Characteristics of fresh Italian ryegrass harvested at the filling and dough stages
The differences in chemical and microbial of two growth stages of Italian ryegrass are shown in Table 1. The contents of pH, DM, NDF and ADF increased significantly (P < 0.05) from the filling to dough stage of Italian ryegrass, while WSC content decreased significantly (P < 0.05). The content of buffering capacity and CP did not change significantly, but decreased slightly with the growth of Italian ryegrass. Among culturable microbes, only LAB count increased with the growth of Italian ryegrass.
Table 1
The chemical components and epiphytic microbiota of fresh Italian ryegrass at two growth stages
Items
|
The filling stage
|
The dough stage
|
SEM
|
P value
|
pH
|
6.12
|
6.75
|
0.143
|
< 0.001
|
Dry matter (g/kg FW)
|
226
|
347
|
7.089
|
< 0.001
|
Water-soluble carbohydrates (g/kg DM)
|
145
|
84.8
|
3.964
|
0.002
|
Buffering capacity (mEq/kg DM)
|
49.0
|
42.6
|
1.891
|
0.084
|
Neutral detergent fiber (g/kg DM)
|
527
|
570
|
2.182
|
0.014
|
Acid detergent fiber (g/kg DM)
|
322
|
347
|
5.905
|
0.007
|
Crude protein (g/kg DM)
|
62.7
|
59.1
|
1.200
|
0.142
|
Lactic acid bacteria (Log10 cfu/g FW)
|
4.33
|
5.42
|
0.198
|
0.032
|
Aerobic bacteria (Log10 cfu/g FW)
|
8.54
|
8.00
|
0.270
|
0.374
|
Yeasts (Log10 cfu/g FW)
|
5.29
|
3.07
|
0.587
|
< 0.001
|
Enterobacteria (Log10 cfu/g FW)
|
5.34
|
6.01
|
0.168
|
0.016
|
FW, fresh weight; TN, total nitrogen; cfu, colony-forming unit.
Fermentation Characteristics Of Italian Ryegrass Silage
Italian ryegrass harvested at the filling and dough stages were ensiled respectively, the corresponding changes of chemical composition and fermentation products during ensiling are shown in Table 2 and Fig. 1. The interaction of growth stage and ensiling days significantly (P < 0.05) affected the pH, WSC, LA, AA and ethanol contents, but not the DM and NH3-N contents (P > 0.05). The ratio of lactic to acetic acid (LA/AA) was only affected (P < 0.05) by ensiling day, and BA content was only affected (P < 0.05) by the growth stage.
DM content in FSN decreased with the ensiling days, while there were no significant changes in DSN. The rapid decline of WSC in FSN occurred in the first 7 days of ensiling, while that in DSN occurred in the first day of ensiling. There was more WSC consumed in FSN during ensiling. Except for the first day of ensiling, the pH of FSN was lower than that of DSN (Fig. 1A). In the first 15 days of ensiling, the pH decreased to the lowest value of each group, thereafter, the pH presented different growth trend. In FSN, the pH increased slightly at the end of ensiling, while in DSN it dramatically increased from 15 to 30 days of ensiling. The LA content of FSN was significantly higher than that of DSN during ensiling (Fig. 1B). However, the overall trend of LA content in FSN and DSN was similar. The LA content of FSN reached the plateau after 15 days of ensiling, and the decrease occurred after 60 days of ensiling. While in DSN, the plateau appeared after 7 days of ensiling, and began to decrease after 30 days of ensiling. As shown in Fig. 1C, FSN had a higher AA content than DSN throughout the ensiling process. The AA content in FSN increased during ensiling, although after 30 days of ensiling the increase became slow. While in DSN, the AA content increased gradually in the first 15 days of ensiling, then decreased until the end of ensiling. The ratio of LA/AA reflects the type of lactic acid fermentation. In the present study, the ratio of LA/AA in FSN and DSN was both lower than 2.0, indicating that hetero-fermentation was prevalent in both groups (Fig. 1D). The BA content of FSN and DSN was stabilized at a narrow range during ensiling, but the former had a higher content than the latter. The changing trend of ethanol content is shown in Fig. 1F. The ethanol content in FSN reached the peak in the first 15 days of ensiling, then decreased slightly. In DSN, the ethanol content increased significantly in the first 30 days of ensiling and decreased rapidly after 60 days of ensiling. While in the whole ensiling process, the ethanol content in FSN was always higher than that in DSN.
