Abstract
BACKGROUND AND AIM: Proteases are key enzymes that hydrolyze peptide bonds enhancing the utilization of feed protein, improving nutrient efficiency, and reducing the need for costly protein ingredients. Despite their growing use in animal nutrition, comparative studies between fungal and microbial proteases in ruminants remain scarce. This study aimed to evaluate the influence of fungal and microbial proteases (25 U/g each) on the microbial composition of rumen fluid and feces, and on the chemical composition of digestive contents in Kazakh White Head bulls. MATERIALS AND METHODS: Twenty bulls (14-15 months old; 310-320 kg) fitted with rumen fistulas were divided into three groups: a control group and two treatment groups, each receiving a basal diet supplemented with either fungal or microbial protease. Rumen fluid and fecal samples were analyzed for taxonomic profiles using next-generation sequencing (MiSeq, Illumina) of the 16S ribosomal RNA V3-V4 region. Chemical composition (dry matter [DM], crude protein [CP], crude fat [CF], crude fiber, and ash) was determined according to GOST mass fraction of DM 31640, mass fraction of CP 13496.4, mass fraction of CF 13496.15, mass fraction of crude fiber 31675, and mass fraction of crude ash 26226 standards. Statistical analysis was performed using the Mann-Whitney U-test (p ≤ 0.05). RESULTS: Microbial protease supplementation increased the abundance of beneficial phyla Bacillota (70.1%) and Bacteroidota (19.5%) in rumen fluid, with a corresponding rise in DM (+6.3%), CF (+9.4%), and CP (+7.9%) relative to control. In feces, Bacillota (70.7%) and Bacteroidota (15.5%) predominated. No opportunistic pathogens (e.g., Pseudomonas and Sutterella) were detected in the microbial protease group, indicating improved microbial balance and intestinal protection. Fungal protease exerted milder effects, with modest increases in nutrient fractions. CONCLUSION: Microbial protease was more effective than fungal protease in optimizing rumen microbiota and enhancing nutrient digestibility in bulls. Its use may support environmentally sustainable livestock production by reducing nitrogen excretion and dependence on high-protein feed ingredients. These findings provide a scientific basis for breed-adapted enzymatic feeding strategies in ruminants.