Sample size calculation is crucial in animal studies to ensure sufficient statistical power while avoiding unnecessary resource use and ethical issues. Two main methods are recommended: power analysis and the resource equation method. Power analysis is the preferred method, involving parameters like effect size, standard deviation, type 1 error, power, and statistical tests. Effect size is the difference between groups, while standard deviation measures variability. Type 1 error is typically 5%, and power is usually 80%. The resource equation method uses the formula E = Total number of animals - Total number of groups, with E ideally between 10 and 20. For quantitative data, the sample size formula is Sample size = 2 SD² (Zα/2 + Zβ)² / d². For qualitative data, the formula is Sample size = 2 (Zα/2 + Zβ)² × P(1-P) / (p1 - p2)². Software like G*Power is recommended for complex calculations. Adjustments for attrition should be made by dividing the calculated sample size by (1 - attrition rate). Researchers should report sample size justification and calculation methods in their manuscripts. Six animals per group is not scientifically justified. Free software and calculators are available for sample size calculations. Proper sample size calculation ensures reliable results and ethical compliance in animal studies.Sample size calculation is crucial in animal studies to ensure sufficient statistical power while avoiding unnecessary resource use and ethical issues. Two main methods are recommended: power analysis and the resource equation method. Power analysis is the preferred method, involving parameters like effect size, standard deviation, type 1 error, power, and statistical tests. Effect size is the difference between groups, while standard deviation measures variability. Type 1 error is typically 5%, and power is usually 80%. The resource equation method uses the formula E = Total number of animals - Total number of groups, with E ideally between 10 and 20. For quantitative data, the sample size formula is Sample size = 2 SD² (Zα/2 + Zβ)² / d². For qualitative data, the formula is Sample size = 2 (Zα/2 + Zβ)² × P(1-P) / (p1 - p2)². Software like G*Power is recommended for complex calculations. Adjustments for attrition should be made by dividing the calculated sample size by (1 - attrition rate). Researchers should report sample size justification and calculation methods in their manuscripts. Six animals per group is not scientifically justified. Free software and calculators are available for sample size calculations. Proper sample size calculation ensures reliable results and ethical compliance in animal studies.