time curve and area under the first moment curve were extrapolated from the first measurement to infinite time (AUC0-∞ and AUMC0-∞, respectively). All plasma SA concentrations were above LLOQ. Time versus concentration figures were produced using a commercially available software (GraphPad Prism 9.0).

      Estimation of Milk Withdrawal Interval

      A milk WDI for SA was estimated using the US FDA Milk Discard App in R (version 1.1.0, R Studio) adapting methods from
      • Smith J.S.
      • Marmulak T.L.
      • Angelos J.A.
      • Lin Z.
      • Rowe J.D.
      • Carlson J.L.
      • Shelver W.L.
      • Lee E.A.
      • Tell L.A.
      Pharmacokinetic parameters and estimated milk withdrawal intervals for domestic goats (Capra aegagrus hircus) after administration of single and multiple intravenous and subcutaneous doses of flunixin meglumine.
      and
      • Meira Jr., E. B. de S.
      • Wiloch E.E.
      • Nixon E.
      • Yeatts J.L.
      • Sheela F.F.
      • Smith G.W.
      • Baynes R.E.
      The pharmacokinetics of transdermal flunixin in lactating dairy goats.
      . A WDI was calculated for a whole-herd treatment scenario and an individual animal entering the bulk tank. In the whole-herd treatment scenario, the tolerance in the application was set to the LOD of 0.0025 µg/mL, as we observed no established tolerance for SA in milk in the United States. In the second scenario, a tolerance limit of 0.0075 µg/mL was used for the individual animal WDI, where milk enters a commingled bulk tank. This tolerance limit was 3 times the assay LOD and is based on the allowed correction set forth by the FDA (
      • US Food and Drug Administration, Center for Veterinary Medicine (US FDA CVM)
      CVM Guidance for Industry (GFI) #3: General principles for evaluating the human food safety of new animal drugs used in food-producing animals.
      ) which assumes that no more than one-third of the milk in a bulk tank will come from treated cows. To satisfy linearity and homoscedasticity assumptions, certain time points were excluded from the analysis per the US FDA guidelines (
      • Smith J.S.
      • Marmulak T.L.
      • Angelos J.A.
      • Lin Z.
      • Rowe J.D.
      • Carlson J.L.
      • Shelver W.L.
      • Lee E.A.
      • Tell L.A.
      Pharmacokinetic parameters and estimated milk withdrawal intervals for domestic goats (Capra aegagrus hircus) after administration of single and multiple intravenous and subcutaneous doses of flunixin meglumine.
      ). For cows 1, 4, and 9, time points 10, 15, 24, and 34 h after the second ASA dose were used. For cow 2, time points 10, 15, 24, and 39 h after the second ASA dose were used. For cows 3, 7, and 10, time points 10, 15, and 24 h after the second ASA dose were used. For cows 5, 6, and 8, time points 10, 15, and 87 h after the second ASA dose were used.
      The application was set to estimate a milk WDI using the methods described in the US FDA Guidance for Industry #3: General Principles for Evaluation the Human Food Safety of New Animal Drugs Used in Food-Producing Animals (US FDA CVM, 2018). A 99th percentile tolerance limit with a 95% confidence was used. The US FDA Milk Discard App requires a minimum of 10 animals with samples analyzed in triplicate. To satisfy this requirement, Monte Carlo simulation was performed in Excel using the mean concentrations and the standard deviations from each time point to generate 2 replicate values. Samples with concentrations above the LOD were used in the model, including 2 samples that had concentrations between the LOD and LLOQ. Six samples had concentrations below the LOD; these were not included in the milk withdrawal analysis or when calculating concentration means and standard deviations for Monte Carlo simulation. The concentrations of these 6 samples were recorded as 0 µg/mL.

      Prostaglandin E2 Metabolite Analysis

      Plasma PGEM concentrations were determined using a commercially available competitive ELISA (Prostaglandin E2 Metabolite ELISA Kit; Cayman Chemical). The protocol supplied by the manufacturer was followed except for the following modifications: (1) during sample purification, samples were centrifuged for 5 min at 3,000 × g and 4°C; (2) 300-µL sample volume and corresponding buffer volumes were used for derivatization; (3) the ethyl acetate extraction step was not performed. The inter-assay CV and intra-assay CV were 9.26 and 18.7%, respectively. The average LLOQ was 1.80 pg/mL. Data were analyzed using a commercially available data analysis tool (MyAssays Desktop). Average PGEM concentrations, average percent changes in PGEM concentrations from the baseline, and the associated standard deviations were calculated using Microsoft Excel. The 80% inhibition concentration of plasma SA was determined using a nonlinear regression technique (GraphPad Prism 9.0).

      RESULTS

      No adverse events were noted in any of the cows throughout the study period.
      The log-transformed mean values for milk and plasma SA concentrations versus time are shown in Figure 1; plasma SA T1/2λz is reported as the harmonic mean. Pharmacokinetic parameters are summarized in Table 1.
      Figure thumbnail gr1
      Figure 1Log-transformed average salicylic acid (SA) concentrations in plasma and milk in 10 postpartum Holstein dairy cattle following oral administration of acetylsalicylic acid at 200 mg/kg of BW. Acetylsalicylic acid was administered after the 0- and 24-h time points. Due to the logarithmic scale, no average concentration icon is shown for time points at which the average concentration was 0 µg/mL. Error bars represent 95% confidence intervals. Error bars were not shown if they were smaller than the average concentration icon or had a value of zero. Data points below the limit of detection were not included. No SA was detected in any milk samples at 0, 72, 82, 96, and 106 h.
      Table 1Pharmacokinetic (PK) parameters of salicylic acid in plasma and milk in 10 postpartum Holstein dairy cattle following 2 oral administrations of acetylsalicylic acid at 200 mg/kg of BW
      Acetylsalicylic acid was administered at 0 and 24 h; plasma and milk were collected through 120 h for PK analysis.
      Item
      Pharmacokinetic results are presented as the geometric mean (minimum–maximum), except for half-life, which is presented as the harmonic mean (minimum–maximum); Cmax = maximum plasma concentration; Tmax = time to Cmax; AUC0–120 = area under the curve for the 120 h after the first treatment; AUC0-∞ = AUC extrapolated to infinite time; AUC % = portion of the AUC0-∞ that is extrapolated after the final concentration measurement; AUMC0-∞ = area under the first moment curve extrapolated to infinite time; λz = slope of the terminal phase; and T1/2λz = terminal half-life.
      PlasmaMilk
      Cmax (μg/mL)96.637 (50.412–139.577)0.229 (0.158–0.337)
      Tmax (h)2.4 (2.0–6.0)
      AUC0–120 (h × μg/mL)977.17 (784.56–1,403.51)
      AUC0-∞ (h × μg/mL)978.38 (786.16–1,403.99)
      AUC % extrapolated0.13 (0.03–0.25)
      AUMC0-∞ (μg/mL × h2)13,211.85 (10,046.87–17,965.85)
      λz (per hour)0.061 (0.054–0.071)
      T1/2λz (h)11.49 (9.70–12.79)
      1 Acetylsalicylic acid was administered at 0 and 24 h; plasma and milk were collected through 120 h for PK analysis.
      2 Pharmacokinetic results are presented as the geometric mean (minimum–maximum), except for half-life, which is presented as the harmonic mean (minimum–maximum); Cmax = maximum plasma concentration; Tmax = time to Cmax; AUC0–120 = area under the curve for the 120 h after the first treatment; AUC0-∞ = AUC extrapolated to infinite time; AUC % = portion of the AUC0-∞ that is extrapolated after the final concentration measurement; AUMC0-∞ = area under the first moment curve extrapolated to infinite time; λz = slope of the terminal phase; and T1/2λz = terminal half-life.
