Direct-fed microbials (DFM) have emerged as a promising dietary strategy for enteric methane abatement. However, it is unclear whether in vitro studies trialing DFM should use ruminal fluid previously adapted to the DFM of interest or if the DFM can be directly added to an unadapted inoculum. Ten lactating, multiparous, rumen cannulated Holstein-Friesian cows were randomly allocated to one of two groups: 1) adapted, basal diet plus 4 g/d of a blend of Bacillus subtilis and Bacillus licheniformis (1.6 × 10⁹ CFU/g each), delivered via the rumen canula; and 2) naive, basal diet only. Ruminal fluid from both groups was incubated in an in vitro 24-h batch culture system with two rates of Bacillus spp. and three feed substrates (hay, pasture, cereal grain), resulting in 12 treatments. Methane production was 16% greater, and total volatile fatty acid concentration was 7% greater in incubations using adapted ruminal fluid compared to those using naive ruminal fluid; however, neither parameter was affected when Bacillus spp. was added to the batch incubation system. Future in vitro studies evaluating DFM should consider including a period of in vivo adaptation to mimic their potential impact under in vivo feeding conditions.

Both in vitro and in vivo studies have shown the potential of Lactobacillus spp. to mitigate enteric methane. However, their mitigation impact on early-lactation dairy cows grazing high-pasture diets has not been studied. This study evaluated the effect of supplementing freeze-dried or liquid Lactobacillus spp. products on enteric methane emission from early lactation dairy cows offered a high forage diet. Forty spring calving Holstein Friesian dairy cows, grazing a perennial ryegrass (PRG) (Lolium perenne L.) based pasture, milked twice daily, were randomly allocated to one of the 3 treatment diets offered for 35 d: 1) CON: Control, fed a basal diet (n = 14); 2) FLA: basal diet plus freeze-dried Lactobacillus spp. (n = 13) delivering 5.0 × 10¹⁰ cfu/milking, and 3) LLA: basal diet plus Liquid Lactobacillus spp. (n = 13) delivering 5.75 × 10¹⁰ cfu/milking. The basal diet comprised 6.9 kg DM/day of a grain mix plus PRG pasture at an allowance of approximately 25 kg DM/cow per day. Covariate observations were made for the first 5 d of the 40-d experiment, before commencing treatments. During the last 5 d of the experiment, individual forage dry matter intake for each cow was estimated using the n-alkane technique, and methane emissions from individual cows were measured using the modified sulfur hexafluoride (SF₆) tracer technique. Data were analyzed using a completely randomized design based ANCOVA. Methane production (g/day), yield (g/kg DMI) and intensity (g/kg energy corrected milk) were not affected by the inclusion of either form of Lactobacillus spp. in the diet, although a numerical reduction of 6% in methane yield was observed in the FLA group, compared with the control group. Methane emissions from the CON cows were approximately 38% lower than those estimated using inventory factors. This is likely due to the low fiber concentration and low DM concentration of the PRG offered likely resulting in a high ruminal passage rate, reducing ruminal retention time and extent of fermentation. The high passage rate could have also restricted the colonization potential of supplemented Lactobacillus spp. This, combined with the large rumen volume of dairy cows and low dose rate may in part explain the limited methane mitigation. Notably, production responses to Lactobacillus spp. supplementation have been more evident in small ruminants, suggesting species-specific differences. These findings highlight the need for tailored probiotic strategies, particularly in pasture based dairy systems.

• A bromoform-based additive did not affect dry matter intake or eating behaviour. • Daily eating, ruminating, and activity patterns were unaffected by the diets. • Lying, standing, and walking were not affected by the treatment diets. • A bromoform-based additive had no negative effect on dairy cow welfare.