Nanodrop one c microvolume spectrophotometer

For the measurement of serum corticosterone and protein concentration, the animals were decapitated 30 min after icv. treatment. For the assessment of serum LH, decapitation was performed 15 min after icv. treatment. Trunk blood was collected into test tubes and left at room temperature for 30 min to clot, then it was centrifuged for 10 min at 3500 rpm. The samples were stored at −80 °C until the assays were performed. Serum corticosterone concentration was measured using a competitive corticosterone ELISA kit (Cayman Chemical, Ann Arbor, MI, USA), according to the manufacturer’s instruction. Serum LH concentration was determined using a sandwich LH ELISA kit (Wuhan Xinquidi Biological Technology Co., Wuhan, China), according to the manufacturer’s instructions. The Pierce Coomassie (Bradford) Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used, according to the manufacturer’s instructions for the measurement of total serum protein concentration. The absorbance was measured at 595 nm with a NanoDrop One^C microvolume spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

Three biological replicates of fresh-frozen sperm samples and cryopreserved sperm samples were used for RNA extraction, which underwent the same thawing procedure (40 °C water bath for 20 s). To achieve high reproducibility, the sperm samples were homogenized using a PowerLyzer24 instrument (Qiagen, Redwood City, CA, USA). Total RNA was extracted using AllPrep DNA/RNA Kit (Qiagen, Redwood City, CA, USA) following the manufacturer’s instructions. RNA concentrations were measured with the NanoDrop OneC Microvolume Spectrophotometer (Thermo Scientific, Waltham, MA, USA). Evaluation of RNA quality was conducted using the LabChip GX Touch HT (PerkinElmer, Hopkinton, MA, USA). RNA-seq library construction was performed following the procedure described in our previous publication [96 (link)] with 500 ng of total RNA input. The size distributions of cDNA libraries were checked using the TapeStation 4200 D1000 ScreenTape (Agilent Technologies, Santa Clara, CA, USA), and library concentrations were determined by a Qubit 3.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). The libraries were commercially sequenced on the Illumina NovaSeq 6000 platform with a 2 × 150 Paired-End configuration at Novogene (Novogene Corporation Inc., Sacramento, CA, USA).

Fecal samples were collected under sedation immediately after blood collection to prevent interference of epinephrine-mediated hyperglycemia (Table S1). Plastic fecal loops were coated with mineral oil and inserted into the rectum and descending colon of the cats until an adequate amount of feces was collected. The samples in this study reflect the fecal composition of the rectum and descending colon, which is representative of the lower gut. We referred to the microbiota characterized in the fecal samples in this research as cat gut microbiota.
Genomic DNA samples were extracted from 200 mg fecal samples using the Qiagen Allprep PowerFecal DNA/RNA kit (Qiagen, MD) following the manufacturer’s protocols. To achieve homogeneous results, the homogenization step was performed by the Qiagen PowerLyzer24 instrument (Qiagen, MD) in the same batch. DNA and total RNA concentrations were measured by a Qubit 3 Fluorometer (Invitrogen, CA), and the A260/A280 absorption ratios were assessed using a NanoDrop One C Microvolume Spectrophotometer (Thermo Fisher Scientific, MA).