This is an early access version, the complete PDF, HTML, and XML versions will be available soon.
Effects of 5-Hydroxymethylfurfural on In Vitro Rumen Fermentation Characteristics and Bacterial Community Structure of Diets with Different Concentrate-to-Roughage Ratios
State Key Laboratory of Animal Nutrition and Feeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China
*
Author to whom correspondence should be addressed.
Animals 2026, 16(15), 2446; https://doi.org/10.3390/ani16152446
Submission received: 6 July 2026
/
Revised: 1 August 2026
/
Accepted: 4 August 2026
/
Published: 6 August 2026
(This article belongs to the Special Issue Alternative Feeds for Ruminants: Sustainability and Nutritional Innovation)
Simple Summary
When feed ingredients are processed at high temperatures during manufacturing, a compound called 5-hydroxymethylfurfural (5-HMF) is produced and ends up in animal feed. While this compound is known to interfere with fermentation in other systems, very little is known about how it affects the rumen—the specialized stomach of cattle and sheep where microbes digest fibrous feed. In this study, we added different amounts of 5-HMF to two types of feed (one high in fiber and one high in grain) and incubated them with rumen fluid in the laboratory. We found that 7500 mg/kg DM 5-HMF modulated fiber digestion and influenced the abundance of certain bacterial taxa, while 30,000 mg/kg DM reduced overall fermentation activity. The effects also depended on the type of feed, with fiber-rich diets showing more pronounced responses. These findings suggest that 5-HMF is not simply harmful, and that its effects depend on both the amount present and diet composition. This knowledge can help the feed industry better understand how heat-processing by-products affect ruminant nutrition and guide the development of safer, more efficient feeding strategies.
Abstract
To investigate the effects of 5-hydroxymethylfurfural (5-HMF) on ruminal fermentation characteristics and bacterial community structure, 5-HMF was supplemented at the levels of 0, 7500, and 30,000 mg/kg DM under two dietary concentrate-to-roughage ratios (20:80, T1; 80:20, T2) in in vitro rumen incubation, mainly focusing on gas production, fermentation parameters, nutrient digestibility and bacterial community. The results showed that 24 h gas production (GP24), 72 h gas production (GP72), the gas production rate (c) and dry matter digestibility (DMD) in T2 were higher than those in T1 (p < 0.01). With increasing levels of 5-HMF supplementation, GP72 and asymptotic gas production (B) increased linearly and quadratically (p < 0.05). T2 also had a higher butyrate concentration and a lower acetate/propionate (A/P) ratio than T1 (p < 0.05). With the increase in 5-HMF supplementation level, ruminal ammonia nitrogen (NH3-N) concentration showed a quadratic response. In contrast, the concentrations of total volatile fatty acids (TVFAs), acetate and isobutyrate decreased linearly. Propionate concentration showed linear and quadratic decreasing trends, while the A/P ratio exhibited linear and quadratic increasing trends (p < 0.05). The supplementation of 5-HMF at 30,000 mg/kg DM reduced propionate and isobutyrate concentrations under both dietary conditions (p < 0.05). No differences were observed in the alpha diversity indices of the bacterial community among different 5-HMF levels or substrates (p > 0.05). PCoA and PERMANOVA revealed that community structure differed between the 7500 mg/kg DM group and the 0 mg/kg DM group in the T1 diet (p = 0.011), whereas no difference was detected among T2 treatments. LEfSe revealed that 6 and 13 differential bacterial taxa were identified in T1 and T2 at the 5-HMF level of 7500 mg/kg DM, respectively. Correlation analysis revealed that, in T1, GP72 was positively correlated with Pseudomonadota, and the A/P ratio was positively correlated with fibrolytic taxa including Rikenellaceae_RC9_gut_group and Christensenellaceae_R-7_group. In T2, c was positively correlated with Fibrobacterota and Fibrobacter, while NH3-N was positively correlated with Desulfovibrio, indicating diet-specific microbial associations with rumen fermentation (p < 0.05). In conclusion, the high-concentrate diet exhibited stronger rumen fermentation activity. 5-HMF exerted diet-specific effects: In the low-concentrate diet, 5-HMF at 7500 mg/kg DM enriched fibrolytic microbes and shifted fermentation toward an acetate-type pattern. In the high-concentrate diet, it modulated hydrogen-metabolizing bacteria while selectively enriching Pseudomonadota capable of participating in carbohydrate metabolism. The 30,000 mg/kg DM level inhibited rumen fermentation. The findings provide a theoretical reference for the application of 5-HMF in ruminant feeds. However, further in vivo trials are required to validate its effects, and future studies with refined dose gradients based on realistic 5-HMF concentrations in feedstuffs are needed to comprehensively evaluate its efficacy and safety.
