How to determine obesity in a rat

Why Assess Obesity in Rats?

Assessing adiposity in laboratory rats provides essential data for biomedical research. Excess body fat in rodents influences metabolic pathways, cardiovascular function, and endocrine signaling, thereby affecting the validity of experimental outcomes. Quantifying obesity enables researchers to:

Correlate diet‑induced weight gain with insulin resistance, hyperlipidemia, and hepatic steatosis.
Evaluate the efficacy of pharmacological agents targeting weight reduction or metabolic improvement.
Standardize animal models for translational studies, ensuring reproducibility across laboratories.
Detect genotype‑specific susceptibility to obesity, supporting genetic investigations of energy balance.

Accurate measurement of rat obesity also informs ethical considerations. Monitoring body composition prevents unintended severe obesity, which can compromise animal welfare and introduce confounding stress responses. Consequently, systematic assessment of adiposity is a prerequisite for rigorous, reproducible, and humane scientific inquiry.

Key Metrics for Obesity Determination

Body Weight and Growth Curves

Body weight provides the most immediate indicator of excess adiposity in laboratory rats. Accurate assessment requires repeated measurements under standardized conditions, typically using a calibrated analytical balance with a precision of at least 0.01 g. Animals should be weighed at the same time of day, preferably after a brief acclimation period, to minimize variability caused by recent food intake or activity.

To generate a reliable growth trajectory, record body weight at consistent intervals (e.g., weekly) from weaning (post‑natal day 21) through the experimental endpoint. Compile the data into a longitudinal dataset and plot individual or cohort averages against age. The resulting curve serves as a reference for normal development and highlights deviations indicative of obesity.

Interpretation of growth curves relies on comparison with established normative values for the specific strain, sex, and housing conditions. Common criteria for classifying obesity include:

Body weight exceeding the mean of the reference population by ≥20 % at a given age.
Persistent upward shift of the growth curve relative to the 95th percentile of normative data.
Accelerated weight gain rate (Δweight/Δtime) surpassing the upper confidence interval of normal growth.

When a rat meets any of these thresholds, classify the subject as obese and consider corroborating measurements (e.g., body composition analysis) to confirm excess fat accumulation. Continuous monitoring of weight and curve progression enables early detection, facilitates timely intervention, and ensures reproducible classification across studies.

Body Mass Index (BMI) in Rats

Body Mass Index (BMI) offers a rapid, non‑invasive estimate of adiposity in laboratory rats. The index is calculated by dividing body weight (grams) by the square of the naso‑anal length (centimetres), yielding a value expressed in g · cm⁻².

Measure body weight with an analytical balance; record to the nearest 0.01 g.
Determine naso‑anal length using a calibrated ruler or digital caliper; record to the nearest 0.1 cm.
Compute BMI: BMI = weight (g) / [length (cm)]².

Reference ranges differ among strains, ages, and sexes, but typical thresholds for adult Sprague‑Dawley rats are:

BMI < 0.45 g · cm⁻² – lean
BMI ≈ 0.45–0.55 g · cm⁻² – normal
BMI > 0.55 g · cm⁻² – obese

Values exceeding the upper limit indicate excess adipose tissue and warrant further investigation.

BMI does not distinguish between lean mass and fat mass; therefore, confirmatory methods such as dual‑energy X‑ray absorptiometry, magnetic resonance imaging, or the Lee index (body weight^(1/3) / naso‑anal length) should be employed when precise quantification of fat is required.

Consistent measurement technique, appropriate reference data, and complementary assessments ensure reliable evaluation of obesity in rat models.

Adiposity Indices

Adiposity indices provide quantitative estimates of excess body fat in laboratory rats and serve as primary criteria for classifying obesity. These measures combine easily obtained morphometric data with organ‑specific fat assessments to produce reproducible values that reflect total adiposity.

