The tissues and samples used in the present study were obtained from the cohort of male Sprague-Dawley rats used in Subias-Gusils et al. [27 (link)]. Briefly, two dietary interventions based on the CAF-R diet alone or supplemented with OLE (CAF-RO diet) at a dose of 25 mg/kg·day of an Olea europaea leaf extract (Benolea®, Frutarom Health BU, Wädenswil, Switzerland) were administered for 14 weeks to CAF diet-induced obese animals. The CAF and CAF-R diets were composed of the following items: bacon, biscuit with pâté, biscuit with cheese, muffins, carrots, jellied sugared milk and standard chow. The amount of each cafeteria food item administered to the CAF-R and CAF-RO animals was readjusted every week based on a 30% reduction in calories relative to the energy consumed by the CAF group. A control (STD) group was fed standard chow ad libitum during the entire experiment [27 (link)]. The CAF diet was provided ad libitum to the animals after weaning, starting at the age of 24 days old and continuing for 8 weeks. When the animals were 80 days-old, the obese CAF-fed animals were subdivided into three groups CAF, CAF-R and CAF-RO according to the diet administered until the end of the experiment.
The average daily intake of macronutrients during the dietary treatments, expressed as the percentage of the average total daily energy intake in kilocalories coming from each macronutrient type, as well as the feed efficiency, are indicated inTable S1 .
The animals were sacrificed at an age of 26 weeks, and the body weights were: STD, 465.9 ± 8.8; CAF, 564.9 ± 15.4; CAF-R, 537.5 ± 15.9; and CAF-RO, 536.6 ± 12.0 (mean ± SEM, grams). Euthanasia was carried out by decapitation after 8 h of fasting, which started in the morning at 6 a.m., leaving 20 min between animals, and sacrifices were performed sequentially between 2 p.m. and 5 p.m. for a total of 3 days. The following tissues were removed: HPT, WAT depots (retroperitoneal -rWAT-, mesenteric -mWAT-, epididymal -eWAT- and inguinal -iWAT-), interscapular brown adipose tissue (iBAT), liver, cecum, gastrocnemius and soleus muscles. Once removed, they were weighed, frozen in liquid nitrogen and stored at −80 °C until further analysis. A macroscopic examination of all tissues was performed before storage. The tissue exeresis was carried out by the same qualified experimenters through the procedure to decrease variability.
The adiposity index was determined as the sum of the eWAT, iWAT, mWAT and rWAT weights (in grams) and expressed as a percentage of body weight (g/kg·100). Abdominal WAT referred to the sum of rWAT, mWAT and eWAT weights, while subcutaneous WAT was considered as the iWAT.
The experimental protocol was approved by the Generalitat de Catalunya (DAAM 9978), following the ‘Principles of laboratory animal care’ and was carried out in accordance with the EU Directive 2010/63/EU for animal experiments and following ARRIVE guidelines.
The average daily intake of macronutrients during the dietary treatments, expressed as the percentage of the average total daily energy intake in kilocalories coming from each macronutrient type, as well as the feed efficiency, are indicated in
The animals were sacrificed at an age of 26 weeks, and the body weights were: STD, 465.9 ± 8.8; CAF, 564.9 ± 15.4; CAF-R, 537.5 ± 15.9; and CAF-RO, 536.6 ± 12.0 (mean ± SEM, grams). Euthanasia was carried out by decapitation after 8 h of fasting, which started in the morning at 6 a.m., leaving 20 min between animals, and sacrifices were performed sequentially between 2 p.m. and 5 p.m. for a total of 3 days. The following tissues were removed: HPT, WAT depots (retroperitoneal -rWAT-, mesenteric -mWAT-, epididymal -eWAT- and inguinal -iWAT-), interscapular brown adipose tissue (iBAT), liver, cecum, gastrocnemius and soleus muscles. Once removed, they were weighed, frozen in liquid nitrogen and stored at −80 °C until further analysis. A macroscopic examination of all tissues was performed before storage. The tissue exeresis was carried out by the same qualified experimenters through the procedure to decrease variability.
The adiposity index was determined as the sum of the eWAT, iWAT, mWAT and rWAT weights (in grams) and expressed as a percentage of body weight (g/kg·100). Abdominal WAT referred to the sum of rWAT, mWAT and eWAT weights, while subcutaneous WAT was considered as the iWAT.
The experimental protocol was approved by the Generalitat de Catalunya (DAAM 9978), following the ‘Principles of laboratory animal care’ and was carried out in accordance with the EU Directive 2010/63/EU for animal experiments and following ARRIVE guidelines.
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