Animals : an Open Access Journal from MDPI · 12 authors, 8 centres
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This study developed and validated a humane endpoint scoring system for a rat model of type 2 diabetes induced by fructose feeding and streptozotocin injection. The most useful parameters for monitoring welfare were dehydration, lack of grooming, posture, abdominal visualization/palpation, and fecal appearance. The scoring system successfully identified signs of suffering without any animal reaching the critical score, supporting its use for ethical monitoring in diabetes research.
**Background:** The use of laboratory animals in diabetes research raises ethical concerns, particularly regarding pain and distress. Humane endpoints are physiological or behavioral signs that define when an animal's suffering must be mitigated or ended. This study aimed to develop and validate a humane endpoint scoring system for a rat model of type 2 diabetes induced by fructose feeding and streptozotocin (STZ) injection, a non-genetic model that mimics the pathogenesis of type 2 diabetes.
**Methods:** Twenty-four male Sprague-Dawley rats were randomly assigned to a control group (n=8) or an induced group (n=16). The induced group received 10% fructose in drinking water for 14 days, followed by a single intraperitoneal injection of STZ (40 mg/kg). The control group received vehicle (0.1 M citrate buffer). Weekly monitoring included body weight, food and water consumption, and a scoring sheet with 14 parameters (body condition/weight, posture, hair/tail appearance and grooming, grimace scale, position of ears and whiskers, nose/cheeks, walk, skin, mental status, response to external stimuli, hydration status, stool appearance, convulsions, and abdominal visualization/palpation). Each parameter was scored 0–3, with a critical total score of 4 indicating need for euthanasia. Blood glucose was measured at weeks 4 and 6 (fasting and 2 h post-feeding). After 7 weeks, animals were euthanized; serum glucose, albumin, cholesterol, and triglycerides were measured, and kidneys were examined histologically. Murinometric (Lee index, BMI, nasal-anal length, thoracic and abdominal perimeters) and nutritional (specific rate of weight gain, food efficiency coefficient) parameters were calculated.
**Key Results:** No deaths occurred during the protocol. Induced animals showed significantly lower body weight from week 2 onward (p<0.05 at week 2; p<0.0001 weeks 3–7). After STZ injection, 81.25% of induced animals lost weight, increasing to 93.75% by the end. Food consumption was lower in induced animals during fructose feeding (p<0.0001) but higher after STZ (p<0.0001). Water consumption was significantly higher in induced animals (p<0.001). Humane endpoint changes were observed only in the induced group: dehydration (week 2), lack of grooming (weeks 3,4,6,7), narrowing of the orbital area (week 3), curved posture (weeks 5,6,7), liquid/pasty diarrhea (week 7), and abdominal distension. No animal reached the critical score of 4, but maximum scores were significantly higher in induced animals (p<0.01). Blood glucose levels were significantly higher in induced animals 2 h post-feeding at weeks 4 (568.92 ± 71.43 mg/dL vs. 141.00 ± 15.22 mg/dL) and 6 (600.00 ± 0.00 mg/dL vs. 141.63 ± 9.55 mg/dL) (p<0.0001). Fasting glucose at necropsy was also higher in induced animals (251.73 ± 136.84 mg/dL vs. 107.87 ± 25.23 mg/dL, p<0.05). Kidney relative weights were significantly increased in induced animals (right: 4.952 ± 0.676 g/kg vs. 4.053 ± 0.165 g/kg, p<0.01; left: 4.802 ± 0.593 g/kg vs. 3.861 ± 0.266 g/kg, p<0.001), with histological evidence of focal cell necrosis and tubular vacuolation. Murinometric and nutritional parameters (final body weight, nasal-anal length, thoracic and abdominal perimeters, Lee index, BMI, specific rate of weight gain, food efficiency coefficient) were all significantly lower in induced animals (p<0.01 to p<0.0001). No significant differences were found in body temperature, serum albumin, cholesterol, or triglycerides.
**Clinical Implications:** This study provides a validated, practical humane endpoint scoring system for the fructose-fed and STZ-injected rat model of type 2 diabetes. The most sensitive parameters were dehydration, lack of grooming, posture, abdominal visualization/palpation, and fecal appearance. The system allows objective, reproducible monitoring of animal welfare, reducing subjectivity and ensuring ethical compliance. The model successfully induced diabetes (hyperglycemia, polyuria, polyphagia, polydipsia, weight loss, and renal changes) without causing severe suffering or mortality. This scoring table can be used by other researchers to monitor welfare in this and potentially other diabetes models, supporting the 3R principles (Replacement, Reduction, Refinement).