Serum gel tubes

At the end of the experiment mice were anaesthetized with isoflurane (3%) delivered in oxygen (1 L/min). They were then sacrificed by terminal cardiac puncture and exsanguination (removal of blood) with a G25 needle. Approximately 0.7 ml of blood was collected from each mouse and decanted immediately into 1.3 ml serum gel tubes (Sarstedt, Nümbrecht, Germany). All organs were collected and snap frozen in liquid nitrogen and were stored at -80°C. Blood serum was isolated after 3 min centrifugation at 9900 rpm, before snap-freezing in liquid nitrogen and storing at -80°C. 100 μl of serum samples were then analyzed in a blinded fashion by a commercial veterinary testing laboratory (MRC Harwell Institute, Oxfordshire, UK) to evaluate the biomarkers serum urea, and creatinine for renal dysfunction, serum alanine aminotransferase (ALT), and serum aspartate aminotransferase (AST) for hepatocellular injury, and lactate dehydrogenase (LDH) for general organ injury.

All blood samples were collected from random-cycling mice after 3–4 hours of fasting. Blood was collected in serum gel tubes (Sarstedt, Nümbrecht, Germany), and serum was obtained and stored at −80°C. Serum levels of estradiol were analyzed using a radioimmunoassay (Siemens Healthcare Diagnostics, Tarrytown, NY, USA). Serum testosterone levels were measured by a radioimmunoassay after diethylether extraction (16 (link)). Levels of cholesterol, triglycerides, and serum glucose were analyzed by colorimetric assays using Infinity reagents (TR13421, TR22421, and TR15421; Thermo Fisher Scientific, Waltham, MA, USA). The distribution of lipids within the plasma lipoprotein fractions was assessed in pooled serum (6 mice per pool) by fast-performance liquid chromatography gel filtration using a Superose 6HR 10/30 column (Pharmacia, Uppsala, Sweden) (17 (link)). Insulin levels were measured using a mouse insulin ELISA (Ultra Sensitive Mouse Insulin ELISA; Crystal Chem, Downers Grove, IL, USA).

Blood was collected via cardiac puncture in 500 Serum-Gel tubes (Sarstedt). Serum was separated by centrifugation at 10,000 rpm for 4 min at room temperature and kept at −80°C until analysis. Serum concentration of IL-6 and CCL5 were determined by using PEPROTECH kit following the manufacturer's kit protocol. In addition serum concentration of cytokines and chemokines were quantified by LUMINEX based mouse cytokine 23-plex assay following manufacturer's instruction (Bio-Rad, USA).

One blood sample per animal was taken from the V. caudalis mediana (serum gel tubes, Sarstedt AG & Co. KG, 51588 Nümbrecht, Germany). After clotting (30 min), the samples were centrifuged with the SERVOspin next benchtop centrifuge (servoprax GmbH, 46485 Wesel, Germany) at 4,000 rpm for 10 min; serum was separated and then stored at −20°C until analysis.
The samples were analyzed for blood chemistry, especially for urea (BUN), ß-hydroxy butyrate (BHB), glutamate dehydrogenase (GLDH), and gamma-glutamyl transferase (GGT) using the non-accredited method FMUAA 172 2019-03 (bichromatic measurement) by the LKS–Landwirtschaftliche Kommunikations- und Service GmbH (Lichtenwalde, Germany).
The analysis of free fatty acids (FFA) was carried out to obtain information about metabolic situation using the NEFA test kit from Randox Laboratories Ltd. (Crumlin, UK).

At the end of the experiment mice were anaesthetized with isoflurane (3%) delivered in oxygen (1 L/min). They were then sacrificed by terminal cardiac puncture and exsanguination (removal of blood) with a G25 needle. Approximately 0.7 ml of blood was collected from each mouse and decanted immediately into 1.3 ml serum gel tubes (Sarstedt, Nümbrecht, Germany). All organs were collected and snap frozen in liquid nitrogen and were stored at -80°C. A section of the kidney and liver were placed in 2-methylbutane and then transferred to liquid nitrogen and stored at -80°C. A section of the kidney and liver were fixed in formalin for 48 h and was then transferred to 70% ethanol for long-term storage. The rest of the organ collected and placed in liquid nitrogen and were stored at -80°C. Blood serum was isolated after being centrifuged for 3 min at 9900 rpm, snap frozen in liquid nitrogen and stored at -80°C. 100 μl of serum samples were then analyzed in a blinded fashion by a commercial veterinary testing laboratory (MRC Harwell Institute, Oxfordshire, UK) to evaluate the following biomarker: total cholesterol, triglycerides, glucose (non-fasting) and alanine aminotransaminase (ALT).

For both fecal- and blood samples, piglets were sampled in random order and weighed shortly before sample collection. Fecal samples were collected in cryotubes on days 4, 8, 14, 26, 35, 43, 59, and 69 by gentle rectal stimulation with cotton swabs (PurFlock Ultra, Puritan) pre-wetted with sterile water. The cryotubes were immediately placed on dry ice and stored at −80°C until further processing.
Blood samples were collected from the jugular vein of sixteen animals on days 14, 26, 43, and 69 using Serum Gel tubes (S-Monovette^®, Sarstedt) that were centrifuged at 2,500 × g for 10 min to separate serum. Serum was stored at −20°C until further use. Blood samples for flow cytometry were collected in Sodium heparin tubes (S-Monovette^®, Sarstedt) and stored at room temperature (RT) until further processing.