Micro- and Macro-Algae Combination as a Novel Alternative Ruminant Feed with Methane-Mitigation Potential

**Background:** The livestock industry faces challenges from greenhouse gas emissions, land degradation, and feed costs. Enteric methane from ruminants has a global warming potential 28 times that of CO₂. Microalgae like Euglena gracilis (EG) offer high protein (26.1%) and fat (17.7%) content and can partially replace expensive concentrates. The red seaweed Asparagopsis taxiformis (AT) can reduce methane by >80% via bromoform, but safety concerns (iodine, bromoform in milk) and mass production limitations restrict its use. This study hypothesized that combining minimum effective levels of EG (as a feed) and AT (as a feed additive) would synergistically reduce methane without harming ruminal fermentation.

**Methods:** The 24 h in vitro batch culture used rumen fluid from two ruminally cannulated non-lactating Holstein cows (894 kg BW) fed orchard grass hay. The basal diet was 50% Kleingrass hay and 50% concentrate mixture. Nine treatments were tested: control, AT at 1% and 2.5% of diet, EG at 10% and 25% (partially replacing concentrate), and four combinations (EG 10%+AT 1%, EG 10%+AT 2.5%, EG 25%+AT 1%, EG 25%+AT 2.5%). Each treatment had 4 replicates across 3 runs. Total gas, methane, CO₂, pH, volatile fatty acids (VFA), ammonia-N, and in vitro dry matter digestibility (IVDMD) were measured. Bromoform in AT was 953.8 μg/g DM. Data were analyzed using PROC MIXED in SAS with orthogonal contrasts.

**Key Results:** AT at 1% and 2.5% reduced methane yield by 21.3% and 80.1%, respectively (p<0.01). EG at 10% and 25% reduced methane by 4.4% and 11%, respectively (not significant, p>0.05). The combination EG 10%+AT 1% reduced methane by 29.9% (p<0.01), and EG 25%+AT 1% reduced methane by 40.1% (p<0.01), showing synergy. Combining 2.5% AT with EG did not significantly improve reduction beyond AT 2.5% alone (75.9–81.7% vs 80.2%, p=0.09). The EG 25%+AT 1% combination shifted fermentation toward more propionate and less acetate (p<0.01) without adversely affecting total VFA or IVDMD. Total VFA was significantly decreased only in the EG 25%+AT 2.5% group (312.64 mmol/L vs control 340.56 mmol/L, p<0.01). Ammonia-N was higher with EG 25% alone (11.52 mg/dL) and in combination with AT (10.83–11.87 mg/dL) vs control (8.73 mg/dL, p<0.05).

**Clinical Implications:** This study provides the first evidence that combining EG and AT can achieve meaningful methane reduction (up to 40%) using lower AT doses (1% vs 2.5%), potentially reducing safety risks from bromoform and iodine. EG serves as a high-quality protein and fat source (26.1% CP, 17.7% EE) that can replace up to 25% of concentrate without harming fermentation. The synergistic effect likely arises from EG's fatty acids (especially myristic and palmitic) and AT's bromoform acting via different anti-methanogenic mechanisms. However, these are in vitro results; in vivo trials are needed to confirm efficacy, animal health, and product safety before commercial application.