Body weight (g): raw mass; baseline for all subsequent calculations.
Lee index: (\sqrt[3]{\text{body weight (g)}} / \text{nasal‑to‑anus length (cm)}); values > 0.300 typically indicate obesity.
Adiposity index: ((\text{sum of epididymal, retroperitoneal, and mesenteric fat pads (g)} / \text{body weight (g)}) \times 100); percentages > 10 % denote elevated fat accumulation.
Body mass index (BMI for rats): (\text{body weight (g)} / \text{nasal‑to‑anus length (cm)}^{2}); thresholds vary with strain and age.
Dual‑energy X‑ray absorptiometry (DEXA): provides whole‑body fat mass and lean mass percentages; values > 25 % fat mass are frequently used as an obesity cut‑off.

Accurate determination of these indices requires standardized animal handling. Body weight should be recorded after a 12‑hour fast to minimize gastrointestinal content influence. Nasal‑to‑anus length must be measured with a calibrated caliper while the animal is lightly anesthetized to prevent tension artifacts. Fat pads are dissected post‑mortem, cleaned of connective tissue, and weighed on an analytical balance with a precision of 0.001 g. When using imaging modalities such as DEXA, calibration phantoms must be employed for each session to ensure inter‑session consistency.

Interpretation of adiposity indices depends on strain‑specific reference ranges. For commonly used strains (e.g., Sprague‑Dawley, Wistar), a Lee index above 0.300, an adiposity index exceeding 10 %, or a DEXA‑derived fat mass greater than 25 % reliably differentiate obese from lean phenotypes. Researchers should report the chosen index, calculation method, and reference values to allow direct comparison across studies.

Lee Index

The Lee Index provides a rapid, non‑invasive estimate of adiposity in laboratory rats. It combines body mass and linear size into a single dimensionless value, allowing researchers to classify subjects as lean, overweight, or obese without sacrificing the animal.

The index is calculated with the formula

[ \text{Lee Index} = \frac{\sqrt[3]{\text{body weight (g)}}}{\text{naso‑anal length (cm)}} \times 10 ]

where body weight is obtained from an analytical balance and naso‑anal length is measured with a calibrated ruler or digital caliper while the rat is gently restrained.

Interpretation guidelines commonly applied to adult rats are:

Lee Index ≤ 0.300 – lean phenotype
0.300 < Lee Index ≤ 0.340 – overweight
Lee Index > 0.340 – obese

Values may vary with strain, age, and sex; researchers should establish baseline ranges for each experimental cohort.

Procedural steps:

Weigh each rat to the nearest 0.01 g.
Measure naso‑anal length to the nearest 0.1 mm.
Compute the cubic root of the weight, divide by the length, and multiply by ten.
Record the result and compare it with the established thresholds.

Advantages of the Lee Index include minimal equipment requirements, rapid data acquisition, and a strong correlation with total body fat determined by chemical extraction. Limitations involve reduced sensitivity for extreme body sizes and inability to distinguish visceral from subcutaneous fat. Consequently, the index is most effective when paired with complementary methods such as dual‑energy X‑ray absorptiometry, magnetic resonance imaging, or post‑mortem fat pad weighing.

Adopting the Lee Index as a primary screening tool streamlines the assessment of obesity in rat models, supporting consistent classification across studies while preserving animal welfare.

Retroperitoneal and Epididymal Fat Pad Weight

Retroperitoneal and epididymal fat pads are widely accepted as reliable indicators of adiposity in laboratory rodents. After euthanasia, the animal is placed in dorsal recumbency, the abdominal cavity opened along the midline, and the peritoneum reflected to expose the retroperitoneal fat surrounding the kidneys. The epididymal fat is located adjacent to the testes; it is carefully excised, freed from connective tissue, and weighed on an analytical balance with a precision of at least 0.1 mg.