      Salicylic acid was present in the plasma of all cows at 0 h at an average concentration of 0.1 µg/mL. Following oral administration, plasma SA was present above LLOQ in all cows through 96 h after the last treatment; average SA concentrations were similar to baseline concentrations at 48 h after the last treatment and below baseline concentrations at 72 and 96 h after the last treatment. Secondary SA peaks were observed at 58 and 82 h after the last treatment.
      Salicylic acid in the milk of all cows was undetectable at 0 h. Following oral administration, SA was present above the LLOQ in the milk of all cows through 15 h from the last treatment and was undetected by 48 h after the last treatment. A secondary SA peak was observed at 87 h after the last treatment in 3 cows (average concentration across all cows: 0.0019 µg/mL; average concentration for the 3 cows with detectable SA: 0.0063 µg/mL). In one cow, SA was undetected at 34 h after the last treatment but was detected again at low levels at 39 h. The estimated milk WDI using the US FDA application was determined to be 156 h after the last treatment in the whole-herd treatment scenario and 120 h for the second scenario, in which milk from an individually treated cow is diluted in the bulk tank (Figure 2).
      Figure thumbnail gr2
      Figure 2Two milk withdrawal interval (WDI) models for salicylic acid (SA) in postpartum dairy cattle after the last of 2 administrations of acetylsalicylic acid at 200 mg/kg of BW, 24 h apart. Open circles represent actual data points included in the models. Vertical black dotted lines represent the estimated WDI. (A) The first withdrawal calculation was based on a whole-herd treatment scenario where the codified tolerance was equal to the limit of detection of 0.0025 µg/mL for the marker residue SA in milk. The estimated WDI for this scenario was 156 h after the last treatment. (B) The second withdrawal calculation included a 3× correction factor for the limit of detection to account for milk from individually treated cows entering a bulk tank; the codified tolerance was equal to 0.0075 µg/mL SA. The estimated WDI for this scenario was 120 h after the last treatment. Figure created with BioRender.com (version 1.1.0).
      Plasma PGEM data are summarized in Table 2; percent changes in PGEM concentrations are shown in Figure 3. The 80% inhibitory concentration (IC80) for plasma SA was determined to be 67 µg/mL.
      Table 2Plasma prostaglandin E2 metabolite (PGEM) concentrations in 10 postpartum Holstein dairy cattle following 2 oral administrations of acetylsalicylic acid at 200 mg/kg of BW
      Acetylsalicylic acid was administered at 0 and 24 h; plasma was collected through 120 h for PGEM analysis.
      PGEM concentrationChange in PGEM concentration from baseline
      Time (h)Mean ± SD (μ/mL)Time (h)Mean ± SD (%)
      0106.11 ± 73.890
      235.33 ± 20.372−49.31 ± 54.75
      657.97 ± 32.326−18.87 ± 84.48
      1084.09 ± 49.991013.03 ± 129.55
      2482.23 ± 56.552412.20 ± 145.86
      1 Acetylsalicylic acid was administered at 0 and 24 h; plasma was collected through 120 h for PGEM analysis.
      Figure thumbnail gr3
      Figure 3Average percent changes in plasma prostaglandin E2 metabolite (PGEM) concentrations compared with baseline (0 h) values in 10 postpartum Holstein dairy cattle following the first of 2 oral administrations of acetylsalicylic acid at 200 mg/kg of BW. Error bars represent standard deviations. The dotted line represents no change from baseline.

      DISCUSSION

      The main findings of the present study were as follows: (1) plasma SA reached an average peak concentration of 96.6 µg/mL at 2.4 h after ASA administration; (2) milk SA reached an average peak concentration of 0.23 µg/mL; (3) milk withdrawal modeling indicated SA levels were below the tolerance limit by 156 h after final ASA treatment, considering whole-herd treatment, and below the tolerance limit by 120 h after the final ASA treatment, considering individually treated animals and a bulk tank factor adjustment; (4) plasma PGEM concentrations were reduced for up to 12 h after ASA administration; (5) the IC80 of SA in plasma was 67 µg/mL.
      The
      • US Food and Drug Administration, Center for Veterinary Medicine (US FDA CVM)
      Guidance for Industry #207: Studies to evaluate the metabolism and residue kinetics of veterinary drugs in food-producing animals: Marker residue depletion studies to establish product withdrawal periods.
      recommends that 20 dairy animals be used for milk residue studies for establishing WDI for veterinary drugs approved for use in food-producing animals. However, based on the existing literature describing ASA pharmacokinetics in nonlactating dairy cattle, the widespread use of ASA in the dairy industry and in recent research, and the relative safety of ASA (
      • Damian P.
      • Craigmill A.L.
      • Riviere J.E.
      Extralabel use of nonsteroidal anti-inflammatory drugs.
      ), we elected to enroll 10 cows.
      Salicylic acid has been detected in the plasma of cows who have not been treated with ASA, likely due to consumption of forages, which contain salicylates (
      • Gingerich D.A.
      • Baggot J.D.
      • Yeary R.A.
      Pharmacokinetics and dosage of aspirin in cattle.
      ;
      • Anderson J.G.
      • Oehme F.W.
      • Moore W.E.
      The effect of oral salicylate (aspirin) on hematologic and biochemical profiles of dairy cattle.
      ).
      • Gingerich D.A.
      • Baggot J.D.
      • Yeary R.A.
      Pharmacokinetics and dosage of aspirin in cattle.
      state that salicyluric acid is normally found in the urine and milk of cows. The plasma SA concentrations we observed before ASA administration were lower than previously reported values (
      • Gingerich D.A.
      • Baggot J.D.
      • Yeary R.A.
      Pharmacokinetics and dosage of aspirin in cattle.
      ), potentially due to differences in diet composition or SA detection methods.
      • Anderson J.G.
      • Oehme F.W.
      • Moore W.E.
      The effect of oral salicylate (aspirin) on hematologic and biochemical profiles of dairy cattle.
      reported detection of SA before ASA administration, but did not indicate the SA concentration. However, visual comparison of the SA concentration graphs suggests that the baseline SA concentrations detected by
      • Anderson J.G.
      • Oehme F.W.
      • Moore W.E.
      The effect of oral salicylate (aspirin) on hematologic and biochemical profiles of dairy cattle.
      were lower than those detected by
      • Gingerich D.A.
      • Baggot J.D.
      • Yeary R.A.
      Pharmacokinetics and dosage of aspirin in cattle.
      . In the study by
      • Gingerich D.A.
      • Baggot J.D.
      • Yeary R.A.
      Pharmacokinetics and dosage of aspirin in cattle.
