Simple SummaryPreclinical studies evaluating the safety and efficacy of new drugs require experimental animals, which is important to ensure both adequate dosage and animal welfare. Administration through voluntary ingestion can improve animal wellbeing. We aimed to develop a protocol using small gelatin cubes to train rats, assessing the influence of age, sex, fasting, flavors (vanilla), and sweeteners (sucralose) to accept the new food. We tested the usefulness of the protocol to supplement rats during lactation. We demonstrated that most animals were easily trained to accept the gelatin cube in 2–3 days. However, some rats refused the new food even after 8 days. The proportion of rats that did not train was higher in adult males compared to young males, adults, and young females, suggesting the influence of sex. Four-hour food deprivation reduced the time for acceptance only in females, but flavoring or sweeteners in the gelatin did not modify it. Rats trained prior to gestation remembered training 2 months later and ate a gelatin containing a supplement daily during lactation for 1–5 min, without problems with the pups. We conclude that gelatin-based supplementation can be used for drug studies in rats, ensuring adequate dosage and wellbeing, which is important for the detection of non-trained rats.AbstractDrug safety and efficacy studies frequently use oral gavage, but repetitive usage may cause problems. Administration through voluntary ingestion represents an opportunity for refinement. We aimed to develop a protocol for voluntary ingestion of gelatin-based supplements in rats, assessing the influence of age, sex, fasting (4 h), and additives (vanilla, VF; sucralose, S), and to test it in lactating dams. Three-week-old and 5-month-old Sprague-Dawley rats were placed individually in an empty cage containing a gelatin cube and trained daily (5 days/week), recording the day the whole cube was consumed (latency). Rats trained prior to gestation were offered a gelatin containing 250 mg/kg cocoa shell extract (CSE) during lactation. Rats that did not eat the cube after 8 training days were considered non-habituated, with a proportion similar in young males (7.1%), young females (11.1%), and adult females (10.3%), but significantly higher in adult males (39.3%). Excluding non-habituated rats, latency was 2–3 days, without differences between young and adult rats (p= 0.657) or between males and females (p= 0.189). VF or VF + S in the gelatin did not modify latency, while fasting significantly reduced it in females (p= 0.007) but not in males (p= 0.501). During lactation, trained females ate the CSE-gelatin within 1–5 min without litter problems. Conclusions: Acceptance of a gelatin-based supplement is negatively influenced by male sex, facilitated by fasting, and not modified by additives. Training is remembered after 2 months and does not interfere with lactation. Gelatin-based voluntary ingestion is suitable to administer drugs that need to pass through the digestive system, ensuring adequate dosage, and is important to detect non-habituated rats prior to the study. The current protocol may be implemented by training the rats in their own cage.