Abstract
Weaning is a critical developmental stage in puppies often accompanied by gastrointestinal disturbances and empiric antibiotic use. This double-blind, placebo-controlled trial evaluated the safety and efficacy of two canine milk-derived probiotic strains, Lacticaseibacillus rhamnosus CECT 30021 and Lactiplantibacillus plantarum CECT 30022, in recently weaned Labrador Retriever puppies. Forty-nine healthy puppies were enrolled and allocated to receive a daily capsule containing either strain alone, a combination of both, or placebo for six weeks, followed by a six-week observation period. Probiotic supplementation was well tolerated, and clinical, hematological, and biochemical parameters remained within normal ranges. Compared with placebo, the probiotic-treated puppies experienced fewer gastrointestinal infections and required less antibiotic therapy. Supplementation helped maintain normal fecal consistency, prevented increases in fecal calprotectin and inflammatory cytokines, and produced significantly higher fecal short-chain fatty acid concentrations. Systemically, probiotic intake was associated with lower serum cortisol, IL-8, TNF-α, and cholesterol, and stronger antibody responses to vaccination. Probiotic-supplemented puppies also showed faster weight gain during treatment, and this difference persisted after supplementation ended. Overall, these findings indicate that both strains, administered individually or in combination, safely support gastrointestinal, metabolic, endocrine, and immunological health in Labrador Retriever puppies during weaning and may help reduce the need for antibiotics.