Effects of Melatonin Supplementation on the Quality, Bacterial Community, and In Vitro Rumen Fermentation of Whole-Plant Soybean Silage

Abstract

Whole-plant soybean (WPS) is a high-protein forage resource, but its natural ensiling is often unsatisfactory due to low water-soluble carbohydrate content and high buffering capacity. This study investigated the effects of exogenous melatonin (ME) at 0 (CK), 5 (ME1), 10 (ME2), and 20 (ME3) mg/kg fresh matter on fermentation quality, chemical composition, in vitro rumen fermentation, and bacterial community structure of WPS silage. ME2 and ME3 had lower pH values and higher lactic acid contents than CK, with both treatments achieving pH values below 4.2. Crude protein concentration increased from 15.42% in CK to 19.96% in ME3, while neutral detergent fiber was lower in all ME treatments, and acid detergent fiber was lower in ME2 and ME3 than in CK. At 36 h, no overall treatment effect was detected for cumulative gas production, whereas in vitro dry matter digestibility differed only between ME2 and ME3. 16S rRNA gene sequencing revealed that ME altered the bacterial community, with community-weighted rrn copy number elevated in ME2 and ME3. Random forest analysis identified Enterococcus as the genus with the highest importance for treatment classification, and functional predictions indicated higher predicted abundances of amino acid biosynthesis pathways in ME treatment groups. These results indicate that ME has potential as an additive for improving WPS silage fermentation, but practical dosage recommendations require further validation through aerobic stability, animal performance, economic, and safety assessments.