2.2. Animals
All procedures concerning animal care and treatment were approved by the Ethical committee for the use of experimental animals at Umm Al-Qura University. For this study males of C57BL/6J (B6) mice were purchased from Harlan (Charles River Laboratories, Wilmington, Massachusetts, USA). Mice were maintained in a temperature-controlled room (21 ± 1 °C) under a 12-h to 12-h light to dark cycle. Mice were individually housed in standard cages with ad libitum water and standard chow (CRM pellets, SDS diets, U.K.). Male of B6 strains were studied (total n = 21).
We calculated the samples based on one-way ANOVA, the between-subject error DF (that is, the within-subject degree of freedom (DF)) is calculated as: DF = N – k = kn – k = k (n – 1), where N = total number of subjects, k = number of groups, and n = number of subjects per group. By rearranging the formula, n is given as: n = DF/k + 1 Based on the acceptable range of the DF, the DF in the formulas are replaced with the minimum (10) and maximum (20) DFs to obtain the minimum and maximum numbers of animals per group: Minimum n = 10/k + 1 Maximum n = 20/k + 1 In total, the minimum and maximum numbers of animals required are: Minimum N = Minimum n x k Maximum N = Maximum n x k as suggested from (Arifin & Zahiruddin, 2017).
In our study Minimum n = 10/3 + 1 = 4.3 = rounded up to 5 animals/group Maximum n = 20/3 + 1 = 7.7 = rounded down to 7 animals/group. Please note that the minimum and maximum numbers of animals per group are rounded up and down, respectively, to keep the DF for each sample size/group within the limit (e.g., DF = 12 for n = 5, and DF = 18 for n = 7). The total sample sizes are: Minimum N = Minimum n x 3 = 5 x 3 = 15 animals Maximum N = Maximum n x 3 = 7 x 3 = 21 animals. In conclusion, for the proposed study, between 5 and 7 animals per group are required. In other words, a total of 15 to 21 animals are required to keep the DF within the range of 10 to 20.
Measurements then started at the age of 10 weeks and were taken over a period of 16 weeks. Body weight and fat mass were monitored, just prior to lights off, 3 times a week (Mondays, Wednesdays and Fridays) throughout the experimental protocol. After 16 weeks mice were fasted for 4 h and euthanized by CO2 and blood samples were taken by cardiac puncture and liver tissues were preserved.
2.3. Induction and assessment of diabetes
Diabetes was induced in overnight-fasted, mice by a single dose of streptozotocin 45mg/kg, intraperitoneally (Jambart et al., 2011), dissolved in cold citrate buffer (0.1M, pH 4.5). This dose was selected to cause incomplete destruction of pancreatic beta cells, as it has been reported that streptozotocin is capable of producing mild-to-severe types of diabetes according to the dosage used, when administered to adult mice by either a single intravenous or intraperitoneal injection (Alatawi et al., 2018). After the injection, the mice had free access to food and water and were given a 5% glucose solution to drink overnight to counter hypoglycemic shock. Age-matched and weight-matched control mice received citrate buffer at the identical amount and pH. Diabetes was confirmed by measuring the fasting tail blood glucose concentration using a glucose meter device (Accu-chek Go, Roche Diagnostics GmbH, Mannheim, Germany) 96 hours after injection with streptozotocin. Ninety-nine percent of the animals had blood glucose above 235mg/dl and they were considered diabetic; mice with blood glucose below 235mg/dl were excluded from the present study.