      , dietary inclusion of forages with higher salicylate content than the diets in the current study may have been responsible for the higher SA levels detected before ASA administration. Both
      • Gingerich D.A.
      • Baggot J.D.
      • Yeary R.A.
      Pharmacokinetics and dosage of aspirin in cattle.
      and
      • Anderson J.G.
      • Oehme F.W.
      • Moore W.E.
      The effect of oral salicylate (aspirin) on hematologic and biochemical profiles of dairy cattle.
      used a method of extraction and spectrophotometry to quantify plasma salicylate and were able to detect concentrations at approximately 5 µg/mL or greater. Delayed analysis (
      • MacDonald R.P.
      • Boutwell J.H.
      • Wilkes W.
      • Slifer E.D.
      • Solow E.B.
      • Stern M.
      Salicylate.
      ) or the presence of interfering substances in the samples could have altered the values reported by
      • Gingerich D.A.
      • Baggot J.D.
      • Yeary R.A.
      Pharmacokinetics and dosage of aspirin in cattle.
      , but these possibilities cannot be confirmed. The method employed by
      • Gingerich D.A.
      • Baggot J.D.
      • Yeary R.A.
      Pharmacokinetics and dosage of aspirin in cattle.
      and
      • Anderson J.G.
      • Oehme F.W.
      • Moore W.E.
      The effect of oral salicylate (aspirin) on hematologic and biochemical profiles of dairy cattle.
      was likely much less sensitive than the method implemented in the current study and therefore may not have been able to detect SA at the levels reported in this study. In the present study, ultra performance liquid chromatography triple quadrupole MS (UPLC-MS/MS) was used to quantify plasma SA; the LOD and LLOQ were 0.0025 µg/mL and 0.0043 µg/mL, respectively. Improving assay sensitivity is important for providing more accurate drug pharmacokinetic profiles, which can inform decisions such as drug dosing protocols and withdrawal recommendations.
      The secondary peaks observed in milk and plasma may be due to environmental contamination by urine or feces containing SA, consumption of salicylate-containing forages, or both (
      • Gingerich D.A.
      • Baggot J.D.
      • Yeary R.A.
      Pharmacokinetics and dosage of aspirin in cattle.
      ;
      • Anderson J.G.
      • Oehme F.W.
      • Moore W.E.
      The effect of oral salicylate (aspirin) on hematologic and biochemical profiles of dairy cattle.
      ;
      • Bates J.L.
      • Karriker L.A.
      • Rajewski S.M.
      • Lin Z.
      • Gehring R.
      • Li M.
      • Riviere J.E.
      • Coetzee J.F.
      A study to assess the correlation between plasma, oral fluid and urine concentrations of flunixin meglumine with the tissue residue depletion profile in finishing-age swine.
      ). Considering that the secondary peaks in plasma were consistently observed at the evening milkings—after the morning feeding time—they are perhaps more likely due to forage consumption than environmental contamination and subsequent drug transfer. Even though drug transfer has been reported for other NSAID in swine and horses (
      • Popot M.A.
      • Menaut L.
      • Boyer S.
      • Bonnaire Y.
      • Toutain P.L.
      Spurious urine excretion drug profile in the horse due to bedding contamination and drug recycling: The case of meclofenamic acid.
      ;
      • Hairgrove T.B.
      • Mask J.W.
      • Mays T.P.
      • Fajt V.R.
      • Bentke A.L.
      • Warner J.L.
      • Baynes R.E.
      Detection of flunixin in the urine of untreated pigs housed with pigs treated with flunixin meglumine at labeled doses.
      ;
      • Bates J.L.
      • Karriker L.A.
      • Rajewski S.M.
      • Lin Z.
      • Gehring R.
      • Li M.
      • Riviere J.E.
      • Coetzee J.F.
      A study to assess the correlation between plasma, oral fluid and urine concentrations of flunixin meglumine with the tissue residue depletion profile in finishing-age swine.
      ), acetylsalicylic acid resulting in detectable plasma or milk SA concentrations in dairy cattle has no drug transfer data.
      • Anderson J.G.
      • Oehme F.W.
      • Moore W.E.
      The effect of oral salicylate (aspirin) on hematologic and biochemical profiles of dairy cattle.
      did report urine salicylate concentrations in cattle for 84 h following ASA administration, so it is possible that urinary contamination of the environment resulted in the observed rebound SA concentrations. This would help explain the relatively high rebound milk SA levels at 96 h after the last treatment. The smaller secondary peaks we observed in milk at 39 and 87 h after the last treatment are more easily explained by forage consumption. Future studies could be designed to determine the exact reason for these late secondary SA peaks. This information could guide the actions of regulatory officials if low SA residues are detected in milk samples of cows reportedly meeting ASA withdrawal recommendations.
      A comparison of plasma salicylate PK profiles in 7 publications is presented in Table 3. These previous studies investigated the PK profiles of SS following a variety of treatment regimens and used different quantification methods than in the present study. The 2 earliest publications (
      • Gingerich D.A.
      • Baggot J.D.
      • Yeary R.A.
      Pharmacokinetics and dosage of aspirin in cattle.
      ;
      • Anderson J.G.
      • Oehme F.W.
      • Moore W.E.
      The effect of oral salicylate (aspirin) on hematologic and biochemical profiles of dairy cattle.
      ) used extraction and spectrophotometry to determine salicylate concentrations, whereas some later studies have used fluorescence polarization immunoassay (
      • Coetzee J.F.
      • Gehring R.
      • Bettenhausen A.C.
      • Lubbers B.V.
      • Toerber S.E.
      • Thomson D.U.
      • Kukanich B.
      • Apley M.D.
      Attenuation of acute plasma cortisol response in calves following intravenous sodium salicylate administration prior to castration.
      ;
      • Kotschwar J.L.
      • Coetzee J.F.
      • Anderson D.E.
      • Gehring R.
      • KuKanich B.
      • Apley M.D.
      Analgesic efficacy of sodium salicylate in an amphotericin B induced bovine synovitis-arthritis model.
      ;
      • Baldridge S.L.
      • Coetzee J.F.
      • Dritz S.S.
      • Reinbold J.B.
      • Gehring R.
      • Havel J.
      • Kukanich B.
      Pharmacokinetics and physiologic effects of intramuscularly administered xylazine hydrochloride-ketamine hydrochloride-butorphanol tartrate alone or in combination with orally administered sodium salicylate on biomarkers of pain in Holstein calves following castration and dehorning.
      ;
      • Bergamasco L.
      • Coetzee J.F.
      • Gehring R.
      • Murray L.
      • Song T.
      • Mosher R.A.
      Effect of intravenous sodium salicylate administration prior to castration on plasma cortisol and electroencephalography parameters in calves.
      ). These methods of determining SA concentrations have higher LOD than the method described in this study; the results reported by
      • Gingerich D.A.
      • Baggot J.D.
      • Yeary R.A.
      Pharmacokinetics and dosage of aspirin in cattle.
      suggest that the extraction protocol had a sensitivity around 5 µg/mL, which was also the LOD of the fluorescence polarization immunoassay used in the later studies. The low LOD of the method used in the present study improves the resolution and utility of the PK data. In the previous studies describing the PK profile of salicylate, the drug administered was often different (SS versus SA), doses administered were smaller (50 or 100 mg/kg of BW), or the doses were administered via a different route (e.g., intravenous, free-choice through the water) than in the present study. The more recent studies investigating the effects of aspirin (
      • Farney J.K.
