Abstract The aim of the current study was to investigate the effects of incubating the tested feed with three dual combinations of two strains each at two doses: 2 × 10 9 (low; 2) and 4 × 10 9 (high; 4) CFU/g feed/combination on nutrient degradability, gas production, methane emission, fermentation parameters, and protozoa count using a sheep model. The three combinations were Lactobacillus acidophilus + Lactobacillus bulgaricus (AB 2 and AB 4 ) , Lactobacillus casei + Lactobacillus plantarum (CP 2 and CP 4 ), and Bacillus licheniformis + Bacillus subtilis (LS 2 and LS 4 ). The in vitro evaluation demonstrated a significant increase in gas production ( P < 0.001) and a significant decrease in methane emissions ( P < 0.001) with all probiotic combinations. Among them, AB and CP c exhibited superiority compared to the control group ( P < 0.001). The AB 2 group had the highest dry matter content compared to CP 4 , LS 2 , LS 4 , and the control group ( P < 0.01). Crude fiber content was highest in AB 2 , AB 4 , CP 2 , and CP 4 compared to the other groups ( P < 0.01). Total gas production (TGP) was improved in all probiotic groups at different time points with the best results in the AB 2 , AB 4 , CP 2 , and CP 4 groups ( P < 0.01). The predictive values of organic matter degradability (OMD), short-chain fatty acids (SCFA), microbial crude protein (MCP), metabolizable energy (ME), and net energy for lactation (NEL) were significantly improved in all tested probiotics, with the greatest improvement observed in the AB 2 group ( P < 0.001). All tested probiotics showed significantly lower values for NH 3 -N, pH, and protozoa count, while TVFA concentrations were significantly higher compared to the basal diet without probiotic supplements ( P < 0.001). The combinations AB and CP c produced the most favorable results among all the in vitro tested parameters. The findings demonstrate the potential of a dual probiotic strategy to enhance in vitro fermentation efficiency. This approach improves nutrient degradability and associated predictive values, while also reducing methane production. This research provides valuable insights for implementing sustainable and efficient dietary interventions in the sheep industry, particularly in light of the challenges posed by climate change.
Saleem et al. (2026) studied this question.