Effects of Probiotic Compounds as Feed Additives on Performance, Rumen Fermentation and Metabolic Blood Indices During the Preweaning Period of Holstein Dairy Calves

Probiotics are promising alternatives to antibiotics in calf rearing, but effects of Lactobacillus species combined with Bacillus subtilis on preweaning development remain unclear. Two probiotic consortia—L. plantarum + B. subtilis (TG1) vs. L. acidophilus + B. subtilis (TG2)—were compared in Holstein calves. Forty-eight newborn calves (initial BW 37.0 ± 2.7 kg) were randomly assigned to control (CG) or probiotic groups (TG1, TG2) from day 3 for 60 days. Growth, fecal scores, serum biochemistry, antioxidant/immune indices, rumen fermentation, 16S rRNA, and untargeted metabolomics were measured. Compared with CG, TG1 increased final body weight (p = 0.002) and average daily gain (ADG, p = 0.002), whereas TG2 did not differ from CG in either parameter. Both TG1 and TG2 decreased feed conversion ratio (FCR, p < 0.001), with TG1 showing a lower value than TG2. At day 60, TG1 had greater body oblique length than TG2 (p = 0.030), whereas neither TG1 nor TG2 differed from CG. Serum analysis: TG1 decreased ALT (p = 0.039) and increased CAT (p = 0.024) and HDL-C (p = 0.047) compared with CG, while TG2 did not differ from CG in these parameters. Both TG1 and TG2 decreased CRE compared with CG (p < 0.001). TG2 increased GSH-Px compared with CG and TG1 (p = 0.044), whereas TG1 did not differ from CG. Rumen fermentation: both TG1 and TG2 increased BCP compared with CG (p = 0.009); TG1 elevated isobutyrate compared with CG (p = 0.030), whereas TG2 did not differ from CG; TG2 reduced NH3-N compared with CG (p = 0.026), whereas TG1 did not differ from CG. No α/β-diversity differences were observed, but taxa were modulated: TG1 enriched Actinobacteria and Olsenella; TG2 enriched Bacteroidetes, Prevotella, and Ruminococcus. Metabolomics: TG1 upregulated steroid hormone and phenylalanine metabolism; TG2 enhanced unsaturated fatty acid biosynthesis. Correlation networks linked Ruminococcus and UCG-005 to antioxidant status and VFA profiles. These findings indicate that different probiotic consortia are associated with distinct physiological outcomes in preweaning calves, supporting consortium selection for targeted feeding.