      • Mamedova L.K.
      • Coetzee J.F.
      • Kukanich B.
      • Sordillo L.M.
      • Stoakes S.K.
      • Minton J.E.
      • Hollis L.C.
      • Bradford B.J.
      Anti-inflammatory salicylate treatment alters the metabolic adaptations to lactation in dairy cattle.
      ,
      • Farney J.K.
      • Mamedova L.K.
      • Coetzee J.F.
      • Minton J.E.
      • Hollis L.C.
      • Bradford B.J.
      Sodium salicylate treatment in early lactation increases whole-lactation milk and milk fat yield in mature dairy cows.
      ;
      • Carpenter A.J.
      • Ylioja C.M.
      • Vargas C.F.
      • Mamedova L.K.
      • Mendonça L.G.
      • Coetzee J.F.
      • Hollis L.C.
      • Gehring R.
      • Bradford B.J.
      Hot topic: Early postpartum treatment of commercial dairy cows with non-steroidal anti-inflammatory drugs increases whole-lactation milk yield.
      ;
      • Barragan A.A.
      • Bauman L.M.
      • da Costa L.
      • Velez J.
      • Gonzalez J.D.R.
      • Schuenemann G.M.
      • Menichetti B.
      • Piñeiro J.
      • Bas S.
      Administration of acetylsalicylic acid after parturition in lactating dairy cows under certified organic management: Part I. Milk yield, milk components, activity patterns, fertility, and health.
      ,
      • Barragan A.A.
      • Bauman L.M.
      • Schuenemann G.M.
      • Velez J.
      • Lakritz J.
      • Coetzee J.F.
      • Gonzalez J.D.R.
      • Piñeiro J.M.
      • Menichetti B.
      • Bas S.
      Administration of acetylsalicylic acid after parturition in lactating dairy cows under certified organic management: Part II. Biomarkers of nociception, inflammation, and stress.
      ,
      • Barragan A.A.
      • Hovingh E.
      • Bas S.
      • Lakritz J.
      • Byler L.
      • Ludwikowski A.
      • Takitch S.
      • Zug J.
      • Hann S.
      Effects of postpartum acetylsalicylic acid on metabolic status, health, and production in lactating dairy cattle.
      ,
      • Barragan A.A.
      • Bas S.
      • Hovingh E.
      • Byler L.
      Effects of postpartum acetylsalicylic acid on uterine diseases and reproductive performance in dairy cattle.
      ) have not described PK profiles of SA.
      Table 3Comparison of the plasma pharmacokinetic parameters of salicylate between the present study and 6 previous studies
      StudyItem
      Cmax = maximum plasma concentration; Tmax = time to Cmax; AUC0-∞ = area under the curve extrapolated to infinite time; T1/2 λz = terminal half-life; N/A = not applicable.
      Cmax (μg/mL)Tmax (h)AUC0-∞ (h x μg/mL)T1/2 λz (h)
      Current study
      Aspirin (acetylsalicylic acid) administered at 200 mg/kg of BW at 0 and 24 h. Pharmacokinetic results are presented as the geometric mean (minimum–maximum), except for half-life, which is presented as the harmonic mean (minimum–maximum).
      96.637 (50.412–139.577)2.4 (2.0–6.0)978.38 (786.16–1,403.99)11.49 (9.70–12.79)
      • Gingerich D.A.
      • Baggot J.D.
      • Yeary R.A.
      Pharmacokinetics and dosage of aspirin in cattle.
      Cmax value is based on results from the multiple dose oral aspirin experiment (100 mg/kg of BW every 12 h for 5 d). This value was visually estimated from Figure 5 in Gingerich et al. (1975). The Tmax and T1/2 values are based on results from the single-dose oral aspirin experiment (100 mg/kg of BW). Standard deviation is listed with T1/2 value. Salicylate concentrations determined using an extraction and spectrophotometry protocol.
      632–4N/A3.7 ± 0.44
      • Anderson J.G.
      • Oehme F.W.
      • Moore W.E.
      The effect of oral salicylate (aspirin) on hematologic and biochemical profiles of dairy cattle.
      Sodium salicylate (SS) administered orally at 100 mg/kg of BW at 0, 12, 25, and 36 h. Cmax value was visually estimated from Figure 1 in Anderson et al. (1979). Salicylate concentrations determined using an extraction and spectrophotometry protocol.
      1172–8N/A5.8
      • Coetzee J.F.
      • Gehring R.
      • Bettenhausen A.C.
      • Lubbers B.V.
      • Toerber S.E.
      • Thomson D.U.
      • Kukanich B.
      • Apley M.D.
      Attenuation of acute plasma cortisol response in calves following intravenous sodium salicylate administration prior to castration.
      SS administered intravenously at 50 mg/kg of BW immediately before castration. Results are presented as mean ± SE, except Cmax, which was estimated visually from Figure 1 in Coetzee et al. (2007). Salicylate concentrations determined using fluorescence polarization immunoassay.
      200–230N/A219.30 ± 18.230.63 ± 0.04
      • Coetzee J.F.
      • Gehring R.
      • Bettenhausen A.C.
      • Lubbers B.V.
      • Toerber S.E.
      • Thomson D.U.
      • Kukanich B.
      • Apley M.D.
      Attenuation of acute plasma cortisol response in calves following intravenous sodium salicylate administration prior to castration.
      Aspirin (acetylsalicylic acid) administered orally at 50 mg/kg of BW immediately before castration. Results were extracted from the discussion.
      ≤10N/AN/AN/A
      • Kotschwar J.L.
      • Coetzee J.F.
      • Anderson D.E.
      • Gehring R.
      • KuKanich B.
      • Apley M.D.
      Analgesic efficacy of sodium salicylate in an amphotericin B induced bovine synovitis-arthritis model.
      SS administered intravenously at 50 mg/kg of BW 4 min after lameness induction. Results are presented as mean ± SE. Salicylate concentrations determined using fluorescence polarization immunoassay.
      226.9 ± 4.9N/A201.65 ± 8.500.62 ± 0.02
      • Baldridge S.L.
      • Coetzee J.F.
      • Dritz S.S.
      • Reinbold J.B.
      • Gehring R.
      • Havel J.
      • Kukanich B.
      Pharmacokinetics and physiologic effects of intramuscularly administered xylazine hydrochloride-ketamine hydrochloride-butorphanol tartrate alone or in combination with orally administered sodium salicylate on biomarkers of pain in Holstein calves following castration and dehorning.
      SS administered for 5 d surrounding the time of castration and dehorning via free-choice consumption of water containing SS (SS concentrations in water: 2.5–5 mg/mL). Results are presented as mean ± SEM (SEM not reported for Tmax). Salicylate concentrations determined using fluorescence polarization immunoassay.
      61.134 ± 10.31241.782.05 ± 14.27N/A
      • Bergamasco L.
      • Coetzee J.F.
      • Gehring R.
      • Murray L.
      • Song T.
      • Mosher R.A.
