This study evaluated the effects of different dietary n-6/n-3 polyunsaturated fatty acid (PUFA) ratios on in vitro ruminant fermentation. Methane production, fatty acid composition, and microbial profiles were compared after the in vitro fermentation of rumen fluid collected from cows that had been fed isoenergetic and isoproteic experimental diets at three different n-6/n-3 ratios: 3.04 (HN6, high n-6 source), 2.03 (MN6, medium n-6 source), and 0.8 (LN6, low n-6 source). The fermented rumen fluid pH and total volatile fatty acid (VFA) levels were significantly decreased (p < 0.05) in the HN6 group as compared with those in the MN6 and LN6 groups. Additionally, the HN6 group produced a significantly lower (p < 0.05) proportion of methane than the MN6 group during in vitro fermentation. The MN6 and LN6 groups had significantly increased (p < 0.05) levels of C18:2n6 and C18:3n3 in the fermented rumen fluid, respectively, as compared with the HN6 group. The Chao 1 diversity index value was lower (p < 0.05) in the HN6 group than in the MN6 and LN6 groups. The observed species richness was significantly lower (p < 0.05) in the HN6 group than in the MN6 group. The reduced relative abundances of Lachnospiraceae UCG-006 and Selenomonas in the HN6 group resulted in lower pH and VFA levels (i.e., acetate, propionate, butyrate, and total VFA) during in vitro fermentation. Furthermore, n-6 and n-3 PUFAs were toxic to Butyrivibrio_2 growth, resulting in high levels of incomplete biohydrogenation. Taken together, the study findings suggest that supplementation of high-forage diets with high levels of n-6 PUFAs could reduce methane emissions, whereas both VFA concentration and pH are reduced.

It is well known that dairy cows are fed diets with high fat content, which can adversely affect rumen fermentation. However, whether the effects of high fat content on rumen fermentation are related to the composition of fatty acids (FA) is for further study. We explored the effects of unsaturated fatty acid (UFA) ratios in vitro on rumen, methane concentration and microbial composition under the same fat levels. The experiment included a low-unsaturated group (LU, UFA proportion: 42.8%), a medium-unsaturated group (MU, UFA proportion: 56.9%), and a high-unsaturated group (HU, UFA proportion: 70.9%). The incubation fluid pH and NH₃-N levels were not significantly different in the three groups. Total volatile fatty acid (TVFA), acetate, propionate, butyrate, and valerate in the MU group had a decreased trend compared to the LU group (0.05 < p < 0.1), and no difference was found in other volatile fatty acids (VFAs) among the three groups. Furthermore, gas production kinetic parameters among the three groups did not differ significantly. The LU group’s CH₄ concentration was significantly higher than the HU group (p < 0.05). The CO₂ concentration in the LU group was also significantly higher than the MU and LU groups (p < 0.05). Additionally, 16S rRNA microbial sequencing results showed that the Shannon diversity value significantly increased in the MU group (p < 0.05) compared to the LU group. Other alpha diversity indices (Chao 1, observed species, and ACE) did not differ among the three groups. The increased proportion of UFA significantly decreased the relative abundance of Succinivibrionaceae_UCG_001 and Fibrobacter (p < 0.05). Meanwhile, the multiple Lachnospiraceae bacteria significantly increased in the MU group (p < 0.05). Overall, our findings indicated that the microbial community in the incubation system could be affected by elevating proportions of UFA, affecting the yield of VFA, whereas the CH₄ concentration was reduced.

Lipid metabolism plays an important role in the energy economy of ruminants. However, its interactions of fat, rumen fermentation, gas emission, and microorganisms are not yet clear. This study evaluated the effect of adding raw oilseeds to high-forage diets on in vitro ruminal fermentation, gas composition, and microbial profile. Three isoenergetic and isoproteic experimental diets were designed and used as fermentation substrate: control treatment (CON group) was the basal diet lacking oilseeds, the other two treatments were the basal diet supplemented by 100 g/kg dry matter (DM) raw whole soybean (S group) and 50 g/kg DM raw flaxseed (F group), respectively. Data showed that the acetate, butyrate, and total VFA concentration of culture fluids in the S group were lower (p < 0.05) than in the F group. There was a tendency to a higher level (p = 0.094) of propionate concentration in the F group compared with the other two groups. The gas production in the F group was higher (p < 0.05) than in the control group. There was a lower abundance of Sutterella (p < 0.05) and a greater abundance of Butyrivibrio (p < 0.05) in both of the two oilseed treatments. Methanobrevibacter (p = 0.078) in the F group was the lowest. Our results suggested that CH₄ emission could be inhibited with flaxseed supplementation by propionate production metabolism, biohydrogenation of unsaturated fatty acid (FA), and toxicity to Methanobrevibacter, while regarding soybean seed supplementation, the emission of CH₄ was more likely to be reduced through biohydrogenation of unsaturated FA modulated by Butyrivibrio.