Abstract

   This study aimed to investigate the effect of storage time on the
   fermentation quality, bacterial community structure, and metabolic
   profiles of Jinmu grain grass silage. It was ensiled in vacuum bags for
   60 days. Samples were collected after 0, 3, 7, 15, 30, and 60 days of
   ensiling. Nutritional analysis revealed no significant differences in
   dry matter (DM), ether extract (EE), crude protein (CP), neutral
   detergent fiber (NDF), or acid detergent fiber (ADF) across storage
   periods (p > 0.05), but relative feeding value (RFV) significantly
   increased at 30 and 60 days (p < 0.05). Fermentation quality improved
   with prolonged storage, pH values declined to 4.01 at 60 days, while
   lactic acid (LA) and acetic acid (AA) increased significantly (p <
   0.05). Butyric acid(BA) was undetected. 16S rDNA sequencing showed
   bacterial diversity (Chao1, Simpson, and Shannon indices) increased
   significantly at 30 and 60 days (p < 0.01); The relative abundance of
   Lacticaseibacillus, and Amylolactobacillus at 30 days were
   significantly higher than 0 and 60 days (p < 0.05); The relative
   abundance of Stenotrophomonas, Serratia, Comamonas,
   GKS98_freshwater_group, and Sphingobium at 60 days were significantly
   higher than 0 and 30 days (p < 0.05). Comprehensive targeted
   metabolomics identified 2958 metabolites. There were 256 differential
   metabolites shared by the comparison groups at 0, 30, and 60 days. The
   pathways for enrichment of differential metabolites mainly include
   plant hormone signal transduction, Histidine metabolism, arginine
   biosynthesis, etc. In conclusion, the storage time of Jinmu grain grass
   silage can enhance its fermentation quality by influencing microbial
   communities and metabolic pathways.

   Keywords: Jinmu grain grass silage, storage time, nutritional
   composition, fermentation quality, bacterial community, metabolomics

1. Introduction

   As a global powerhouse in animal husbandry, China’s livestock sector is
   of significant international importance. However, the nation faces the
   persistent challenge of supporting a vast population with limited
   arable land, resulting in low per-capita grain availability [[36]1].
   This constraint has led to a critical shortage of feed resources, which
   now acts as a major bottleneck constraining the sustainable development
   of both the animal husbandry and feed industries in China [[37]2].
   Consequently, enhancing the development and utilization of alternative
   feed resources is imperative to ensure the nation’s food security and
   agricultural sustainability.

   Jinmu grain grass is a plant species belonging to the genus Pennisetum
   of the Poaceae family. It is rich in nutrients, containing more than 18
   essential amino acids and various vitamins that are beneficial to
   animal growth [[38]3]. Moreover, its leaves have no burrs (steel
   hairs), which is conducive to digestion by livestock and poultry. Under
   the suitable climatic conditions for growing farmland in the western
   region, the annual output of fresh grass is 40 to 60 tons per mu. The
   Jinmu grain grass grown in Xinjiang can be harvested three times a
   year, usually from August to November [[39]4]. The feed after ensiling
   is soft, juicy, fragrant, and palatable, and the nutritional value of
   the feed is improved, which is conducive to increasing the feed intake
   and digestion and utilization rate of livestock and poultry [[40]5].
   Therefore, ensiling processing can ensure the year-round supply of
   Jinmu grain grass for livestock and poultry and reduce nutrient loss
   during feed storage. In addition, the plants of Jinmu grain grass are
   tall, upright, and clustered, with well-developed root systems, wide
   adaptability, high stress resistance, fast growth rate, strong
   regenerative capacity, high yield, and are resistant to acid, high
   temperature, drought, and cold, as well as diseases and pests. They
   will not die within −30 °C and can be planted in most areas of China.
   Meanwhile, Jinmu grain grass is a typical four-carbon plant with a high
   photosynthetic rate, which can prevent soil erosion and is suitable for
   desertification control [[41]6]. To sum up, Jinmu grain grass is an
   ideal feed resource for livestock and poultry.

   However, the application of Jinmu grain grass as a feed resource has
   not been widely promoted, and there are few reports on the mechanism of
   its silage in enhancing nutritional value and the fermentation
   mechanism of the micro-ecosystem. Therefore, in this study, Jinmu grain
   grass was processed into silage to explore the changes in its
   nutritional components and silage quality at different storage times.
   The effects at 30 and 60 days of ensiling on the bacterial community of
   Jinmu grain grass were analyzed with 16Sr DNA sequencing, and
   comprehensive targeted metabolomics was conducted to screen the
   differential metabolites and metabolic pathways of each comparison
   group between 0, 30, and 60 days. By studying the nutritional
   composition, fermentation quality, microbial quantity, bacterial
   community structure, and metabolic characteristics of Jinmu grain grass
   at different storage times, the changes in its microbial community and
   metabolites were revealed. The aim of this study was to investigate the
   effects of storage time on the fermentation quality, bacterial
   community structure, and metabolic profiles of Jinmu grain grass
   silage. The results are expected to provide theoretical references for