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Objectives: Previous studies investigated the effect of non-steroidal anti-inflammatory drugs (NSAID) on stress and pain in cows with various disease by evaluating behavioral changes and measuring cortisol. The efficacy of NSAID to lower cortisol and reduce behavioral changes has been proven. However, it remains unclear how much the injection effected the stress parameters and if NSAID influenced cortisol only by reducing pain or by effecting the cortisol metabolism itself. Therefore, the aim of this study was to investigate the effect of a single injection of NSAID on fecal cortisol concentration and standing and lying behavior in healthy dairy cows.
Materials and Methods: Twenty-four healthy (i.e., without metabolic or infectious disease or lameness) Holstein-Friesian dairy cows were enrolled 14 – 21d after dry-off in a double-blind, placebo-controlled, crossover trial. Upon enrolment all cows were fitted with a HOBO Pendant G Data Logger (Onset Computer Corporation, Pocasset, MA) to continuously record standing and lying behavior in 1-min intervals. Daily time lying and number of lying bouts was calculated starting with the day after enrolment. Four days after enrolment (t0) cow were fixed in tie stalls and received an intravenous injection in the jugular vein of either NSAID (i.e., 2.2 mg/kg flunixin, 2ml/45kg Paraflunixin RPS, Bayer Animal Health, Monheim, Germany) or placebo (PLA; i.e., 2 ml/45kg normal saline) based on a randomization list. Fecal samples were collected to measure fecal cortisol metabolites (i.e., 11,17 dioxoandrostanes; DOA). DOA measurement is a well-established and feedback free method (i.e., minimal stress due to restraining and handling) to quantify stress in cows. Fecal samples were obtained from the rectum before treatment (t0) as well as 24, 36, 48, 60, 72 and 96h after treatment and frozen at – 26° C till analysis. DOA was measured utilizing an 11-oxoetiocholanolone enzyme immunoassay. 1 wk after first treatment cows received an injection of the substance that was not used in the 1st study week. Fecal samples were again collected on t0, t24, t36, t48, t60, t72 and t96. Data were analyzed utilizing a generalized linear mixed model. Time after treatment was included as repeated factor and cows as random effect.
Results: Baseline DOA on t0 did not differ between 1st and 2nd study week (93.2 ± 38.4 ng/g; P = 0.256). While there was no difference between NSAID and PLA treated cows (P = 0.284), DOA concentration in all cows increased between 24 and 36h after injection (115.5 ± 51.8 ng/g; P = 0.009) and remained on an elevated level until 72h (111.7 ± 59.1 ng/g) after injection (P > 0.05). DOA baseline levels of t0 and t24 DOA were reattained 96h after injection (P > 0.05). Time lag between injection and an increased DOA level was consistent with previous studies that described a minimum of 8 to 16 h between an increase in blood cortisol coinciding with the stressor and an elevated concentrations of fecal DOA. There was no effect of week (i.e., 1st or 2nd study week; P = 0.997) or parity (P = 0.692) on DOA concentration. Before treatment average daily time lying was 750 ± 170 min with an average of 8.7 ± 3.6 lying bouts. On the day of injection average daily time lying decreased to 669 ± 165 min (P = 0.003) with an average number of lying bouts of 8.0 ± 3.2 (P = 0.039). Total time lying and the number of lying bouts reattained baseline values 1 day after injection and remained on these level throughout the study. While there was no effect of treatment (i.e., NSAID or PLA; P = 0.241) and study week (P = 0.707) on daily time lying, an interaction between parity and time relative to treatment was found (P = 0.06). Cows in 3rd or higher lactation lay 98 min less on the day of injection compared to the days before and after (P < 0.001). There was no effect in 1st or 2nd lactation cows (P > 0.05).
Conclusions: NSAID have no effect on cortisol metabolism in health dairy cows and do not influence standing and lying behavior. While a single intravenous injection and the associated fixation of the cows effect standing and lying behavior on the day of treatment, fecal glucocorticoid metabolites remain elevated up to 72h after injection. Further studies are necessary to investigate, why standing and lying behavior on the day of injection was only affected in older cows.