# In Vitro Evaluation of Ruminal Fermentation and Methane Production in Response to the Addition of Modified Nano-Bentonite with or Without Saccharomyces cerevisiae to a Forage-Based Diet

## TL;DR

This study explores how modified nano-bentonite, with or without yeast, can reduce methane emissions in ruminants without harming nutrient digestion in a high-fiber diet.

## Contribution

The novel use of modified nano-bentonite combined with yeast as a sustainable alternative to antibiotics for methane mitigation in ruminant diets.

## Key findings

- Modified nano-bentonite combined with yeast significantly reduced methane emissions compared to the control.
- MNBHigh with yeast increased short-chain fatty acid concentrations and improved ruminal properties.
- MNBHigh without yeast reduced hemicellulose degradation and protozoal counts in rumen fermentation.

## Abstract

Reducing greenhouse gas (GHG) emissions from farm animals is important for protecting the environment. In this study, we developed a special form of natural clay, called modified nano-bentonite (MNB), and combined it with a type of yeast (Saccharomyces cerevisiae) commonly used in animal diets. We tested whether this mixture could reduce ruminal methane (CH4) production without adverse effects on ruminal nutrient degradability in a high-fiber diet. In this study, MNB was developed by organically modifying natural bentonite with sodium dodecyl sulfate (SDS). Our results showed that the combination of MNB with yeast helps lower CH4 emissions and improves nutrient degradability, which could be a natural and eco-friendly alternative to traditional antibiotics used in animal farming.

Modified nano-clays, alone or combined with probiotics, may offer a novel and sustainable approach to improve ruminal fermentation and mitigate CH4 emissions in high-fiber diets. This study evaluated the properties and effects of modified nano-bentonite (MNB), with or without yeast (Saccharomyces cerevisiae), compared to natural bentonite (NB) and monensin, using the in vitro gas production (GP) technique. The substrate used was a basal diet composed primarily of forage (Trifolium alexandrinum clover) in a 70:30 forage-to-concentrate ratio. The treatments were a control group receiving the basal diet without additives; a monensin-added diet containing 40 mg/kg of dry matter (DM); a yeast-added diet with Saccharomyces cerevisiae at 2 × 108 CFU/g of DM; a NB clay-added diet at 5 g/kg of DM; and MNB diets added at two levels (0.5 g/kg of DM (MNBLow) and 1 g/kg of DM (MNBHigh)), with or without S. cerevisiae. MNB showed a smaller particle size and improved properties, such as higher conductivity, surface area, and cation exchange capacity, than NB. Sulfur and related functional groups were detected only in MNB. No differences were observed in total GP, while both the monensin diet and the MNBHigh-with-yeast diet significantly reduced CH4 emissions compared to the control (p < 0.05). The MNBHigh-without-yeast combination significantly (p < 0.05) reduced hemicellulose degradation, as well as total protozoal counts, including Isotricha and Epidinium spp. (p < 0.05), compared to the control. Ammonia levels did not differ significantly among treatments, while NB and MNBHigh diets tended to have (p = 0.063) the highest short-chain fatty acid (SCFA) concentrations. These findings suggest the potential modulatory effects of yeast and MNB on rumen fermentation dynamics and CH4 mitigation.