# Effect of dual bacterial combinations on in vitro nutrient degradability, gas production, methane emission, ruminal fermentation parameters and predictive values in sheep

## TL;DR

This study shows that combining two types of bacteria in sheep feed improves digestion and reduces methane emissions, offering a sustainable solution for the sheep industry.

## Contribution

The study introduces a dual probiotic strategy that enhances in vitro fermentation efficiency in sheep feed.

## Key findings

- Dual bacterial combinations significantly increased gas production and decreased methane emissions.
- AB2 and CP4 groups showed the highest improvements in nutrient degradability and fermentation parameters.
- Probiotics reduced ammonia and protozoa count while increasing volatile fatty acids.

## 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 × 109 (low; 2) and 4 × 109 (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 (AB2 and AB4), Lactobacillus casei + Lactobacillus plantarum (CP2 and CP4), and Bacillus licheniformis + Bacillus subtilis (LS2 and LS4). 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 CPc exhibited superiority compared to the control group (P < 0.001). The AB2 group had the highest dry matter content compared to CP4, LS2, LS4, and the control group (P < 0.01). Crude fiber content was highest in AB2, AB4, CP2, and CP4 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 AB2, AB4, CP2, and CP4 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 AB2 group (P < 0.001). All tested probiotics showed significantly lower values for NH3-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 CPc 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.