Animals

This study adhered to the guidelines of the Animal Research: Reporting of In Vivo Experiments [12] and Planning Research and Experimental Procedures on Animals: Recommendations for Excellence [13]. Inclusion criteria would be healthy sheep, aged 2 to 4 years, weighing 30 to 45Kg. Any sheep showing disease or not meeting the age and weight criteria would be excluded from the study. We used ten adult Santa Ines sheep with a mean age of three years (range: 2–4 years) and a mean weight of 37.05 kg (31–42 kg). To minimize the effects of subjective bias when allocating animals to treatment, they were randomly allocated to two experimental periods of two and four weeks using the coin toss method.

All procedures were performed in accordance with the National Institutes of Health (NIH) guide for the care and use of laboratory animals [14] and Brazilian guidelines for the care and use of animals in teaching or scientific research activities (DBCA) of the National Council for the Control of Animal Experimentation [15]. A veterinarian with over 20 years of experience conducted the nutritional care, fasting, and pre- and post-operative care of animals. The study protocol was approved by The Ethics Committee on Animal Use of the Universidade Federal Fluminense (No. 9.531.061.119). All experiments were conducted between March and July 2020. No animals were euthanized at the end of the study in accordance with the guidelines of the Reduction, Refinement, and Replacement (3Rs) Program, whose goal is to reduce the number of animals used in experiments, minimize their pain and discomfort, and avoid euthanasia at their end [16]. All animals were immunized against common sheep diseases and were monitored for good physical condition.

The animals were housed at the Universidade Federal Fluminense’s farm in a semi-extensive fenced system with native forage and brachiaria grass (Brachiaria humidicola and Brachiaria decumbens). In the preoperative period, animals received feed composed of the pastures, and in the postoperative period, pastures with nutritional supplementation for sheep and mineral water ad libitum. To reduce the preoperative stress levels of animals, the veterinarian transferred the sheep from the farm to the research center two weeks before surgery to allow for proper acclimatization [17]. The animals fasted for eight hours before surgery.