Materials and chemicals

The experiment was conducted from June to August 2022 at a research base located in Hohhot, Inner Mongolia Autonomous Region. The geographical coordinates range from 110°46’ to 112°10’ east and from 40°51’ to 41°8’ north. This region is situated within the Eurasian continent and is characterized by a temperate continental monsoon climate with precipitation ranging from 335.2 to 534.6 mm. The average outdoor temperature in summer is 22 –26 °C, up to 35 °C.The experimental area covered 666 m² of saline-alkali soil. The harvest date was June 28, 2022, and the alfalfa was cut at the early flowering stage of the first cutting. The selected alfalfa variety was WL168.The seeds were supplied by Inner Mongolia Zhengda Co., Ltd. and originated in the United States.

After harvest, a part of the fresh samples were placed in sterilized storage tubes and transported to the Key Laboratory of Grassland and Resources and Environment College of Inner Mongolia Agricultural University for storage at -80 °C.The remaining grass samples were spread on clean plastic sheets and naturally dried to a moisture content of approximately 60%. The dried samples were then cut into 2–3 cm lengths using a shredder.

The processed alfalfa samples were divided into three equal portions and subjected to secondary fermentation by evenly spraying them with a composite inoculant containing Lactiplantibacillus plantarum and Lentilactobacillus buchneri. (Production enterprise: Inner Mongolia Hemei Kesheng Biotechnology Co., Ltd; Production address: Hohhot, Inner Mongolia) The targeted bacterial population in the sprayed inoculant was set at 1 × 10⁶ cfu/g. The microbial inoculant was dissolved in sterile water at a ratio of 30 ml per kilogram of forage. The solution was then evenly sprayed onto the forage samples while continuously mixing the samples to ensure thorough and uniform distribution of the inoculant. The first part had a stacking density of 500 kg/m³ (500 g of grass samples filled into a 1 L polyethylene barrel), the second part had a stacking density of 600 kg/m³ (600 g of grass samples filled into a 1 L polyethylene barrel), and the third part had a stacking density of 700 kg/m³ (700 g of grass samples filled into a 1 L polyethylene barrel). Each density treatment group was further divided into two temperature gradients (3 density treatment groups * 2 temperatures * 3 replicates).

After filling, the top of each barrel was sealed with a sealing cover equipped with a sealing rubber and plunger ring, and the barrel opening was wrapped with plastic film to prevent the entry of oxygen. The ambient temperature treatment group was placed indoors for fermentation, while the high-temperature treatment group was exposed to outdoor fermentation. During the experiment, which took place in summer, the outdoor temperature could reach 35 °C. The ambient temperature treatment group was placed indoors for fermentation, with an average indoor temperature of 20 °C.

After 60 days of ensiling, the barrels were opened to evaluate the quality of alfalfa silage under high-temperature conditions with stacking densities of 500 kg/m³, 600 kg/m³, and 700 kg/m³ (referred to as G500, G600, and G700, respectively), as well as under ambient temperature conditions with stacking densities of 500 kg/m³, 600 kg/m³, and 700 kg/m³ (referred to as C500, C600, and C700, respectively). Additionally, 20 g of each sample was placed in storage tubes and stored at -80 °C. These samples were sent to Majorbio Bio-pharm Technology (Majorbio Bio-pharm Technology Co., Ltd., Shanghai, China) for sequencing analysis.