Rajaraman Bharanidharan ^1* Panyavong Xaysana ² Woohyeong Hong ² Taehoon Kim ³ Ridha Ibidhi ⁴ Jun Suk Byun ⁵ Yookyung Lee ⁶ Byamungu Mayange Tomple ¹ Kyoung Hoon Kim ^1,2*

• ¹ Institute of Green Bio Science and Technology, Seoul National University, Pyeongchang,Gangwon-do, Republic of Korea
• ² Graduate School of International Agricultural Technology, Seoul National University, Pyeongchang 25354, Republic of Korea
• ³ Department of Geography, McGill University, Montreal, QC, H3A 0B9,, Canada
• ⁴ Agroécologie, INRAE, Institut Agro, Univ. Bourgogne, Univ. Bourgogne Franche-Comté, F-21000 Dijon, France
• ⁵ University of Maryland Center for Environmental Science, MD 21613, United States
• ⁶ National Institute of Animal Sciences, Rural Development Administration, Jeonju 54875, Republic of Korea

Two in vivo experiments were conducted to evaluate the potential of Pharbitis nil seeds (PA) as an anti-methanogenic additive to ruminant feed. In experiment 1, six Hanwoo steers (459.0 ± 25.8 kg) were fed either a total mixed ration (TMR; 32-d period) or TMR supplemented with PA at 5% dry matter (DM) intake (TMR-PA; 45-d period) for two consecutive periods. Fecal and urine outputs were measured in an apparent digestibility trial in both periods. Methane (CH4) yield and heat energy (HE) were measured using respiratory chambers equipped with gas analyzers. Although there were no differences (p > 0.05) in nutrients or gross energy intake (GEI) between the groups, an increase (p < 0.05) in the apparent digestibility of DM (9.1%) and neutral detergent fiber (22.9%) was observed in the TMR-PA group. Pronounced decreases (p < 0.05) in CH4 (g/Kg DM; 17.1%) and urinary N excretion (% N intake; 7.6%) were observed in the TMR-PA group, leading to a 14.7% increase in metabolizable energy intake (% GEI). However, only a numerical increase (p > 0.05) in retained energy was observed due to the increase in HE loss. In experiment 2, five rumen cannulated Holstein steers (744 ± 35 kg) were fed the same TMR or TMR-PA diets for 40 days; rumen samples were collected at 0, 1.5, and 3 h after feeding on the last day of the feeding period. In the TMR-PA group, drastic decreases (p < 0.05) in rumen ammonia concentration and pH (56.3%) associated with an increased (p = 0.091) rumen short-chain fatty acid concentration 3 1.5 h after feeding were observed. A 26.6% increase (p < 0.05) in the propionate proportion during the treatment period clearly reflected a shift in the ruminal H2 sink after 3 h of feeding. A 40% reduction (p = 0.067) in the relative abundance of rumen protozoa Entodinium caudatum was also observed. It was concluded that PA could be a natural feed additive for CH4 and N emission abatement.