      Effect of intravenous sodium salicylate administration prior to castration on plasma cortisol and electroencephalography parameters in calves.
      SS administered intravenously at 50 mg/kg of BW immediately before castration. Results are presented as mean ± SEM. Salicylate concentrations determined using fluorescence polarization immunoassay.
      198.720 ± 8.220N/A192.73 ± 21.220.68 ± 0.08
      1 Cmax = maximum plasma concentration; Tmax = time to Cmax; AUC0-∞ = area under the curve extrapolated to infinite time; T1/2 λz = terminal half-life; N/A = not applicable.
      2 Aspirin (acetylsalicylic acid) administered at 200 mg/kg of BW at 0 and 24 h. Pharmacokinetic results are presented as the geometric mean (minimum–maximum), except for half-life, which is presented as the harmonic mean (minimum–maximum).
      3 Cmax value is based on results from the multiple dose oral aspirin experiment (100 mg/kg of BW every 12 h for 5 d). This value was visually estimated from Figure 5 in
      • Gingerich D.A.
      • Baggot J.D.
      • Yeary R.A.
      Pharmacokinetics and dosage of aspirin in cattle.
      . The Tmax and T1/2 values are based on results from the single-dose oral aspirin experiment (100 mg/kg of BW). Standard deviation is listed with T1/2 value. Salicylate concentrations determined using an extraction and spectrophotometry protocol.
      4 Sodium salicylate (SS) administered orally at 100 mg/kg of BW at 0, 12, 25, and 36 h. Cmax value was visually estimated from Figure 1 in
      • Anderson J.G.
      • Oehme F.W.
      • Moore W.E.
      The effect of oral salicylate (aspirin) on hematologic and biochemical profiles of dairy cattle.
      . Salicylate concentrations determined using an extraction and spectrophotometry protocol.
      5 SS administered intravenously at 50 mg/kg of BW immediately before castration. Results are presented as mean ± SE, except Cmax, which was estimated visually from Figure 1 in
      • Coetzee J.F.
      • Gehring R.
      • Bettenhausen A.C.
      • Lubbers B.V.
      • Toerber S.E.
      • Thomson D.U.
      • Kukanich B.
      • Apley M.D.
      Attenuation of acute plasma cortisol response in calves following intravenous sodium salicylate administration prior to castration.
      . Salicylate concentrations determined using fluorescence polarization immunoassay.
      6 Aspirin (acetylsalicylic acid) administered orally at 50 mg/kg of BW immediately before castration. Results were extracted from the discussion.
      7 SS administered intravenously at 50 mg/kg of BW 4 min after lameness induction. Results are presented as mean ± SE. Salicylate concentrations determined using fluorescence polarization immunoassay.
      8 SS administered for 5 d surrounding the time of castration and dehorning via free-choice consumption of water containing SS (SS concentrations in water: 2.5–5 mg/mL). Results are presented as mean ± SEM (SEM not reported for Tmax). Salicylate concentrations determined using fluorescence polarization immunoassay.
      9 SS administered intravenously at 50 mg/kg of BW immediately before castration. Results are presented as mean ± SEM. Salicylate concentrations determined using fluorescence polarization immunoassay.
      The greater average Cmax reported here compared with that reported by
      • Gingerich D.A.
      • Baggot J.D.
      • Yeary R.A.
      Pharmacokinetics and dosage of aspirin in cattle.
      may be appropriate, considering the higher target dose used in this study. However, the multiple dose aspirin experiment conducted by
      • Gingerich D.A.
      • Baggot J.D.
      • Yeary R.A.
      Pharmacokinetics and dosage of aspirin in cattle.
      had minimal blood sampling times, so the peak concentration values presented in the figures may not be an accurate representation of the true plasma salicylate Cmax. In a study conducted by
      • Anderson J.G.
      • Oehme F.W.
      • Moore W.E.
      The effect of oral salicylate (aspirin) on hematologic and biochemical profiles of dairy cattle.
      , 3 nonlactating female Holstein cows were administered SA orally at 100 mg/kg of BW at 0, 12, 25, and 36 h; the plasma salicylate Cmax value was similar to the values reported in this study. Considering the similarity between treatment regimens in the study conducted by
      • Gingerich D.A.
      • Baggot J.D.
      • Yeary R.A.
      Pharmacokinetics and dosage of aspirin in cattle.
      and
      • Anderson J.G.
      • Oehme F.W.
      • Moore W.E.
      The effect of oral salicylate (aspirin) on hematologic and biochemical profiles of dairy cattle.
      , it is likely that blood collection in the study by
      • Gingerich D.A.
      • Baggot J.D.
      • Yeary R.A.
      Pharmacokinetics and dosage of aspirin in cattle.
      was not properly timed to observe the plasma salicylate peaks, and a lower average Cmax was thus observed.
      • Baldridge S.L.
      • Coetzee J.F.
      • Dritz S.S.
      • Reinbold J.B.
      • Gehring R.
      • Havel J.
      • Kukanich B.
      Pharmacokinetics and physiologic effects of intramuscularly administered xylazine hydrochloride-ketamine hydrochloride-butorphanol tartrate alone or in combination with orally administered sodium salicylate on biomarkers of pain in Holstein calves following castration and dehorning.
      reported a lower mean Cmax value than that reported here, which is most likely due to differences in dosage route: free-choice SA was administered in water at a concentration between 2.5 and 5 mg/mL, with doses ranging from 13.62 to 151.99 mg/kg. The higher Cmax values reported by
      • Coetzee J.F.
      • Gehring R.
      • Bettenhausen A.C.
      • Lubbers B.V.
      • Toerber S.E.
      • Thomson D.U.
      • Kukanich B.
      • Apley M.D.
      Attenuation of acute plasma cortisol response in calves following intravenous sodium salicylate administration prior to castration.
      ; intravenous SS group),
      • Kotschwar J.L.
      • Coetzee J.F.
      • Anderson D.E.
      • Gehring R.
      • KuKanich B.
      • Apley M.D.
      Analgesic efficacy of sodium salicylate in an amphotericin B induced bovine synovitis-arthritis model.
      , and
      • Bergamasco L.
      • Coetzee J.F.
      • Gehring R.
      • Murray L.
      • Song T.
      • Mosher R.A.
      Effect of intravenous sodium salicylate administration prior to castration on plasma cortisol and electroencephalography parameters in calves.
      are reasonable, considering that SS was administered intravenously. In Holstein calves administered oral aspirin at 50 mg/kg of BW immediately before castration, plasma salicylate levels did not exceed 10 µg/mL (
      • Coetzee J.F.
      • Gehring R.
      • Bettenhausen A.C.
      • Lubbers B.V.
      • Toerber S.E.
      • Thomson D.U.
      • Kukanich B.
      • Apley M.D.
      Attenuation of acute plasma cortisol response in calves following intravenous sodium salicylate administration prior to castration.
      ). Overall, the differences in Cmax among the different studies likely reflect the variation in dosing and the timing of sample collection. Although AUC is a better indicator of drug exposure than Cmax, it was less frequently reported. Future research should focus on reporting AUC values to more accurately compare drug exposure differences among studies.
      The Tmax reported in this study is similar to the values reported by
      • Gingerich D.A.
      • Baggot J.D.
