Coccyx

Rats were weighed and injected intraperitoneally with 2% (w/v) pentobarbital (40 mg/kg). As described in the previous study,^{42 (link)} the experimental level rat tail disc (Co7/8) was located by digital palpation on the coccygeal vertebrae and confirmed by counting the vertebrae from the sacral region in a trial radiograph. Needles (27G) were used to puncture the whole layer of annulus fibrosus though the tail skin. To make sure that the needle is not punctured too deep, the length of the needle was decided according to the annulus fibrosus and the nucleus pulposus dimensions, which were measured in the preliminary experiment and found to be about 4 mm. All the needles were kept in the disc for 1 min. Metformin was diluted with normal saline, to achieve a final metformin concentration of 20 mg/ml. After surgery, the metformin solution was immediately injected intraperitoneally to deliver a dose of 50 mg/kg/day until the rats were killed. Daily monitoring of the rats was carried out to ensure their well-being, and all animals were allowed free unrestricted weight bearing and activity.

Samples of feedstuffs were collected daily (day −2 to 8) and lyophilized in a Genesis Freeze Dryer 25 (Hucoa Erlöss, SA/Thermo Fisher Scientific) to determine their chemical composition and FA profile. Hay was offered daily at 08:00 h as a single meal in individual troughs, where cows were tied up until they finished their ration, during approximately 2 h. ALPRO feeding stations were programmed to offer 3 kg of concentrate daily (as-fed basis) to all the cows during the basal and refeeding periods. Individual concentrate intake was recorded daily.
The BCS was recorded upon calving, 30 DIM, and on experimental period day −2 and 8. It was determined by a trained person on a 1–5 scale, based on estimating the fat covering ribs, loin, and tailhead (Lowman et al., 1976 ). Cows were weighed on an electronic scale upon calving and then at 07:00 h on 30 and 31 DIM and on experiment day −2, 1, 3, 5, 6, and 8. Milk yield was estimated on the same days by the weight-suckle-weight technique (Le Neindre and Dubroeucq, 1973 (link)). Calves were weighed before and after the two daily 30-min periods in which they had access to suckle their dams. The daily milk yield was estimated as the sum of the milk consumed by the calf in these two suckling periods. Milk samples were manually taken from each dam after the morning suckling. Five minutes before the manual extraction, all cows received an intramuscular injection of oxytocin (40 UI, Facilpart, Laboratorios Syva, León, Spain) to accelerate the letdown of the residual milk. A 100-mL sample was collected to determine milk composition, added with sodium azide (PanReac) as a preservative and refrigerated at 4 °C until the analysis. To determine FA composition, a second 40-mL sample was collected, lyophilized, and stored at −20 °C until analyzed.
Cows were bled on the same experiment days described above to assess their metabolic profile. Blood samples were collected from the coccygeal vein at 07:00 h after suckling and before offering hay. Heparinized tubes (BD Vacutainer Becton-Dickenson and Company) were used for the β-hydroxybutyrate (BHB) and MDA determinations, and the tubes that contained K2 EDTA (BD Vacutainer Becton-Dickenson and Company) were used to analyze glucose, NEFA, and urea concentrations. Immediately after collection, blood samples were centrifuged at 3,500 rpm for 20 min at 4 °C. Plasma was collected and frozen at −20 °C until further analyses.

The farm enrolled in this study conducted pre-calving plasma NEFA analyses every 2 weeks for monitoring transition cow health. For this, ten primiparous and ten multiparous cows were randomly selected (www.graphpad.com/quickcalcs/randomSelect1/) for blood collection among all cows expected to calve in the following 7 to 14 d. Blood samples were collected at the time of daily delivery of fresh total mixed ration. Samples were obtained via puncture of coccygeal vessels using EDTA vacuum tubes (Monoject EDTA K3; Covidien, Minneapolis, MN). Immediately after collection, samples were centrifuged at 2,000 x g for 10 min. Plasma was removed and placed into 2-mL microcentrifuge tubes (Fisher Scientific, Waltham, MA). All samples were refrigerated, and plasma NEFA concentration was determined within 24 h by Central Star Cooperative (Grand Ledge, MI) using a 96-well plate protocol validated for cattle (14 (link)). The test has a sensitivity (95%CI) of 88.9% (67.2–96.9%) and a specificity (95% CI) of 100.0% (97.1–100.0%) for the identification of excessive lipid mobilization pre-calving (14 (link)). A convenience sample size that included the results from all the cows sampled between September 2015 and March 2020 whose actual calving date was between 4 and 14 days [mean (SD) = 7.4 (2.1) days] after sampling (n = 1,532) were included in the study. The details regarding calving date, calf identification, calf health, and calf performance were extracted from the farm record system (DairyComp 305, Valley Agricultural Software, Tulare CA).

Blood was collected via the coccygeal vein using lithium–heparin vacutainer tubes (BD Vacutainer, BD and CO., Franklin Lakes, NJ) in the morning before feeding on d 1, 30, 63, 64, 65, 66, 67, 68, and 69 of the study. Tubes were placed on ice and transported to the laboratory. For plasma, tubes were centrifuged at 2,500 × g at 4 °C for 15 min. Samples were then aliquoted into microcentrifuge tubes and stored at −80 °C until analysis. Plasma non-esterified fatty acids (NEFAs), β-hydroxybutyrate (BHB), glucose, total cholesterol, total bilirubin, creatinine, urea, aspartate aminotransferase (AST), γ-glutamyl transpeptidase (GGT), total plasma reactive oxygen metabolites (ROMs), ferric reducing ability of plasma (FRAP), haptoglobin, ceruloplasmin, paraoxonase (PON) activity, and magnesium (Mg) were analyzed as described previously (Trevisi et al., 2013 (link)). Myeloperoxidase (MPO) was measured as described by Bionaz et al. (2007) (link). In addition, blood samples collected on d 1, 30, 64, 66, and 68 were used to analyze monocyte and neutrophil oxidative burst activity and phagocytosis capacity via a flow cytometry-based assay as described previously by Zhou et al. (2018) (link).