Table 2
Effects of different growth stages on chemical components and microbial compositions during Italian ryegrass ensiling
Items
|
Treatments
|
Ensiling days
|
SEM
|
P value
|
1
|
3
|
7
|
15
|
30
|
60
|
G
|
D
|
G×D
|
Dry matter (g/kg FW)
|
FSN
|
220Ba
|
217Bab
|
213Babc
|
210Bbc
|
211Bbc
|
205Bc
|
12.97
|
< 0.001
|
0.001
|
0.228
|
DSN
|
369A
|
375 A
|
370A
|
354A
|
348A
|
357A
|
Water-soluble carbohydrates (g/kg DM)
|
FSN
|
125Aa
|
71.9Ab
|
55.8Ac
|
46.4Acd
|
38.3Ade
|
31.5Ae
|
5.039
|
< 0.001
|
< 0.001
|
< 0.001
|
DSN
|
48.7Ba
|
40.5Bab
|
36.2Bbc
|
31.0Bbcd
|
25.7Bd
|
28.5cd
|
Ammonia nitrogen (g/kg TN)
|
FSN
|
39.0c
|
55.1b
|
67.7a
|
75.2a
|
75.4Aa
|
74.6a
|
2.374
|
0.039
|
< 0.001
|
0.810
|
DSN
|
40.0c
|
51.7b
|
64.4a
|
72.6a
|
70.9Ba
|
71.5a
|
Lactic acid bacteria (Log10 cfu/g FW)
|
FSN
|
8.96Bab
|
9.90a
|
9.49Aa
|
8.11b
|
6.03c
|
6.91c
|
0.214
|
0.272
|
< 0.001
|
0.005
|
DSN
|
9.55Aa
|
9.18ab
|
8.96Bab
|
8.22bc
|
7.23c
|
7.15c
|
Enterobacteria (Log10 cfu/g FW)
|
FSN
|
7.20Ba
|
4.88Bb
|
ND
|
ND
|
ND
|
ND
|
0.007
|
< 0.001
|
< 0.001
|
0.067
|
DSN
|
7.54Aa
|
6.20Ab
|
ND
|
ND
|
ND
|
ND
|
Aerobic bacteria (Log10 cfu/g FW)
|
FSN
|
8.95Ba
|
7.90Bab
|
6.06Bc
|
6.74Bbc
|
6.04Bc
|
5.81c
|
0.217
|
< 0.001
|
< 0.001
|
0.859
|
DSN
|
9.57Aa
|
8.97Aa
|
7.20Ab
|
7.34Ab
|
6.79Ab
|
6.66b
|
Yeasts (Log10 cfu/g FW)
|
FSN
|
7.07Ba
|
4.99b
|
4.75bc
|
3.90bc
|
4.03bc
|
3.36c
|
0.268
|
0.004
|
< 0.001
|
0.904
|
DSN
|
7.56Aa
|
6.11ab
|
5.41bc
|
4.54bc
|
ND
|
4.02c
|
A−B Values with different capital letters within the same column indicate significant differences between treatments in the same ensiling day (P < 0.05). a−c Values with different lowercase letters indicate significant differences among ensiling days in the same treatment (P < 0.05).
1 FW, fresh weight; TN, total nitrogen; cfu, colony-forming units.
2 FSN, natural fermentation of Italian ryegrass harvested at the filling stage; DSN, natural fermentation of Italian ryegrass harvested at the dough stage; ND, not detected.
3 G, effect of growth stage; D, effect of ensiling day; G×D, interaction effect of growth stage and ensiling day.
Characteristics Of Silage Microorganisms
The counts of typical microorganisms in silage are shown in Table 2. Except for LAB, other microorganisms were significantly affected by the growth stage of Italian ryegrass (P < 0.05). All microorganisms significantly decreased by the effect of the ensiling day (P < 0.05). The interaction of growth stage and ensiling days only significantly affected the LAB counts (P < 0.05). The LAB count in FSN increased at the first 7 days of ensiling, then decreased. While in DSN, its count decreased throughout the ensiling process. The enterobacteria count in DSN was significantly higher than that in FSN in the first 3 days of ensiling (P < 0.05). However, after 7 days of ensiling, no enterobacteria was detected in FSN and DSN. Throughout the whole ensiling process, the number of aerobic bacteria and yeasts in DSN was always higher than that in FSN.
Bacterial Community By Illumina Analysis
In order to better understand the role of microorganisms in silage, bacterial community as a whole were detected by high-throughput sequencing.
Bacterial community diversities of fresh Italian ryegrass and their changes during ensiling are illustrated in Fig. 2. The epiphytic microbiota of FDS was more diverse than that of FFS. Ensiling process decreased the alpha diversity of the epiphytic microbiota of FDS, and narrowed the difference in alpha diversity between the two bacterial communities. The beta diversity was illustrated by the PCoA plot. The plot shows that on the PC1 axis, the bacterial community structure of FSN and DSN was clearly separated by growth stage, indicating that bacterial communities from the same growth stage were more similar during ensiling.
At the phylum and genus levels, the epiphytic microbiota and their changes during ensiling are shown in Figs. 3 and 4. The relative abundance of Proteobacteria and Actinobacteriota in FFS was higher than that in FDS. While, the relative anbundance of Firmicutes in FDS was higher than that in FFS. Pseudomonas, Sphingomonas and Microbacterium were the three most abundant bacteria in FFS, of which the first two belong to Proteobacteria, accounting for about 60% of the whole bacterial community, and the latter belongs to Actinobacteriota. In FDS, the number of genera was obviously higher than that in FFS, so the relative abundance of dominant genus was lower than that in FFS. Exiguobacterium belonging to Firmicutes, Allorhizobium and Pantoea belonging to Proteobacteria were the three most abundant bacteria in FDS. Lactococcus was the epiphytic LAB presented in FDS, but no LAB in FFS was higher than 0.1%.