      • Yeary R.A.
      Pharmacokinetics and dosage of aspirin in cattle.
      and
      • Anderson J.G.
      • Oehme F.W.
      • Moore W.E.
      The effect of oral salicylate (aspirin) on hematologic and biochemical profiles of dairy cattle.
      ; any numerical differences are likely due to random variation or different sampling times. The Tmax reported by
      • Baldridge S.L.
      • Coetzee J.F.
      • Dritz S.S.
      • Reinbold J.B.
      • Gehring R.
      • Havel J.
      • Kukanich B.
      Pharmacokinetics and physiologic effects of intramuscularly administered xylazine hydrochloride-ketamine hydrochloride-butorphanol tartrate alone or in combination with orally administered sodium salicylate on biomarkers of pain in Holstein calves following castration and dehorning.
      is significantly longer, which may be explained by the dosage route (free-choice through the water versus via oral bolus in the present study). Because calves were self-medicating, the timing of peak water intake may have artificially increased Tmax.
      A summary by the Center for Veterinary Medical Products (
      • Center for Veterinary Medical Products (CVMP)
      Acetylsalicylic acid, sodium acetylsalicylate, acetylsalicylic acid dl-lysine and carbasalate calcium: Summary report (1).
      ) reported an absorption half-life of 2.9 h for cattle administered oral aspirin (20 to 100 mg/kg of BW); in the same report, following intravenous administration of ASA dl-lysine to cattle at 90 mg/kg of BW, the T1/2λz was found to be 36.5 min. Comparing the values in Table 3, the T1/2λz reported in the studies evaluating oral administration of ASA or SS are considerably longer than the T1/2λz of salicylate following intravenous administration of SS. The relatively slow absorption of aspirin from the rumen—the absorption half-life is approximately 3 h (
      • Gingerich D.A.
      • Baggot J.D.
      • Yeary R.A.
      Pharmacokinetics and dosage of aspirin in cattle.
      ;
      • Anderson J.G.
      • Oehme F.W.
      • Moore W.E.
      The effect of oral salicylate (aspirin) on hematologic and biochemical profiles of dairy cattle.
      ;
      • Center for Veterinary Medical Products (CVMP)
      Acetylsalicylic acid, sodium acetylsalicylate, acetylsalicylic acid dl-lysine and carbasalate calcium: Summary report (1).
      )—is one potential reason for the prolonged T1/2λz in these studies. Even though a flip-flop phenomenon is possible (the rate of absorption, rather than the rate of elimination, is the limiting step in final drug elimination;
      • Toutain P.L.
      • Bousquet-Mélou A.
      Plasma terminal half-life.
      ), this does not fully explain the greatly extended T1/2λz we observed in the present study. We did observe a much higher T1/2λz in the present study than was reported by
      • Gingerich D.A.
      • Baggot J.D.
      • Yeary R.A.
      Pharmacokinetics and dosage of aspirin in cattle.
      or
      • Anderson J.G.
      • Oehme F.W.
      • Moore W.E.
      The effect of oral salicylate (aspirin) on hematologic and biochemical profiles of dairy cattle.
      . Because the dose used in the present study was double the dose used in the earlier studies, it is possible that a metabolism pathway was saturated and we thus observed a longer T1/2λz. (Salicylate and SA are primarily metabolized through glucuronidation pathways in the liver;
      • Center for Veterinary Medical Products (CVMP)
      Acetylsalicylic acid, sodium acetylsalicylate, acetylsalicylic acid dl-lysine and carbasalate calcium: Summary report (1).
      .) However, the authors feel two other explanations for the extended T1/2λz are more robust. First, improved assay sensitivity may have allowed detection of a secondary terminal phase in the present study.
      • Toutain P.L.
      • Bousquet-Mélou A.
      Plasma terminal half-life.
      have shown that terminal half-life is heavily influenced by assay sensitivity; as assay sensitivity improves, terminal half-life will appear to increase due to detection of late terminal phases. Second, decrease in drug clearance due to altered postpartum physiology may have increased T1/2λz. In a comparison of the pharmacokinetic profiles of meloxicam in postpartum and mid-lactation cows,
      • Gorden P.J.
      • Burchard M.
      • Ydstie J.A.
      • Kleinhenz M.D.
      • Wulf L.W.
      • Rajewski S.J.
      • Wang C.
      • Gehring R.
      • Mochel J.P.
      • Coetzee J.F.
      Comparison of milk and plasma pharmacokinetics of meloxicam in postpartum versus mid-lactation Holstein cows.
      reported that Cmax, Tmax, and mean residence time were increased and clearance was decreased in postpartum cows; the reduction in clearance resulted in an increase in drug exposure and relative bioavailability. In addition, the authors detected higher levels of meloxicam in milk in postpartum cows compared with mid-lactation cows at all time points. However, they did not detect any difference in T1/2λz. Furthermore,
      • Warner R.
      • Ydstie J.A.
      • Wulf L.W.
      • Gehring R.
      • Coetzee J.F.
      • Mochel J.P.
      • Gorden P.J.
      Comparative pharmacokinetics of meloxicam between healthy post-partum vs. mid-lactation dairy cattle.
      reported similar findings in a study evaluating the pharmacokinetics of both intravenous and oral meloxicam in postpartum versus mid-lactation cows. In this study, the authors also reported a significantly longer T1/2λz in the postpartum cows. The authors postulated that these clearance differences could have been due to changes in plasma protein binding or reduced liver enzyme levels in postpartum cows. Considering this evidence for change in NSAID clearance in postpartum cows, it is likely that the postpartum cows used in this study had decreased clearance of SA compared with cows involved in previous studies evaluating PK profiles after oral ASA administration. Because we did not administer ASA intravenously, we were unable to quantify clearance or bioavailability in this study (
      • Toutain P.L.
      • Bousquet-Mélou A.
      Plasma clearance.
      ). The bioavailability of oral ASA in dairy cattle has been reported to be 70% (
      • Gingerich D.A.
      • Baggot J.D.
      • Yeary R.A.
      Pharmacokinetics and dosage of aspirin in cattle.
      ). The present study was also not designed to measure changes in plasma protein, protein binding, or liver enzymes. Further research investigating changes in plasma protein-drug interactions and metabolic enzyme expression in the liver could determine the differences in NSAID pharmacokinetics in postpartum dairy cows compared with cows at other lactation stages. Such research would be useful in optimizing NSAID treatment protocols for cows in different production stages. In summary, increases in assay sensitivity and postpartum decreases in drug clearance in the present study are likely responsible for the longer T1/2λz observed. Future research is needed to compare clearance and bioavailability of SA in postpartum and mid-lactation cows following oral ASA administration.
      No data are currently available on SA in the milk of postpartum dairy cattle, so the data reported here are novel. Postpartum cows were specifically selected as the study population based on recent efforts by
      • Barragan A.A.
      • Bauman L.M.
      • da Costa L.
      • Velez J.
      • Gonzalez J.D.R.
      • Schuenemann G.M.
      • Menichetti B.
      • Piñeiro J.
      • Bas S.
      Administration of acetylsalicylic acid after parturition in lactating dairy cows under certified organic management: Part I. Milk yield, milk components, activity patterns, fertility, and health.
