title: Sample 7
organism: Capra hircus
description: Protocols: Milk samples for RNA extraction were collected at the end of the adaptation period (no-MFD, control samples) from all goats, and at the end of the fish-oil (FO) supplementation period from goats selected as RESPO− and RESPO+. Milk samples were collected 1 hour after milking and 10 min after injection of 5 IU of oxytocin/animal (Facilpart, Laboratorios SYVA, León, Spain) to maximize milk somatic cell concentration. To prevent RNA degradation, udders were cleaned with soap and water and disinfected with povidone-iodine, and the nipples were also flushed with RNAseZap (Ambion, Austin, TX, USA). A sterile gauze was used to cover the collection tube to avoid contamination. 25 Murciano-Granadina goats (body weight: 30.5 ± 3.6 kg) at a similar stage of their first lactation (days in milk: 40.1 ± 5.9) were housed in individual tie stalls. During a 3 wk adaptation period, goats were fed a total mixed ration (TMR) formulated with alfalfa hay (particle size > 4 cm) and concentrate (forage:concentrate ratio 50:50) without lipid supplementation. Subsequently, goats were supplemented with 20 g of fish oil (FO; Afampes 121 DHA, Afamsa, Mos, Spain)/kg of diet DM, which is known to cause milk fat depression in dairy goats, for 5 additional weeks to induce a stable inhibition in milk fat synthesis. At the end of this second period, and on the basis of the magnitude of MFD, the 5 most-responsive goats (RESPO+; with a mean decrease in milk fat content of 26.0%) and the 5 least-responsive goats (RESPO−; with a mean decrease in milk fat content of 9.7%) were selected. Animals were milked once daily at approximately 0830 h in a 10-stall milking parlor (DeLaval, Madrid, Spain). They had ad libitum access to diets daily, and clean drinking water was available at all times. For RNA extraction, the MSCs from 50 mL of fresh milk were pelleted by centrifugation at 650×g for 10min at 4 °C in the presence of a final concentration of 0.5 mM of EDTA. Then, the pellet was washed three times with 10, 2 and 1.5 mL of PBS (pH 7.2 and 0.5 mM of EDTA) followed by centrifugation at 650×g for 10 min at 4 °C. The last pellet was used for RNA extraction using 500 μL of TRIzol (Invitrogen, Carlsbad, CA, USA). The RNA sequencing was conducted at CNAG (Centro Nacional de Análisis Genómico, Barcelona, Spain), where the TruSeq Stranded Total RNA Library Prep Kit (Illumina, San Diego, CA, USA) was used for library preparation.