# In vitro screening of dihalomethanes as potential methane inhibitors in dairy cows

## TL;DR

This study tested dihalomethanes as methane inhibitors in dairy cows and found that some significantly reduced methane production but also affected rumen fermentation.

## Contribution

The study identifies specific dihalomethanes with high methane-mitigating potential while highlighting their impact on rumen fermentation.

## Key findings

- Dibromomethane, bromoiodomethane, and diiodomethane reduced methane production by over 90%.
- Dichloromethane had no significant methane-mitigating effect.
- Methane reduction was accompanied by reduced volatile fatty acid production.

## Abstract

Summary: Halogenated compounds, particularly trihalomethanes, have demonstrated large potential as antimethanogenic feed additives. However, concerns can be raised in relation to the use of these compounds as feed additives due to the transfer of halogenated metabolites into milk. This in vitro study aimed to identify dihalomethanes with CH4-mitigating potential to potentially reduce the transfer of halogens to milk when used as feed additives. When added to corn silage, CH4 production was reduced by the dihalomethanes dibromomethane (>90%), bromoiodomethane (>90%), and chloroiodomethane (58%) compared with corn silage incubated alone in a buffered rumen inoculum, whereas dichloromethane showed no CH4-mitigating effect. However, reductions in CH4 production were accompanied by reductions in total volatile fatty acid (VFA) production; hence, future studies should focus on finding the optimal dose of the dihalomethanes resulting in the largest reduction without having negative effects on overall rumen fermentation.

Summary: Halogenated compounds, particularly trihalomethanes, have demonstrated large potential as antimethanogenic feed additives. However, concerns can be raised in relation to the use of these compounds as feed additives due to the transfer of halogenated metabolites into milk. This in vitro study aimed to identify dihalomethanes with CH4-mitigating potential to potentially reduce the transfer of halogens to milk when used as feed additives. When added to corn silage, CH4 production was reduced by the dihalomethanes dibromomethane (>90%), bromoiodomethane (>90%), and chloroiodomethane (58%) compared with corn silage incubated alone in a buffered rumen inoculum, whereas dichloromethane showed no CH4-mitigating effect. However, reductions in CH4 production were accompanied by reductions in total volatile fatty acid (VFA) production; hence, future studies should focus on finding the optimal dose of the dihalomethanes resulting in the largest reduction without having negative effects on overall rumen fermentation.

•Four out of five dihalomethanes reduced CH4 production from corn silage in vitro.•Dibromomethane, bromoiodomethane, and diiodomethane reduced CH4 by more than 90%.•Dichloromethane did not reduce CH4 production compared with the control treatment.•Reductions in CH4 production were accompanied by reductions in total VFA production.

Four out of five dihalomethanes reduced CH4 production from corn silage in vitro.

Dibromomethane, bromoiodomethane, and diiodomethane reduced CH4 by more than 90%.

Dichloromethane did not reduce CH4 production compared with the control treatment.

Reductions in CH4 production were accompanied by reductions in total VFA production.

Volatile halogenated compounds are known to possess antimethanogenic properties with most in vivo studies focusing on trihalomethanes. However, concerns can be raised in relation to the use of these compounds as feed additives due to the transfer of halogenated metabolites into milk, which may pose a threat to consumer health. Hence, the objective of this in vitro study was to investigate the CH4-mitigating potential of 5 dihalomethanes (DHM) to potentially reduce the transfer of halogenated compounds when used as feed additives in the future. The DHM, dibromomethane (BM), bromoiodomethane (BIM), dichloromethane (CM), chloroiodomethane (CIM), and diiodomethane (DIIM) were dissolved in 99% ethanol, reaching final solutions of 2 mM. In 2 separate runs, 0.1 mL of the solutions were incubated as triplicates in buffered rumen fluid together with 0.5 ± 0.02 g (± SD) of corn silage. Similarly, corn silage and buffered rumen fluid with the addition of 0.1 mL of 99% ethanol served as control and blank samples, respectively. Accumulated gas production was determined using the ANKOMRF system equipped with airtight gasbags. Gas composition in collected gas was determined after 24 h, whereas the concentration of VFA in the fermented rumen fluid was determined after 48 h of incubation. All DHM except CM resulted in significant reductions and increases in CH4 and H2 production, respectively, compared with the standard feed incubated without DHM. Hence, BIM, BM, and DIIM reduced CH4 production per gram of incubated DM by more than 90%, whereas CIM reduced CH4 production by 58% per gram of incubated DM. Reductions in CH4 production were accompanied by significant reductions in total VFA production, proportions of acetate in total VFA, and increases in proportions of propionate compared with the control treatment. In conclusion, BM, BIM, DIIM, and to a lesser extent CIM were efficient CH4 inhibitors; however, the DHM also had negative effects on fermentation parameters.