Dietary methane (CH₄) mitigation is in some cases associated with an increased hydrogen (H₂) emission. The objective of the present study was to investigate the acute and short-term effects of acceptors for H₂ (fumaric acid, acrylic acid, or phloroglucinol) supplemented via pulse-dosing to dairy cows fed CH₄ mitigating diets (using nitrate or 3-nitrooxypropanol), on gas exchange, rumen gas, and VFA composition. For this purpose, 2 individual 4 × 4 Latin square experiments were conducted with 4 periods of 3 d (nitrate supplementation) and 7 d (3-nitrooxypropanol supplementation), respectively. In each study, 4 rumen-cannulated Danish Holstein cows were used. Each additive for CH₄ mitigation was included in the ad libitum-fed diet within the 2 experiments (exp. 1 and exp. 2), to which the cows were adapted for at least 14 d. Acceptors for H₂ were administered twice daily in equal portions through the rumen fistula immediately after feeding of the individual cow. In exp. 1 (nitrate), the treatments were CON-1 (no H₂-acceptor), FUM-1 (fumaric acid), ACR-1 (acrylic acid), and FUM+ACR-1 (50% FUM-1 + 50% ACR-1). In exp. 2 (3-nitrooxypropanol), the 3 treatments, CON-2, FUM-2, and ACR-2, were similar to CON-1, FUM-1 and ACR-1 treatments, however the fourth treatment was PHL-2 (phloroglucinol). Gas exchanges were measured in respiration chambers, and samples of rumen liquid and headspace gas were taken in time series relative to feeding and dosing on specific days. Headspace gas was analyzed for gas composition, and rumen liquid was analyzed for VFA composition and dissolved gas concentrations. Headspace gas composition and dissolved gas concentration were only measured in exp. 2. Dry matter intake was reduced upon acrylic acid supplementation. There were no significant effects of any treatments in any experiments on H₂ emission, except for a decrease in hourly H₂ emission rate (g/h) at 1 h after feeding in both experiments. In exp. 2, H₂ headspace proportions increased with ACR-2 supplementation, whereas dissolved concentrations were unaffected. In exp. 1, cows on ACR-1 increased propionate proportion at 1 h after feeding. In exp. 2, both FUM-2 and ACR-2 increased rumen propionate proportion in the hours after feeding and dosing. There was no effect on rumen acetate for cows on PHL-2. There was a strong positive correlation between rumen dissolved CH₄ and headspace CH₄ (r = 0.84), whereas the equivalent correlation was weaker for H₂ (r = 0.41). For the relationship between dissolved concentrations and emissions of CH₄ and H₂, there was a moderate positive correlation for CH₄ (r = 0.54), whereas it was weak for H₂ (r = 0.28) with zero slope. In conclusion, the results suggested that fumaric acid and acrylic acid to some extent was reduced to propionate without associative effects on measures for H₂ redirection. Furthermore, phloroglucinol seemed not to be metabolized in the rumen in the present study, because no effects on rumen acetate or measures of H₂ were observed. Changes in H₂ headspace and emission may be a poor proxy for actual changes in the rumen fluid concentration of H₂.

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