The absolute weight of each depot provides a direct measure of stored energy. To account for variations in body size, fat pad weight is commonly expressed as a percentage of total body mass or as a ratio to the sum of all measured fat depots. Typical thresholds for classifying an animal as obese are:

Retroperitoneal fat > 5 % of body weight.
Epididymal fat > 3 % of body weight.
Combined retroperitoneal + epididymal fat > 8 % of body weight.

Values exceeding these limits correlate strongly with metabolic disturbances such as hyperglycemia and dyslipidemia. When comparing groups, statistical analysis should include normality testing, followed by parametric or non‑parametric tests appropriate for the distribution of the data. Reporting should specify the method of euthanasia, the time of dissection relative to feeding, and the temperature at which the balance was calibrated.

Consistent handling of these fat pads enhances reproducibility across studies and supports the quantitative assessment of obesity in rodent models.

Dual-energy X-ray Absorptiometry (DEXA)

Dual‑energy X‑ray absorptiometry (DEXA) provides quantitative measurements of total body composition in laboratory rodents. The method distinguishes between bone mineral content, lean tissue, and adipose tissue by comparing attenuation of two X‑ray photon energies. Because fat and lean mass absorb X‑rays differently at each energy level, the resulting dual‑energy ratio yields precise estimates of fat mass without invasive procedures.

To apply DEXA for evaluating excess adiposity in rats, follow a standardized protocol. First, acclimate animals to handling and maintain a consistent fasting period (typically 4–6 hours) to reduce gastrointestinal variability. Anesthetize with an agent that minimally affects metabolic rate (e.g., isoflurane). Place the rat in a supine position on the scanner platform, aligning the spine with the device’s reference line to ensure reproducible positioning. Calibrate the instrument daily using a phantom of known composition; verify linearity across the weight range expected for the study.

During the scan, the system records attenuation data at low (≈40 keV) and high (≈70 keV) energies. Software algorithms convert these data into:

Fat mass (g) – sum of subcutaneous, visceral, and intermuscular fat.
Lean mass (g) – muscle, organ tissue, and body water.
Bone mineral content (g) – skeletal mass.

Calculate the percentage of body fat by dividing fat mass by total body mass and multiplying by 100. Values exceeding strain‑specific thresholds (e.g., >30 % for Sprague‑Dawley males) indicate an obese phenotype. Longitudinal DEXA assessments permit tracking of weight gain, fat redistribution, and response to dietary or pharmacological interventions.

Advantages of DEXA include high precision (coefficients of variation <2 % for fat mass), rapid acquisition (≈1 minute per animal), and the ability to reuse the same subjects for repeated measures. Limitations involve sensitivity to motion artifacts, potential underestimation of visceral fat in very lean animals, and the requirement for anesthesia, which may influence metabolic parameters if not carefully controlled.

In summary, DEXA delivers reliable, non‑destructive quantification of adiposity in rats, supporting rigorous evaluation of obesity‑related studies.

Glucose and Insulin Metabolism Markers

Glucose and insulin metabolism markers provide quantitative evidence of metabolic dysregulation associated with excess adiposity in laboratory rodents. Baseline fasting blood glucose, measured after a 6‑hour deprivation period, establishes the animal’s glycemic set point. An intraperitoneal glucose tolerance test (IPGTT) adds dynamic information: blood samples collected at 0, 15, 30, 60, and 120 minutes after a glucose bolus reveal the rate of glucose clearance. Elevated peak values or delayed return to baseline indicate impaired glucose handling, a hallmark of obesity‑related insulin resistance.

Plasma insulin concentration, determined by ELISA or radioimmunoassay, complements glucose data. Fasting insulin levels above the strain‑specific reference range suggest hyperinsulinemia. The Homeostatic Model Assessment of Insulin Resistance (HOMA‑IR) combines fasting glucose (mmol/L) and insulin (µU/mL) using the formula: (glucose × insulin) / 22.5. Values exceeding the established threshold for the colony identify insulin‑resistant subjects.

