Physiological Parameters

Blood samples were collected via jugular venipuncture into vacuum tubes (BD Vacutainer; Becton Dickinson, Franklin Lakes, NJ) on d −7, −5, −2, and −1; immediately before castration (T0); and 30, 60, 120, and 240 min and 1, 2, 5, 7, 14, 21, and 28 d after castration. Additional samples were collected 60 and 240 min and on d 1, 2, and 5 after castration to determine plasma concentrations of meloxicam. These samples were collected into 10-mL lithium heparin tubes (BD Vacutainer; Becton Dickinson) and centrifuged at 0 °C for 15 min at 1.5 × g, and the serum was decanted and stored at −80°C for further analysis using high-pressure liquid chromatography (Agilent 1100 Pump, Column Compartment, and Autosampler, Santa Clara, CA) with mass spectrometry detection (LTQ, Thermo Scientific, San Jose, CA) at Iowa State University's College of Veterinary Medicine (Ames, IA). The limit of quantitation of the analysis was 1.0 ng/mL, with a limit of detection of 0.3 ng/mL.

Samples to determine concentrations of substance P (SP) were collected on d −1, at T0, and at 30, 60, 120, and 240 min and on d 1, 2, 5, 7, 14, 21, and 28 into a 6-mL vacuum tube with EDTA (BD Vacutainer; Becton Dickinson). The sample was then centrifuged at 0 °C for 15 min at 1.5 × g, and the serum was decanted and frozen at −80°C for further SP analysis, which was previously described by Van Engen et al. (2014) with some modifications at Iowa State University's College of Veterinary Medicine (Ames, IA). Nonextracted plasma samples were analyzed in duplicate with a double-antibody RIA using a purchased primary antibody (Substance-P (3–11) Antibody for Immunohistochemistry Human, Rat, Mouse, #H-061-05, Phoenix Pharmaceutical, Burlingame, CA). The range of detection for SP was between 5 and 320 pg/mL, with an average R² = 0.98. The coefficient of variation for intra-assay variability was 8.2%, and the interassay variability was calculated at 20.6%. The limit of detection was 10 pg/mL, and the limit of quantitation was 20 pg/mL.

Samples to determine complete blood cell count were collected on d −7, −5, −2, and −1; at T0; and at 30, 60, 120, and 240 min and on d 1, 2, 5, 7, 14, 21, and 28 after castration. The sample was collected in a 6-mL EDTA tube (BD vacutainer; Becton Dickinson) for complete blood count using a HemaTrueHematology Analyzer (Heska, Lobeland, CO), which measured white blood cells (WBC), red blood cells (RBC), platelet count, and the neutrophil:lymphocyte (N:L) ratio.

Blood samples to measure haptoglobin were collected on d −1, 1, 2, 5, 7, 14, 21, and 28, whereas serum amyloid A (SAA) was collected on d −1, 1, 2, 5, and 7. The sample was collected into 10-mL nonadditive tubes (BD Vacutainer; Becton Dickinson), which were centrifuged at 4 °C for 15 min at 1.5 × g, and the serum was decanted and frozen at −80°C for further analysis. Haptoglobin concentrations were analyzed using a Roche Cobas c501 biochemistry analyzer (Roche Diagnostics, Laval, QC, Canada) using a Tridelta bovine haptoglobin calibrator (TP801CAL, Tridelta, Maynooth, Ireland) and 2 levels of in-house controls (bovine serum pools) daily and 2 levels of Tridelta controls weekly. Samples to determine SAA concentrations were collected on d −1, 1, 2, 5, and 7 and analyzed using an enzyme-linked immune sorbent assay (Tridelta Phase range SAA kit, TP 807, Tridelta Development, Maynooth, Ireland).

Saliva samples to determine cortisol concentration were collected on d −7, −5, −2, and −1 before castration; at T0; and at 30, 60, 120, and 240 min and 1, 2, 5, 7, 14, 21, and 28 d after castration. Samples were collected with a cotton swab and immediately stored in a plastic tube and frozen at −20°C for further cortisol analysis using an enzyme immunoassay kit (Salimetrics, State College, PA). Interassay and intra-assay variability values were 14.1% and 5.9%, respectively.

Hair from the forehead was clipped and stored in plastic bags at room temperature on d −1 and on d 28 after castration for further cortisol analysis. Hair samples were processed as described in detail by Moya et al. (2014), and cortisol was quantified using an enzyme-linked immunosorbent assay (Salimetrics). Values for interassay and intra-assay variability were 8.3% and 5.8%, respectively.

Thermographic images of the scrotal area were collected on d −7, −5, and −2; at T0; and at 30, 60, 120, 240 min and on d 1, 2, 5, 7, 14, 21, and 28 after castration. Images were taken from behind the calves 1 m from the scrotal area using a FLIR I60 infrared camera (FLIR Systems, Burlington, ON, Canada) and analyzed with FLIR Tools version 5.1 (FLIR Systems) using an emissivity coefficient of 0.98 to measure scrotal temperature (ST).

Rectal temperature was measured on d −7, −5, −2, −1, 0, 1, 2, 5, 7, 14, 21, and 28 using a digital thermometer (GLA Agricultural Electronics, M750 Livestock Thermometer, San Luis Obispo, CA).

Animals were weighed on d −1, 1, 2, 5, 7, 14, 21, and 28 in the squeeze chute. Initial BW was the weight collected on d −1, and the final BW was the weight collected on d 28.

Scrotal swelling was evaluated using an 11-point scale as described by Molony et al. (1995) and then collapsed into a 5-score scale. The scores were as follows: 4, presence of suppurative discharge with inflammatory response that required intervention; 3, presence of pus with increasing inflammatory response; 2, increasing degree of swelling with obvious erythema but without pus; 1, increasing degree of swelling without obvious erythema; and 0, no swelling, inflammation, or infection visible or palpable.