2.2. Feed Intake, Performance, Nutrient Apparent Digestibility and Biopsy Sampling of Intestinal Tissue

To examine the effect of the flavours on feed intake, weight gain, apparent feed digestibility, energy balance and relative gene expression in the small intestines, a metabolic experiment was conducted with eight lambs (4 males and 4 females) using the sucram and capsicum flavours. Sucram and capsicum were selected because their preference was similar to the control during the preference test, and they are reported to influence feed efficiency in mammals [22,23]. The lambs (one male and one female) were assigned to either sucram, capsicum, a mix (sucram and capsicum at 1:1 ratio) or no flavour control supplemented in the feed. The feed consisted of a total mixed ration, whole corn grain and pellets in a 50:25:25 ratio (weight as is), respectively, formulated to ensure 15.7% CP and 2.68 Mcal/kg metabolizable energy (ME) on DM basis (Table 1). Every morning, feed and flavours were weighed and mixed thoroughly by hand for two minutes, partitioned into 12 equal meal portions and loaded into the automatic feeders. Feed quantities were adjusted every day to ensure at least 5% refusals.

Following 14 days of exposure to the flavours, the total collection of urine, faeces and refusals was carried out for five days. Urine was collected using catheters (in females only; Foley catheter 16 Fr.; Degania Silicone Ltd., Hatzor HaGlili, Israel) into 1500 mL urine bags with 10 mL of 20% sulphuric acid as a preservative while faeces were collected on a net fitted under the metabolic cages. Total collected urine and faeces were weighed and stored at −20 °C until chemical analysis. The treatments were switched at the end of the first period and the experiment repeated for the second period. Sheep were weighed at the beginning and at the end of each period of the experiment to determine weight gain (Scales Galore, Brooklyn, NY, USA), and daily weight gain was expressed in grams per metabolic body weight (g/Kg BW^0.75) to control for baseline weight variability between periods.

At the end of each total collection period, the feed was withdrawn from the lambs for 24 h in preparation for the biopsy procedure. The procedure was performed under full anaesthesia, as follows. Lambs were sedated with Xylazine (0.05 mg/kg body weight (BW); Sigma-Aldrich, Rehovot, Israel). Anaesthesia was then initiated by intravenous injection of ketamine (2.2 mg/kg BW; Chanelle, Berkshire, UK) and Assival to affect (TEVA Pharmaceutical Industries Ltd., Petach Tikva, Israel). Anaesthesia was maintained with isoflurane gas (Sigma-Aldrich, Rehovot, Israel) utilizing an anaesthetic machine (Vetland Medical Sales and Services LLc, Louisville, KY, USA) at 2%. For venous catheterisation, the right arm was shaved, scrubbed thoroughly with septal scrub and rinsed with 70% ethanol. A longitudinal incision was made on the right side of the abdominal flank after surgical scrub and preparation. Approximately 3 cm of intestinal tissue was taken from the proximal jejunum (10–15 cm post the duodenal loop) using the full-thickness biopsy technique (resection and anastomosis). Biopsied tissue samples were washed with isotonic saline, immediately snap-frozen in liquid nitrogen and stored at −80 °C awaiting gene expression analysis.

All of the lambs were healthy throughout the experiment, except one male lamb that died after the first period of the experiment. As a result, data for the first period of the experiment consisted of eight lambs, while the second period consisted of seven lambs.