Additional markers reinforce the metabolic profile:

Glycated hemoglobin (HbA1c) – reflects average glucose exposure over several weeks.
Plasma C‑peptide – distinguishes endogenous insulin secretion from exogenous sources.
Adiponectin and leptin concentrations – correlate with adipose tissue expansion and insulin sensitivity.

Collectively, these measurements generate a reproducible metabolic fingerprint that differentiates lean from obese phenotypes in rats, enabling precise classification for experimental interventions.

Fasting Glucose Levels

Fasting glucose concentration provides a quantitative indicator of metabolic alterations associated with excess adiposity in laboratory rats. After an overnight (12–16 h) deprivation of food, blood is collected from the tail vein or saphenous plexus and glucose is measured with a calibrated glucometer or enzymatic assay. Values are expressed in millimoles per liter (mmol/L) and compared with established reference ranges for the specific strain, age, and sex.

Elevated fasting glucose supports the presence of insulin resistance, a hallmark of diet‑induced obesity. Typical thresholds for obese phenotypes include:

Male Sprague‑Dawley, 12 weeks: >7.0 mmol/L
Female Wistar, 10 weeks: >6.5 mmol/L

Values below these limits suggest normal glucose homeostasis, although borderline elevations may indicate early metabolic dysregulation.

Interpretation requires control of confounding variables:

Consistent fasting duration eliminates post‑prandial spikes.
Ambient temperature and stress minimization reduce catecholamine‑driven glucose fluctuations.
Repeated measurements (at least two consecutive fasting days) improve reliability.

Integrating fasting glucose data with body‑weight indices, adipose‑tissue weight, and leptin concentrations yields a comprehensive assessment of obesity status in rats.

Insulin Sensitivity Tests

Insulin sensitivity assays provide quantitative insight into metabolic dysfunction associated with excess body fat in rodents. By measuring the physiological response to administered insulin, researchers can differentiate lean from obese phenotypes and track progression of adiposity‑related insulin resistance.

Commonly employed protocols include:

Insulin tolerance test (ITT) – a single intraperitoneal injection of insulin (0.5–1.0 U/kg) followed by serial blood glucose measurements at 5‑, 15‑, 30‑, 60‑, and 90‑minute intervals. The rate of glucose decline (k‑value) reflects peripheral insulin action.
Glucose tolerance test (GTT) – oral or intraperitoneal glucose load (1.5–2.0 g/kg) with concurrent insulin sampling. Elevated peak glucose and delayed return to baseline indicate impaired insulin‑mediated clearance.
Hyperinsulinemic‑euglycemic clamp – continuous insulin infusion (typically 10 mU·kg⁻¹·min⁻¹) paired with variable glucose infusion to maintain euglycemia. The glucose infusion rate (GIR) directly quantifies whole‑body insulin sensitivity; lower GIR values correspond to higher insulin resistance.

Interpretation relies on comparison with age‑matched control rats. In obese subjects, ITT curves display attenuated glucose reduction, GTT profiles reveal prolonged hyperglycemia, and clamp studies produce reduced GIR. These metrics, when combined with body composition analysis (e.g., DEXA or MRI), confirm the presence and severity of obesity‑related metabolic impairment.

Standardization of fasting duration (4–6 h), ambient temperature (22–24 °C), and animal handling minimizes variability. Repeated testing at defined intervals (weekly or bi‑weekly) enables longitudinal monitoring of insulin responsiveness throughout dietary interventions or pharmacologic treatments.

Methodologies for Data Collection

Animal Handling and Ethical Considerations

Accurate assessment of adiposity in laboratory rodents demands strict adherence to humane handling practices and compliance with regulatory standards. Researchers must obtain approval from an institutional animal care committee before initiating any protocol that involves weight monitoring, diet manipulation, or body composition analysis. The protocol should define species‑specific housing conditions, including temperature, humidity, light‑dark cycle, and enrichment, to prevent stress‑induced metabolic alterations that could confound results.

