EFFECT OF BROMOFORM ON ENTERIC METHANE, AND RUMEN FERMENTATION CHARACTERISTICS

Author

Mohamed G. Embaby, Southern Illinois University CarbondaleFollow

Date of Award

12-1-2024

Degree Name

Doctor of Philosophy

Department

Agricultural Sciences

First Advisor

AbuGhazaleh, Amer

Abstract

Ruminant livestock are a significant contributor to global greenhouse gas emissions, accounting for 6% of total emissions worldwide. Over the past two decades, extensive research has examined the environmental impact of methane (CH4) emissions from these animals. Recent scientific interest has focused on seaweed as a potential CH4 inhibitor, with the compound bromoform identified as the key active component responsible for reducing CH4. A review of the literature, and different mitigation strategies are mentioned in chapter 1 of this dissertation.The second chapter of this dissertation discussed the effects of different bromoform concentration levels on enteric CH4 production and rumen fermentation using 24 h rumen batch culture systems. The doses tested ranged from 0.02 to 13.4 μl bromoform per liter of rumen fluid. The ANKOM batch culture system was utilized, with samples collected after 24 h of incubation for analysis of volatile fatty acids (VFAs) and CH4 levels. Statistical analysis using one-way ANOVA, Student's T-test, and Dunnett's model in JMP Pro revealed a linear reduction in CH4 production as bromoform concentrations increased from 0.02 to 13.4 μl/L (P<0.001). Total CH4 mitigation occurred at doses from 0.20 to 13.4 μl/L (P<0.01), while lower doses of 0.02, 0.05, and 0.10 μl/L reduced CH4 production by 4, 8, and 23% respectively compared to the control (P<0.001). Total VFA concentrations responded quadratically to bromoform treatment. High doses (1.67-13.4 μl/L) caused a reduction in total VFAs (tVFAs), whereas low doses (0.02-0.10 μl/L) had no significant effect. Decreased tVFAs at high doses corresponded with reductions in the molar proportions of acetate and propionate and an increased acetate: propionate ratio (P<0.001). Conversely, low doses decreased the acetate: propionate ratio (P<0.001). Butyrate molar proportions increased across all dose levels (P<0.01).On Chapter 3, we investigated the long-term effects of different doses of bromoform, an active compound from the red seaweed Asparagopsis taxiformis, on CH4 production and rumen fermentation characteristics using a continuous fermenter system. Four treatments were tested - control (no bromoform) and 0.14, 0.27, 0.56 μl/L bromoform doses - in a Latin square design with four 10-day periods. Bromoform doses were divided and dosed with a 50:50 forage: concentrate diet three times daily. Results showed bromoform doses up to 0.56 μl/L reduced enteric CH4 production by 98% compared to control, while increasing hydrogen (H2) concentrations by 550-860% and decreasing ammonia (NH3-N) and pH. Total VFA and dry matter digestibility (DMD) were unaffected, but bromoform altered individual VFA proportions, decreasing acetate, iso-butyrate and acetate: propionate ratio while increasing propionate and butyrate.The withdrawal effect of bromoform was investigated in chapter 4 using four continuous fermenters over two 10-day periods. Treatments were control with no bromoform (T1), bromoform (0.14 μl/L rumen fluid/day) added for the first 5 days (T2), 7 days (T3), or all 10 days (T4) of each period. Bromoform addition reduced CH4 concentrations by 98% and H2 levels compared to control, but these effects nearly disappeared 3-5 days after bromoform dosing ended. The molar proportions of acetate decreased while the molar proportions of propionate increased with bromoform, though these changes were less pronounced 3-5 days post-dosing. Total VFA, NH3-N, and DMD were unaffected by treatment diets. The last chapter of this dissertation discusses the use of Laser Methane Detector (LMD) and Optical Gas Imaging (OGI) for detecting and quantifying enteric CH₄ emissions from ruminants in vitro. Four dietary treatments with varying forage-to-concentrate ratios(F:C) were tested in rumen fermenters. The control treatment (T1) was a 50:50 forage to concentrate diet (F:C), while T2 was the control diet with bromoform added, a known CH4 inhibitor, T3 was a high forage diet (80:20 F:C), and T4 was a low forage diet (20:80 F:C). LMD measurements aligned with the standard gas chromatography (GC) method. Results showed that diet composition significantly impacts CH₄ production: the high-forage diet (T3) produced the most CH₄, while adding bromoform fully inhibited emissions. OGI images analyzed with semantic segmentation models, particularly Gasformer, effectively detected CH₄ plumes. The study concluded that combining OGI with deep learning models can effectively monitor CH₄ emissions to inform dietary and management practices to mitigate climate change. In conclusion, bromoform significantly mitigated CH4 emissions without significantly impairing rumen fermentation however, the effect of bromoform can be reversed with the discontinuation of the treatment suggesting its mode of action is unlikely related to altering the rumen microbial profile. Future research is necessary to evaluate the effect of adding bromoform on feed intake and animal health, and the different aspects of introducing bromoform to the animal feeding industry