Vacutainer precision glide

Blood samples were collected by coccygeal venipuncture into vacuum tubes without anticoagulant (BD Vacutainer Precision Glide, Becton Dickinson, Franklin Lakes, NJ) at 35 DIM. All blood samples were allowed to clot, centrifuged at 1,500 × g for 15 min and serum was stored in aliquots at -20°C. Progesterone was measured using the Progesterone Double Antibody RIA Kit (MPBiomedicals, Inc., Costa Mesa, CA, USA) according to the manufacturer’s instructions. Briefly, 100 μL of each standard, serum sample, and control were added into their respective anti-progesterone coated tubes. Then, 1 mL of progesterone I-125 was added to all tubes and vortexed briefly. Tubes were then incubated in a 37°C-water bath for 120 min. The contents of the tubes were decanted, and tubes were counted in a gamma counter calibrated for I-125 (Biodex Medical Systems, Inc.). The lower limit of detection was 0.1 ng/ml, and the intra-assay coefficient of variation was 14.3%. Samples were analyzed in the same batch.

Animals were weighed and anesthetized with an intraperitoneal injection of ketamine/dexmedetomidine solution (ketamine 150 mg/kg and dexmedetomidine 0.5 mg/kg), injection volume of 0.1 ml/100 g of body weight). Following confirmation of reflex loss, the intraperitoneal cavity and thorax were opened, and blood samples were taken from the left ventricle using BD Vacutainer® PrecisionGlide™ single-used needles (18g x 1.5″, Cat # 360748) and collected in VACUETTE® serum separator tubes. Thereafter, animals were transcardially perfused with a pre-flush of 0.1 M phosphate buffer (PB) followed by 4% paraformaldehyde (PFA) in PB. Tissues of interest were carefully dissected and weighed (brain, pancreas, m. tibialis anterior) and immersion fixed in 4% PFA/phosphate buffered saline (PBS) overnight at 4°C. The superior poles of the eyes were marked by punctuation to allow orientation of the retinas. Retinas from the left eye were dissected and processed as previously described (42 (link)). Samples were rinsed several times with PBS and impregnated in a 30% sucrose/PBS solution for 2-3 days at 4°C. Tissues were frozen at -80°C until further immunostaining; all immunostaining details are provided in Table 1.

Blood sampling was conducted between 0830 h and 1130 h prior to feed distribution. Heifers were restrained in the chute and secured with a nylon rope halter in order to expose the jugular vein region. The jugular area was disinfected with 70% isopropyl alcohol and blood samples were collected using 0.9 × 25 mm blood collection needles (BD Vacutainer^® Precision Glide, BD Inc., Franklin Lakes, NJ, USA). Blood was collected into sodium heparin tubes (BD Vacutainer^®, BD Inc.) for the metabolite profile, EDTA tubes (Monoject^® Blood Collection Tube, Kendall Healthcare, Mansfield, TX, USA) for the CBC analysis, and serum separator tubes (BD Vacutainer^®, BD Inc.) for the specific antibody response. Samples for CBC analysis were taken at the start and end of the performance evaluation, and stored at 4 °C prior to analysis. Samples for blood plasma metabolite profile analysis were taken every 30.6 ± 3.0 days during the performance evaluation, then stored at −80 °C until further processing. Blood samples for immune response were collected and kept at room temperature for 25 min to allow clotting before being further processed. Both the metabolite profile and the immune response samples were centrifuged at 4 °C at 3000× g for 25 min, then the supernatant was decanted into micro tubes and kept frozen until analysis.