Handling techniques must minimize anxiety and injury. Personnel should be trained in gentle restraint methods, such as tail‑pinch or tunnel capture, and should avoid prolonged immobilization. Prior to experimental measurements, rats should undergo a habituation period of at least five days during which they become accustomed to the weighing platform and any imaging devices. Continuous observation for signs of distress—piloerection, reduced grooming, abnormal gait—allows timely intervention.

Ethical considerations encompass:

Selection of the smallest viable sample size to achieve statistical power.
Implementation of humane endpoints, including criteria for excessive weight gain, impaired mobility, or metabolic decompensation.
Provision of analgesia or supportive care when invasive procedures (e.g., dual‑energy X‑ray absorptiometry) are required.
Documentation of all interventions, health status checks, and deviations from the approved protocol.

Data integrity relies on consistent animal care. Regular health assessments, balanced diet formulation, and precise record‑keeping of food intake and body weight ensure that observed obesity metrics reflect experimental variables rather than uncontrolled welfare issues.

Equipment and Protocols

Accurate assessment of adiposity in laboratory rats requires reliable instrumentation and a reproducible measurement schedule.

Essential equipment includes:

Precision analytical balance (0.01 g resolution) for body‑weight determination.
Digital caliper or length board for nose‑to‑anus measurement.
Dual‑energy X‑ray absorptiometry (DEXA) scanner or quantitative magnetic resonance (QMR) system for whole‑body fat quantification.
Metabolic cage equipped with food‑intake and water‑consumption sensors for longitudinal monitoring.
Imaging platform (ultrasound or MRI) for depot‑specific fat visualization when detailed distribution data are needed.

Standard protocol proceeds as follows:

Acclimate animals to handling and measurement environment for at least 48 h.
Record body weight on the analytical balance after a 4‑h fast to reduce gastrointestinal content variability.
Measure nose‑to‑anus length with the digital caliper; calculate the Lee index (cube root of body weight (g) ÷ body length (cm)). Values >0.300 indicate excess adiposity.
Perform DEXA or QMR scanning under light anesthesia; obtain total fat mass and lean mass percentages.
If imaging is employed, acquire cross‑sectional images of visceral and subcutaneous depots; quantify depot volumes using software analysis.
Log daily food intake and calculate feed efficiency (weight gain ÷ caloric intake) to corroborate adiposity trends.

Data from weight‑length indices, body‑composition scans, and intake metrics provide a comprehensive profile of rat obesity, enabling consistent classification across studies.

Interpreting Results and Classification

Establishing Thresholds for Obesity

Determining when a rat is obese requires quantitative cut‑off values that separate normal growth from excess adiposity. The first step is to generate a reference dataset from a healthy cohort matched for strain, sex, and age. Record body weight, length (naso‑anal distance), and body composition using techniques such as dual‑energy X‑ray absorptiometry (DEXA) or quantitative magnetic resonance (QMR). Calculate the following indices for each animal:

Weight‑for‑length ratio: weight (g) ÷ length (cm)².
Percent body fat: fat mass ÷ total mass × 100.
Adiposity index: sum of epididymal, retroperitoneal, and mesenteric fat pads ÷ body weight × 100.

Analyze the distribution of each index in the reference group. Define the obesity threshold as the value exceeding the mean by two standard deviations, or employ receiver‑operating‑characteristic (ROC) analysis to select the point that maximizes sensitivity and specificity for metabolic disturbances (e.g., hyperinsulinemia, dyslipidemia). Report the chosen cut‑off together with confidence intervals to enable reproducibility.

Validate the thresholds by applying them to an independent cohort subjected to a high‑fat diet. Confirm that animals classified as obese exhibit consistent physiological alterations, such as elevated leptin levels or impaired glucose tolerance. Adjust the cut‑offs if the validation cohort shows systematic deviation, ensuring that the final thresholds reliably reflect pathological adiposity across experimental conditions.

