Impact of Unbalanced and Alternative Protein Diets on the Cecal Bacteria and Skeletal Development: A Comparative Study in Rats

Standard methods to assess diet quality provide only limited insights, making holistic approaches essential for exploring the broader effects of specific diets. Previous research has introduced the growing rat as a powerful model—particularly focused on skeletal development—to evaluate various aspects of diet quality. This includes overall weight and length gain, micronutrient-dependent bone mineralization, structural integrity, and functional resistance to fracture, all of which are sensitive to nutritional deficiencies. The diet-gut microbiome-bone axis underscores the microbiome as a key intermediary influencing bone development. However, variations in experimental design across microbiome studies often lead to inconclusive or inconsistent results. This comparative study integrates four 16S rRNA gene amplicon sequencing datasets investigating effects of alternative protein sources and unbalanced Western diets on cecal microbial communities and correlating bone parameters. Although these datasets share similarities in overall experimental design and research aims, they differ in sequencing strategies. Genera such as Bacteroides_H_857956, Lactobacillus, Lepagella, Parabacteroides_B_862066, Phocaeicola_A, UBA3263 and UBA3282 were found to correlate with skeletal parameters. Additional genera—including Acetitomaculum, Turicimonas, Mucispirillum, Turicibacter, Blautia_A_141781, Ligilactobacillus, Staphylococcus , and Paramuribaculum —showed correlations with the severity of skeletal abnormalities. Microbiome functional analyses revealed overlapping enrichment of predicted pathways related to redox reactions, energy metabolism, and amino acid biosynthesis. Here, our joint analysis of four alternative protein and unbalanced diet data sets revealed the severe effects of diet protein level and protein processing on bacterial composition of the caecum and skeletal growth, suggesting that compositional change and specific genera could be used as complex health indicators.