Influences of Growth Stage and Ensiling Time on Fermentation Characteristics, Nitrite, and Bacterial Communities during Ensiling of Alfalfa

Abstract

This study examined the impacts of growth stage and ensiling duration on the fermentation characteristics, nitrite content, and bacterial communities during the ensiling of alfalfa. Harvested alfalfa was divided into two groups: vegetative growth stage (VG) and late budding stage (LB). The fresh alfalfa underwent wilting until reaching approximately 65% moisture content, followed by natural fermentation. The experiment followed a completely randomized design, with samples collected after the wilting of alfalfa raw materials (MR) and on days 1, 3, 5, 7, 15, 30, and 60 of fermentation. The growth stage significantly influenced the chemical composition of alfalfa, with crude protein content being significantly higher in the vegetative growth stage alfalfa compared to that in the late budding stage (p < 0.05). Soluble carbohydrates, neutral detergent fiber, and acid detergent fiber content were significantly lower in the vegetative growth stage compared to the late budding stage (p < 0.05). Nitrite content, nitrate content, nitrite reductase activity, and nitrate reductase activity were all significantly higher in the vegetative growth stage compared to the late budding stage (p < 0.05). In terms of fermentation parameters, silage from the late budding stage exhibited superior characteristics compared to that from the vegetative growth stage. Compared to the alfalfa silage during the vegetative growth stage, the late budding stage group exhibited a higher lactate content and lower pH level. Notably, butyric acid was only detected in the silage from the vegetative growth stage group. Throughout the ensiling process, nitrite content, nitrate levels, nitrite reductase activity, and nitrate reductase activity decreased in both treatment groups. The dominant lactic acid bacteria differed between the two groups, with Enterococcus being predominant in vegetative growth stage alfalfa silage, and Weissella being predominant in late budding stage silage, transitioning to Lactiplantibacillus in the later stages of fermentation. On the 3rd day of silage fermentation, the vegetative growth stage group exhibited the highest abundance of Enterococcus, which subsequently decreased to its lowest level on the 15th day. Correlation analysis revealed that lactic acid bacteria, including Limosilactobacillus, Levilactobacillus, Loigolactobacillus, Pediococcus, Lactiplantibacillus, and Weissella, played a key role in nitrite and nitrate degradation in alfalfa silage. The presence of nitrite may be linked to Erwinia, unclassified_o__Enterobacterales, Pantoea, Exiguobacterium, Enterobacter, and Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium.

Keywords: alfalfa silage; bacterial communities; enzyme activity; growth stage; nitrite.

Figure 1

Nitrite content (A), nitrate content (B), nitrite reductase (C), and nitrate reductase enzyme activities (D) during alfalfa silage at different growth stages. Different lowercase letters (a–f) indicate significant differences in means (p < 0.05).

Figure 2

Number of genera and bacterial communities in alfalfa silage bacterial community.

Figure 3

Principal component analysis (PCoA) of bacterial communities in silage raw material (MR) and alfalfa silage.

Figure 4

Relative abundance of bacterial communities at the phylum (A) and genus (B) levels in silage raw material (MR) and alfalfa silage.

Figure 5

Compare the changes in microorganisms in alfalfa raw materials (A) and ensiling for 1, 3, 5, 7, 10, 30, and 60 days (B–I).

Figure 6

Correlation analysis between nitrite content, nitrate content, and microbial community structure (A) and fermentation characteristics (B) in alfalfa silage, as well as correlation analysis between fermentation quality and microbial community structure (C). LA, lactic acid; AA, acetic acid; PA, propionic acid; BA, butyric acid; NH₃–N, ammonia nitrogen. *, 0.01 < p < 0.05; **, 0.001 < p < 0.01; ***, p < 0.001.

Figure 7

Predicted pathways of bacterial community in alfalfa raw material (VG–MR, LB–MR) and silage on day 60 (VG–60, LB–60). (A) Predicted pathways on level 1; (B) predicted pathways on level 2; (C) predicted pathways on level 3.