Differentiating Obesity from Healthy Weight Gain

Accurate identification of pathological adiposity in laboratory rats requires more than a simple increase in body mass. Normal growth trajectories differ between strains, ages, and sexes; therefore, weight gain must be evaluated against established reference curves for the specific population. When a rat’s weight exceeds the 95th percentile of its expected range, the possibility of obesity emerges, but confirmation demands additional parameters.

Body‑condition scoring provides a rapid, visual assessment. Trained observers grade the prominence of the ribs, scapular region, and lumbar area on a scale of 1 (emaciated) to 5 (severely obese). Scores of 4 or 5 indicate excess fat deposition beyond what is expected for healthy maturation. This qualitative tool complements quantitative measurements.

Quantitative techniques distinguish adipose accumulation from lean tissue growth:

Dual‑energy X‑ray absorptiometry (DEXA) yields precise percentages of fat, lean mass, and bone mineral content.
Magnetic resonance imaging (MRI) or micro‑computed tomography (µCT) maps regional fat distribution, exposing visceral versus subcutaneous deposits.
Dissection of epididymal, retroperitoneal, and mesenteric fat pads, expressed as a percentage of total body weight, offers a direct metric; values above 10 % typically signal obesity.

Metabolic profiling reinforces anatomical data. Elevated circulating leptin, hyperinsulinemia, and impaired glucose tolerance correlate strongly with excess adiposity. Regular blood sampling and oral glucose‑challenge tests detect functional consequences of fat overload, separating it from benign weight gain due to muscle hypertrophy or organ growth.

A practical workflow integrates these elements:

Record baseline weight, length, and BCS at weaning.
Monitor weekly weight and BCS, comparing each point to strain‑specific growth charts.
At predetermined intervals (e.g., 8‑week intervals), perform DEXA or µCT scans to quantify body composition.
Collect fat‑pad samples and blood for hormonal and glucose analyses when body‑fat percentage exceeds 20 % or BCS reaches 4.
Classify the animal as obese only if at least two independent measures—body‑fat proportion, fat‑pad weight, and metabolic disturbance—confirm excess adiposity.

Applying this multidimensional approach prevents misclassification of normal developmental weight gain as obesity, ensuring experimental validity and animal welfare.

Factors Influencing Obesity Assessment

Strain and Genetic Background

Strain selection directly influences the reliability of obesity assessments in laboratory rats. Genetic background determines baseline body weight, fat distribution, and metabolic response to dietary challenges. Consequently, choosing an appropriate strain is essential for reproducible results.

Commonly used strains include:

Sprague‑Dawley – outbred, moderate susceptibility to diet‑induced weight gain; suitable for general obesity models.
Wistar – outbred, similar to Sprague‑Dawley but with slightly higher baseline adiposity; useful for comparative studies.
Zucker (fa/fa) – homozygous for leptin‑receptor mutation; exhibits rapid, severe obesity without high‑fat feeding; ideal for genetic obesity investigations.
Obese Zucker (fa/+) heterozygotes – display intermediate phenotype; allow evaluation of gene‑dose effects.
Fischer 344 – inbred, resistant to diet‑induced obesity; serves as a lean control group.
Brown Norway – inbred, low propensity for excess weight; provides a contrasting genetic background.

When planning experiments, consider the following factors:

Baseline body composition measured by dual‑energy X‑ray absorptiometry (DXA) or magnetic resonance imaging (MRI) for each strain.
Response to high‑fat or high‑calorie diets documented in peer‑reviewed literature; select strains whose weight trajectories match the experimental timeline.
Presence of known metabolic mutations (e.g., leptin‑receptor deficiency) that may confound interpretation of pharmacological interventions.
Age and sex, because genetic background interacts with hormonal status to modify adiposity.

