title: R_MFE_D6h
organism: Ovis aries
description: Protocols: Milk samples for RNA-Seq were collected at the period when FE was evaluated, between Days 40-50 at first lactation.Briefly, for each animal, 50 ml of milk was collected one hour after morning milking and ten minutes after the injection of 5 IU of Oxitocine Facilpart (Syva, León, Spain) to maximize the number of exfoliated milk epithelial cells . To avoid RNA degradation during sampling collection, the udder was disinfected with povidone-iodine, nipples were cleaned with RNAseZap (Ambion, Austin, TX, USA), and sterile gauze was used to cover the collection container (i.e., 50 ml RNAse-free tube). Forty Spanish Assaf female lambs (86.7 days of age; SD=2.5) were bought from a single flock and were transported to the facilities of the Instituto de Ganadería de Montaña (IGM; CSIC- Universidad de León). The animals were managed under standard commercial conditions; ewe lambs, i.e., female lambs, were fed a standard diet for replacement ewe lambs, consisting of 16% crude protein. At three months of age, they were divided into two groups: 20 nutritional protein-restricted ewes (NPR) and 20 control ewes. The protein restriction period was applied in NPR ewes during the mammary gland allometric growth period (64 days, between 3.5 and 5.8 months of age). Thus, the control ewes received the standard diet mentioned above for 64 d, and during the same period, the NPR ewes received the same diet without soybean meal (42.3% reduction in protein content). Both groups had ad libitum access to barley straw. Estrus was synchronized to avoid differences due to the lactation stage, the ewes were artificially inseminated at approximately 10 months of age, and lambing was concentrated within a timeframe of a few days. During the first lactation, milk production was recorded daily, and milk composition (protein and fat content) was analyzed regularly for each ewe from weeks 1 to 12 after lambing. Thus, for each animal, a total of 27 records, including milk yield (MY) and milk protein and fat percentages (PP and FP, respectively), were available. On Day 33 of lactation, the ewes were housed in individual pens. To estimate FE, individual feed intake and milk production were measured over 28 days after the ewes adapting not only to the diet but also to the individual stalls. During lactation, the ewes were fed a total mixed ration (TMR) formulated from alfalfa hay (particle size > 4 cm) and concentrate (50:50 forage:concentrate ratio), including sugar beet molasses, to hinder the selection of dietary components. The ingredients and chemical composition of the TMR are reported in Table 1. Clean drinking water was made consistently available. Feed intake was calculated by weighing the amounts of dry matter (DM) offered and refused by each animal. The ewes were milked twice daily at approximately 08:30 and 18:30 h in a single-side milking parlor with 10 stalls (DeLaval, Madrid, Spain). The total milk produced by each animal at both milkings was collected and weighed to calculate the milk yield. Composite samples of the milk produced by each ewe were prepared daily according to individual yields in both milkings. One aliquot (40 mL) of that composite milk was collected on Days 4, 5, 9, 13, 14, 15, 23 and 28 of the trial, preserved with bronopol (D&F Control Systems Inc., San Ramon, CA, USA) and stored at 4 °C until analyzed for fat, protein, lactose, and total solids concentrations and for somatic cell count. FE was calculated using two indices: RFI and FCR. The FCR was calculated as the ratio between DMI and the energy-corrected milk yield (ECM): FCR = DMI/ECM, where DMI represents the mean dry matter intake during the experimental period (kg/d), and ECM (L/d) was calculated using the formula proposed by INRA [25] for sheep: ECM = L/d milk yield × [(0.0071 × g/L milk fat) + (0.0043 × g/L milk protein) + 0.2224 The RFI as the residuals of the following regression model, using the glm procedure of the R software (v4.2.1): DMI = µ + a × ECM + b × MBW + c × BW×BWC + RFI, where DMI represents the mean dry matter intake during the experimental period (kg/d); µ is the intercept; ECM is the energy-corrected milk yield (kg/d); MBW is the mean metabolic body weight (BW) (BW0.75; kg); BW×BWC is the interaction between the body weight (BW in kg) and the BW change per day (kg/d); RFI is the residual term; and a, b, and c are the regression coefficients. High-FE and Low-FE (phenotype column) are those animals that could be classified as high-FE or low-FE based on FCR and RFI indices After milk collection, the milk somatic cells (MSCs) were pelleted by centrifugation at 650×g for 10 minutes at 4 °C and in the presence of a final concentration of 0.5 mM EDTA to eliminate casein and fat globules. Then, the MSC pellet was washed twice with 10 and 2 mL of PBS (pH 7.2 and 0.5 mM EDTA), followed by centrifugation at 650×g for 10 min at 4 °C. The last pellet was kept in RNAlater (Sigma‒Aldrich, Madrid, Spain) and stored at −80 °C until RNA extraction using 500 μL of TRIzol according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). The RNA quality was evaluated using an Agilent 2,100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Then, 28 samples with enough RNA quality (RIN > 6.4) were sent to be sequenced at Novogene (Cambridge, UK). The UltraTM RNA Library Prep Kit (NEBNext®) was used to construct the libraries.
attributes.tag: phenotype
attributes.value: high_FE
attributes.tag: strain
attributes.value: Assaf
attributes.tag: individual
attributes.value: 536
attributes.tag: organism
attributes.value: Ovis aries
attributes.tag: organism
attributes.value: Ovis aries
attributes.tag: collection date
attributes.value: not collected
attributes.tag: sex
attributes.value: female
attributes.tag: scientific_name
attributes.value: Ovis aries
attributes.tag: common name
attributes.value: sheep
attributes.tag: organism part
attributes.value: mammary gland
attributes.tag: developmental stage
attributes.value: primiparous
attributes.tag: isolate
attributes.value: not applicable
attributes.tag: diet
attributes.value: protein restriction
attributes.tag: cell_type
attributes.value: milk somatic cell
attributes.tag: geographic location (country and/or sea)
attributes.value: not collected
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2023-12-12