ABSTRACT

Background and Aim: Enteric methane emission from dairy cows contributes substantially to greenhouse gas production and represents an inefficient loss of dietary energy. Phytosterols are plant-derived bioactive compounds with lipid-modulating and rumen fermentation-regulating properties; however, their effects on methane emission intensity and rumen microbial ecology in lactating dairy cows remain insufficiently explored. This study evaluated the effects of dietary phytosterols supplementation on lactation performance, nutrient digestibility, serum biochemical parameters, rumen fermentation characteristics, methane emission intensity, and rumen microbial composition in mid-lactation Holstein dairy cows. 

Materials and Methods: Thirty-four multiparous Holstein dairy cows with similar days in milk and milk yield were randomly assigned to either a control (CON) group or a phytosterols (PHY) group receiving 15 g/d of a commercial phytosterols product containing 5% active phytosterols. The experimental period lasted 50 days, including 7 days of adaptation and 43 days of data collection. Feed intake and milk yield were recorded daily. Milk composition, apparent nutrient digestibility, serum biochemical indices, rumen fermentation parameters, methane emission intensity, quantitative polymerase chain reaction, and 16S rRNA gene sequencing were analyzed. Methane and carbon dioxide emissions were measured using an automated head-chamber system. 

Results: Dietary phytosterols supplementation significantly improved milk yield, milk fat percentage, milk protein percentage, energy-corrected milk, and 3.5% fat-corrected milk compared with the CON group (p < 0.05). Apparent digestibility of organic matter, crude protein, neutral detergent fiber, and ether extract was also significantly enhanced. Serum glucose and blood urea nitrogen concentrations increased, whereas total cholesterol and low-density lipoprotein cholesterol concentrations decreased in the PHY group. Phytosterols supplementation significantly reduced methane emission intensity per kilogram of energy-corrected milk. Ruminal acetate proportion and acetate-to-propionate ratio decreased, whereas microbial crude protein and branched-chain volatile fatty acids increased. In addition, phytosterols altered rumen microbial composition by increasing the abundance of beneficial bacterial genera, including Succinivibrionaceae UCG-001 and Prevotella, while reducing methanogenic archaea, particularly Methanobacteriota and Methanimicrococcus. 

Conclusion: High-dose phytosterols supplementation improved lactation performance, enhanced nutrient utilization, modulated rumen microbial communities, and reduced methane emission intensity in mid-lactation dairy cows. These findings indicate that phytosterols may serve as a promising natural feed additive for improving dairy production efficiency while supporting methane mitigation strategies in sustainable dairy farming. 

Keywords: digestibility, lactation performance, methane mitigation, Phytosterols, rumen fermentation, volatile fatty acids.