      ,
      • Barragan A.A.
      • Bauman L.M.
      • Schuenemann G.M.
      • Velez J.
      • Lakritz J.
      • Coetzee J.F.
      • Gonzalez J.D.R.
      • Piñeiro J.M.
      • Menichetti B.
      • Bas S.
      Administration of acetylsalicylic acid after parturition in lactating dairy cows under certified organic management: Part II. Biomarkers of nociception, inflammation, and stress.
      ,
      • Barragan A.A.
      • Hovingh E.
      • Bas S.
      • Lakritz J.
      • Byler L.
      • Ludwikowski A.
      • Takitch S.
      • Zug J.
      • Hann S.
      Effects of postpartum acetylsalicylic acid on metabolic status, health, and production in lactating dairy cattle.
      ,
      • Barragan A.A.
      • Bas S.
      • Hovingh E.
      • Byler L.
      Effects of postpartum acetylsalicylic acid on uterine diseases and reproductive performance in dairy cattle.
      ) that describe positive benefits of ASA given at 200 mg/kg in the postpartum period. The use of ASA for an anti-inflammatory indication following parturition would constitute an extra-label drug use (ELDU). Under the guidance of the Animal Medicinal Drug Use Clarification Act, the prescribing veterinarian would be responsible for determining an appropriate WDI (
      • US Food and Drug Administration (US FDA)
      Extralabel Drug Use in Animals, final rule. Food and Drug Administration, Department of Health and Human Services. 21 CFR part 530.
      ). We determined the Cmax of SA in milk to be 0.23 µg/mL. Our findings suggest that a milk withhold period of 120 h (5 d) would be most appropriate for cattle in the immediate postpartum period to meet US FDA tolerance guidelines for unapproved drugs. Our description of the PK and milk residue profiles of SA in lactating dairy cows will help producers make informed treatment decisions and ensure food supply safety. Though our data can inform withdrawal decisions, the methods employed in this study are not practical for field use due to their time-intensive nature. To facilitate SA residue detection in milk, future research should focus on developing a rapid assay. Although aspirin is considered to be of low regulatory concern, a 24-h milk WDI is currently recommended based on potential risk for individuals with Reye's syndrome (
      • Damian P.
      • Craigmill A.L.
      • Riviere J.E.
      Extralabel use of nonsteroidal anti-inflammatory drugs.
      ). However, the risk of Reye's syndrome is correlated with exposure to high doses of sodium acetylsalicylate, which are unlikely to be obtained through consumption of residues in milk (
      • Center for Veterinary Medical Products (CVMP)
      Acetylsalicylic acid, sodium acetylsalicylate, acetylsalicylic acid dl-lysine and carbasalate calcium: Summary report (1).
      ).
      When estimating milk WDI, we included all data above the assay LOD. This decision was based on recent publications describing the issues with defining LLOQ and excluding data that fall below that level (
      • Jelliffe R.W.
      • Schumitzky A.
      • Bayard D.
      • Fu X.
      • Neely M.
      Describing assay precision—Reciprocal of variance is correct, not CV percent.
      ;
      • Woodward A.
      • Whittem T.
      The lower limit of quantification in pharmacokinetic analyses.
      ). In general, assay LLOQ is determined based on an arbitrary threshold for sample variation (CV%; often set at >20%); when measurements lie below this threshold, they are often discarded form the analysis (
      • Jelliffe R.W.
      • Schumitzky A.
      • Bayard D.
      • Fu X.
      • Neely M.
      Describing assay precision—Reciprocal of variance is correct, not CV percent.
      ). However, CV% increases as measurements approach zero, so the establishment of an LLOQ based on this information is erroneous and may exclude valuable data (
      • Jusko W.J.
      Use of pharmacokinetic data below lower limit of quantitation values.
      ;
      • Jelliffe R.W.
      • Schumitzky A.
      • Bayard D.
      • Fu X.
      • Neely M.
      Describing assay precision—Reciprocal of variance is correct, not CV percent.
      ).
      • Woodward A.
      • Whittem T.
      The lower limit of quantification in pharmacokinetic analyses.
      proposed 3 options for handling data below LLOQ: (1) discard data below LLOQ, (2) censor data using maximum likelihood estimates, and (3) use data below LLOQ without adjustment.
      • Keizer R.J.
      • Jansen R.S.
      • Rosing H.
      • Thijssen B.
      • Beijnen J.H.
      • Schellens J.H.M.
      • Huitema A.D.R.
      Incorporation of concentration data below the limit of quantification in population pharmacokinetic analyses.
      reported improved model performance and precision and decreased bias in population PK analyses when using all data above LOD compared with other methods of censoring data below LLOQ. This effect was more pronounced when the proportion of data below LLOQ was larger. In the present study, only 2 milk samples were below LLOQ and above LOD. Compared with a model that excluded data below LLOQ, our final model that included all data above LOD resulted in a longer bulk tank factor WDI (120 vs. 108 h) and a shorter whole-herd WDI (156 vs. 168 h). Two other possible combinations of time points were able to be modeled, but these models had a poorer fit and were thus not selected over the final model.
      Prostaglandin E2, or PGEM, concentrations in postpartum dairy cattle have been minimally reported in the literature.
      • Farney J.K.
      • Mamedova L.K.
      • Coetzee J.F.
      • Kukanich B.
      • Sordillo L.M.
      • Stoakes S.K.
      • Minton J.E.
      • Hollis L.C.
      • Bradford B.J.
      Anti-inflammatory salicylate treatment alters the metabolic adaptations to lactation in dairy cattle.
      reported an elevation in the total concentration of plasma eicosanoids after cessation of oral treatment with sodium salicylate in drinking water, but they did not provide reports on all individual eicosanoids evaluated. prostaglandin E2 as a primary mediatory of inflammation and pain (
      • Myers M.J.
      • Scott M.L.
      • Deaver C.M.
      • Farrell D.E.
      • Yancy H.F.
      Biomarkers of inflammation in cattle determining the effectiveness of anti-inflammatory drugs.
      ), is of particular interest in pharmacodynamic analysis. We observed reduction in PGEM concentrations at plasma SA levels of approximately 700 and 425 µM at 2 and 6 h, respectively.
      • Myers M.J.
      • Scott M.L.
      • Deaver C.M.
      • Farrell D.E.
      • Yancy H.F.
      Biomarkers of inflammation in cattle determining the effectiveness of anti-inflammatory drugs.
      reported that 300 µM of aspirin resulted in reduction in prostaglandin E2 (
      • Myers M.J.
      • Scott M.L.
      • Deaver C.M.
      • Farrell D.E.
      • Yancy H.F.
      Biomarkers of inflammation in cattle determining the effectiveness of anti-inflammatory drugs.
      ). The discrepancy between our results and those of
      • Myers M.J.
      • Scott M.L.
      • Deaver C.M.
      • Farrell D.E.
      • Yancy H.F.
      Biomarkers of inflammation in cattle determining the effectiveness of anti-inflammatory drugs.
      can be explained by the weaker inhibition of cyclooxygenase by SS compared with ASA (
      • Mitchell J.A.
      • Akarasereenont P.
      • Thiemermann C.
      • Flower R.J.
      • Vane J.R.