Compared to epiphytic microbiota, bacteria community structure has changed greatly after 3 days of ensiling. Both in FSN and DSN, Firmicutes became the dominant phylum (Fig. 4a), while the shift from Proteobacteria to Firmicutes in the former was faster than that in the latter (Fig. 4b and c). After 3 days of ensiling, the relative abundance of most bacteria belonging to Proteobacteria in FSN decreased, while there were some bacteria belonging to Enterobacteriaceae increased slightly, such as Serratia, Pantoea and Hafnia. But their relative abundance decreased to a very small amount in the later ensiling process. In DSN, bacteria belonging to Proteobacteria decreased after 3 days of ensiling, except for Pantoea and Enterobacter, which increased at the early stage of ensiling, and decreased to a small amount in the later stage of ensiling. Compared to FSN, the slow shift of bacteria community in DSN was also reflected in the change of LAB genera (cocci-type LAB to rod-type LAB). In FSN, Weissella became the dominant bacteria after 3 days of ensiling, and after nearly 30 days of ensiling, Lactobacillus replaced Weissella as the dominant genus, accompanied by the increase of Pediococcus. At the end of ensiling, the relative abundance of Lactobacillus and Pediococcus did not obviously change, but the relative abundance of Weissella further decreased, and the relative abundance of Clostridium and Bifidobacterium increased instead. In DSN, Weissella and Lactococcus were the two dominant LABs after 3 days of ensiling. After 30 days of ensiling, the relative abundance of Weissella further increased, while the relative abundance of Lactococcus decreased greatly. Lactobacillus and Pediococcus were the two LABs that increased at the later of ensiling. At the end of ensiling, the relative abundance of Weissella began to decrease, and Lactobacillus and Pediococcus further increased. However, the relative abundance of Weissella was still higher than that of Lactobacillus and Pediococcus. These three LABs dominated the bacterial community of DSN after 60 days of ensiling. Compared to FSN, Lactobacillus failed to become the dominant LAB. Although the relative abundance of epiphytic LAB in FFS was lower than that in FDS, the total relative abundance of LABs in FSN was higher than that in DSN in the first 30 days of ensiling (Fig. 5).
Metabolic Prediction Of Bacterial Community
Metabolisms changed by ensiling are illustrated in Fig. 6a. Compared to silage, epiphytic microbiota of fresh Italian ryegrass had more amino acid, energy, cofactors, vitamins, xenobiotics, terpenoids and polyketides metabolisms, and the biosynthesis of other secondary metabolites. Among them, metabolisms of terpenoids, polyketides and Xenobiotics in FFS were significantly higher than that in FDS (P < 0.05). The metabolisms of carbohydrate, glucan, and nucleotide were upregulated in silage. Among them, the glycan metabolism in FFS was significantly lower than that in FDS (P < 0.05). Carbohydrate and amino acid were the two metabolisms with the highest proportion in fresh and ensiled Italian ryegrass, so we specifically analyzed the carbohydrate and amino acid metabolisms (Fig. 6b and c). In terms of carbohydrate metabolism, the citrate cycle, butanoate, propanoate, glyoxylate and dicarboxylate metabolisms in fresh Italian ryegrass were higher than that in their corresponding silage. Ensiling upregulated the metabolisms of starch, sucrose, amino and nucleotide sugar, galactose, glycolysis/ gluconeogenesis, fructose and mannose. Most amino acid metabolisms were downregulated in silage, such as glycine, serine, threonine, arginine and proline metabolisms, Lysine biosynthesis was the only obviously upregulated amino acid metabolism in silage. Carbohydrate and amino acid metabolisms in FFS and FDS were similar, while amino acid metabolisms downregulated by ensiling were higher in FFS than those in FDS. Carbohydrate metabolism in the first 3 days of ensiling of FSN was similar to that in the first 30 days of ensiling of DSN, while amino acid metabolism of FSN in the late stage of ensiling was quite different from DSN. Thus, for the metabolic level, it also showed that the fermentation degree of DSN was lower than that of FSN.
In order to find out the contribution of bacteria to the metabolisms, the correlation between bacteria and the metabolism was analyzed (Fig. 7). Bacteria which was abundant in epiphytic microbiota were positively correlated to the downregulated carbohydrate and amino acid metabolisms during ensiling, and negatively correlated to the upregulated carbohydrate and amino acid metabolisms. Lactobacillus, Weissella, Pediococcus, Bifidobacterium, Serratia and Hafnia were positively correlated to the upregulate carbohydrate and amino acid metabolic pathway during ensiling, indicating that these bacteria exerted the main function in Italian ryegrass silage.