Documentation of strain provenance, breeding history, and any sub‑line variations is mandatory. Genetic drift can alter phenotypic traits over generations; periodic genotyping ensures consistency. For studies requiring precise control of genetic variables, use congenic or consomic lines where a single chromosome segment from an obesity‑prone donor is introgressed onto a lean recipient background.

In summary, strain and genetic background dictate the threshold for classifying a rat as obese, shape the pattern of weight gain, and affect the validity of metabolic endpoints. Careful selection, thorough phenotypic baseline, and rigorous genetic verification constitute the foundation for accurate obesity determination in rat models.

Age and Sex

Age critically influences the interpretation of body weight and composition data in laboratory rats. Juvenile animals exhibit rapid growth; their weight increases reflect skeletal and muscle development rather than excess adiposity. Consequently, reference weight ranges must be age‑specific, typically derived from growth curves that define the 95 % confidence interval for each postnatal day. Adult rats (≥ 12 weeks) display relatively stable weight, allowing direct comparison of absolute body mass to established obesity thresholds. When evaluating older rats (> 18 months), age‑related sarcopenia can mask adipose accumulation, necessitating complementary measurements such as fat pad mass or imaging‑based body composition.

Sex determines baseline body mass and fat distribution. Male rats normally weigh 20–30 % more than females of the same age, and their visceral fat proportion is higher. Female rats tend to accumulate subcutaneous fat and exhibit estrous‑cycle fluctuations in body weight. These physiological differences require separate reference values for each sex. Failure to segregate data by sex leads to misclassification of obesity status, especially when using simple weight‑to‑length ratios.

Accurate obesity assessment combines weight‑based indices with direct quantification of adipose tissue. Recommended practices include:

Record body weight weekly; compare to age‑ and sex‑matched growth charts.
Measure naso‑anal length; calculate the Lee index (cube root of body weight (g) ÷ nasal‑anal length (cm) × 10). Apply sex‑specific cut‑off values.
Harvest and weigh epididymal (male) or peri‑ovarian (female) fat pads for terminal studies.
Employ non‑invasive techniques (DEXA, MRI, micro‑CT) to estimate total and regional fat percentages; calibrate against sex‑specific standards.

Experimental designs must incorporate age‑matched and sex‑matched control groups, stratify data accordingly, and apply statistical models that include age and sex as covariates. This approach ensures that obesity classifications reflect true adiposity rather than normal developmental or sexual dimorphism.

Dietary Interventions

Dietary manipulation provides a reliable means to provoke and evaluate adiposity in laboratory rats. Researchers induce weight gain by altering macronutrient ratios, caloric density, and feeding schedules, then compare physiological and morphological markers against control cohorts.

A high‑fat diet (HFD) typically contains 45–60 % of calories from fat, with a modest increase in total energy density (4.5–5.5 kcal g⁻¹). A high‑sugar diet (HSD) supplies 30–40 % of calories from simple carbohydrates, often combined with moderate fat content. Calorie‑restricted regimens maintain intake at 70–80 % of ad libitum levels to establish a baseline for lean phenotypes. Each protocol requires precise formulation, batch verification, and consistent storage conditions to avoid nutrient degradation.

Key steps in the assessment process include:

Baseline measurement: Record body weight and body length before diet initiation to calculate the Lee index (cube root of body weight (g) ÷ body length (cm)).
Daily monitoring: Weigh animals at the same time each day, document food consumption per cage, and adjust feed to maintain the intended caloric surplus or restriction.
Endpoint analysis: At the study’s conclusion, determine fat pad weights (epididymal, retroperitoneal, mesenteric) and perform magnetic resonance imaging or dual‑energy X‑ray absorptiometry for whole‑body composition.
Biochemical profiling: Measure serum leptin, insulin, triglycerides, and glucose to corroborate phenotypic classification.

Consistent application of these dietary interventions, coupled with rigorous phenotypic measurements, enables accurate classification of obese versus non‑obese rats and supports downstream investigations of metabolic dysfunction.