      Selectivity of nonsteroidal antiinflammatory drugs as inhibitors of constitutive and inducible cyclooxygenase.
      ). However, considering the short half-life of ASA in plasma (
      • Gingerich D.A.
      • Baggot J.D.
      • Yeary R.A.
      Pharmacokinetics and dosage of aspirin in cattle.
      ), the active metabolite SA may play a larger role than ASA in inhibiting prostaglandin E2 production following oral administration of ASA. We observed large standard deviations in both PGEM concentrations and change in PGEM concentrations following ASA administration. Thus, although we did report at least 6 h of prostaglandin inhibition, this result may not be truly representative of the response of postpartum dairy cattle.
      • Wischral A.
      • Verreschi I.T.
      • Lima S.B.
      • Hayashi L.F.
      • Barnabe R.C.
      Pre-parturition profile of steroids and prostaglandin in cows with or without foetal membrane retention.
      reported that, compared with cows without retained fetal membranes, those with retained fetal membranes had significantly higher PGEM concentrations at 24, 48, 72, and 120 h before and 12 h after calving and lower PGEM concentrations 1 h after calving. Even though none of the cows in this study had retained fetal membranes, it is possible that other, undetected health events could have similarly altered PGEM concentrations, resulting in the large standard deviations observed. It is also unclear whether the transient reduction in prostaglandin production reported here is biologically significant. Future research should focus on establishing reference ranges for PGEM concentrations in postpartum dairy cattle and determining the long-term effects of ASA on postpartum inflammation and whole-lactation production and health. Research such as that conducted by
      • Vailati Riboni M.
      • Meier S.
      • Priest N.V.
      • Burke C.R.
      • Kay J.K.
      • McDougall S.
      • Mitchell M.D.
      • Walker C.G.
      • Crookenden M.
      • Heiser A.
      • Roche J.R.
      • Loor J.J.
      Adipose and liver gene expression profiles in response to treatment with a nonsteroidal antiinflammatory drug after calving in grazing dairy cows.
      analyzing adipose and liver gene expression could help elucidate long-term effects of ASA administration and help determine whether the transient inhibition of prostaglandin production reported here is clinically meaningful.
      Literature suggests that IC80 is more closely correlated with analgesia than half-maximal inhibitory concentration (IC50;
      • Huntjens D.R.
      • Danhof M.
      • Della Pasqua O.E.
      Pharmacokinetic-pharmacodynamic correlations and biomarkers in the development of COX-2 inhibitors.
      ); thus, we chose to calculate IC80. The IC80 value of 67 µg/mL determined in the present study is more than twice the 30 µg/mL therapeutic level previously reported (
      • Gingerich D.A.
      • Baggot J.D.
      • Yeary R.A.
      Pharmacokinetics and dosage of aspirin in cattle.
      ).
      • Gingerich D.A.
      • Baggot J.D.
      • Yeary R.A.
      Pharmacokinetics and dosage of aspirin in cattle.
      stated that they selected the 30 µg/mL therapeutic level based on therapeutic minimum levels in humans, the presumed difficulty of achieving greater levels in cattle, and the alleviation of pain observed in 2 of the animals enrolled in the study. In the previous study (
      • Gingerich D.A.
      • Baggot J.D.
      • Yeary R.A.
      Pharmacokinetics and dosage of aspirin in cattle.
      ), no lactating cows were enrolled and no pharmacodynamic evaluation was performed. Lactating and nonlactating dairy cattle likely exhibit different NSAID PK profiles (
      • Gorden P.J.
      • Burchard M.
      • Ydstie J.A.
      • Kleinhenz M.D.
      • Wulf L.W.
      • Rajewski S.J.
      • Wang C.
      • Gehring R.
      • Mochel J.P.
      • Coetzee J.F.
      Comparison of milk and plasma pharmacokinetics of meloxicam in postpartum versus mid-lactation Holstein cows.
      ;
      • Warner R.
      • Ydstie J.A.
      • Wulf L.W.
      • Gehring R.
      • Coetzee J.F.
      • Mochel J.P.
      • Gorden P.J.
      Comparative pharmacokinetics of meloxicam between healthy post-partum vs. mid-lactation dairy cattle.
      ) and the therapeutic level of SA in lactating dairy cattle may differ from that in other cattle. Our analysis of PGEM concentrations and calculation of IC80 is a more objective pharmacodynamic measure than lameness evaluation, which was used by
      • Gingerich D.A.
      • Baggot J.D.
      • Yeary R.A.
      Pharmacokinetics and dosage of aspirin in cattle.
      . Hence, the therapeutic level we report here may be more accurate and reliable than that previously reported. More comprehensive analyses of the pharmacodynamic effects of NSAID in postpartum dairy cattle are needed to refine treatment recommendations for managing postpartum inflammation.

      CONCLUSIONS

      The results of the present study suggest that the current 24-h milk withdrawal recommendation for cattle treated with ASA may require revision to 120 h after ASA treatment. Furthermore, these data suggest that ASA administration transiently reduces prostaglandin production for up to 12 h. Given that aspirin is not approved for use in lactating dairy cattle in the United States, producers must consult with a veterinarian and demonstrate a valid veterinarian-client-patient relationship before initiating use of ASA in postpartum dairy cattle. Furthermore, because aspirin is not approved by the FDA, ELDU requires adherence with Animal Medicinal Drug Use Clarification Act, which stipulates that ELDU is permitted only if the well-being of the animal is threatened, and that ELDU for production purposes is strictly prohibited. Further research should focus on determining the etiology of the secondary SA peaks following ASA administration and expounding on the pharmacodynamics of ASA and its long-term effects on dairy cow health. Evaluating the differences in NSAID pharmacokinetics between postpartum cows and cows at other production stages and determining reference values for PGEM in postpartum dairy cattle may allow for more robust research and refinement of current NSAID treatment protocols.

      ACKNOWLEDGMENTS

      This project was supported by the College of Veterinary Medicine at Kansas State University (Manhattan, KS). Kleinhenz and Coetzee are supported by the Agriculture and Food Research Initiative Competitive, grant nos. 2017-67015-27124, 2020-67030-31479, 2020-67015-31540, 2020-67015-31546, and 2021-67015-34084 from the USDA National Institute of Food and Agriculture (Washington, DC). The authors acknowledge the Boehringer Ingelheim Veterinary Scholars Program and the Kansas State University College of Veterinary Medicine Office of Research for support of Bailey Fritz (Manhattan, KS). The authors thank Conrad Schelkopf and Alyssa Leslie (Kansas State College of Veterinary Medicine) for assistance with sample collection. Special thanks to Mike Scheffel (Kansas State Dairy Unit) and the staff at the Kansas State Dairy Unit (Manhattan, KS) for their assistance with this study. The following is a summary of author responsibility. BRF: sample collection and analysis, method development, manuscript preparation; MDK: study design, funding, sample collection and analysis, manuscript preparation; SRM: sample analysis, method development, manuscript preparation; GM: method development, manuscript preparation; MSM: sample collection, manuscript preparation; MW: sample collection, manuscript preparation; AKC: sample collection, manuscript preparation; JFC: study design, funding, manuscript preparation. The authors have not stated any conflicts of interest.

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