Effects of inactivated Lactobacillus acidophilus on growth performance, blood parameters, fecal fermentation and microbiota in neonatal Holstein calves

Inactivated probiotics can exhibit beneficial properties similar to live probiotics while offering greater stability and safety. However, the understanding of how inactivated probiotics affect the growth, health, and gut microbiota of neonatal calves is still limited. This study investigated the effect of inactivated <i>Lactobacillus acidophilus</i> (ILA, ≥1 × 10⁸ CFU equivalents/mL) on growth performance, blood parameters, and fecal fermentation and microbiota of neonatal dairy calves. Forty female Holstein calves (37.92 ± 2.04 kg; 2.45 ± 0.96 d) were randomly assigned to a basal diet supplemented with 0 (CK), 2, 4, or 6 mL/d of ILA (ILA2, ILA4, and ILA6) until 54 d of age. The results showed that while ILA supplementation did not affect body weight or feed efficiency, it linearly increased structural growth indices (body length and heart girth) at both 30 and 54 d of age ( <i>P</i> < 0.05). Crucially, ILA significantly reduced fecal scores (<i>P</i> < 0.001) and diarrhea incidence (<i>P</i> = 0.019), particularly in the ILA4 group. Serum analysis revealed that ILA elevated the levels of immunoglobulins A, G, and M (<i>P</i> < 0.001), as well as total protein, globulin, glucose, and superoxide dismutase (<i>P</i> < 0.05), indicating improved immune status and metabolic health. Furthermore, ILA modulated fecal fermentation, inducing a quadratic response in acetic acid (<i>P</i> = 0.005) and total volatile fatty acids (<i>P</i> = 0.010). Microbiome analysis indicated linear increases in Firmicutes, Proteobacteria, and the genus <i>Faecalibacterium</i>, coupled with linear decreases in Bacteroidota, Spirochaetota, and <i>Rikenellaceae_RC9_gut_group</i> (<i>P</i> < 0.05). This ILA-induced microbial shift enriched functional pathways associated with transport, transcription, replication, and butanoate metabolism compared with the CK group (<i>P</i> < 0.05). Spearman correlation analysis identified significant correlations between fecal microbiota composition and host growth, metabolism, and health. Collectively, the beneficial effects of ILA on structural growth, diarrhea reduction, fecal fermentation, and metabolic health in calves are closely related to dynamic changes in fecal microbiota. These beneficial effects may stem from ILA's ability to modulate the composition, diversity, and function of the gut microbiota. This study offers a scientific basis for the application of ILA in calf production.