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Article

Fermentation Characteristics, Chemical Composition, and Aerobic Stability in Whole Crop Corn Silage Treated with Lactic Acid Bacteria or Artemisia argyi

by
Huili Pang
,
Pilong Zhou
,
Zishan Yue
,
Zhenyu Wang
,
Guangyong Qin
,
Yanping Wang
,
Zhongfang Tan
* and
Yimin Cai
*
School of Agricultural Sciences, Zhengzhou University, Zhengzhou 450052, China
*
Authors to whom correspondence should be addressed.
Agriculture 2024, 14(7), 1015; https://doi.org/10.3390/agriculture14071015
Submission received: 2 May 2024 / Revised: 6 June 2024 / Accepted: 21 June 2024 / Published: 27 June 2024
(This article belongs to the Section Farm Animal Production)
Browse Figures
Versions Notes

Abstract

:
Whole crop corn silage (WCCS) been an important source of roughage for confined ruminants. However, at the silage feed-out phase, the rise in temperature and relative humidity under aerobic conditions breeds the production of undesirable microorganisms, such as yeast and mold. In order to investigate the conservation characteristics and aerobic stability underlying the effects of additives in whole crop corn silage (WCCS), whole crop corn (WCC) at the milk-ripe stage was ensiled with Lentilactobacillus (L.) buchneri (LB) and different proportions of Artemisia argyi (AA) for 90 days (d) at room temperature, respectively, and aerobic exposure after 90 d fermentation was also conducted. The study found that AA as an additive improved the fermentation quality and enhanced aerobic stability of WCCS, for which the addition of 60% AA increased the lactic acid fermentation rate, with the lactic acid concentration at the end of aerobic exposure significantly higher than in all other treatment groups, at 98.21 g/kg DM (p < 0.01), which decreased the relative abundance of none wanted microorganisms and reduced the content of fungal toxins (p < 0.05). After 90 d of fermentation, LB also increased the organic acids and reduced the pH compared with control, thereby improving fermentation quality. Furthermore, we also discovered that the relative abundance of Candida within the 60% AA was the highest. Candida have the ability to convert WSC into organic acids and lower pH, thus improving the quality of silage. Particularly, 60% AA could improve the fermentation quality and aerobic stability of silage through the biosynthetic pathways of phenylalanine, tyrosine and tryptophan, as well as by participation in the hydrolysis of glycoside hydrolases (GHs). Unexpectedly, the addition of AA was found to reduce the relative abundance of antibiotic resistance genes. WCC, ensiled with 60% AA, exhibited excellent fermentation quality and aerobic stability, providing a theoretical basis for understanding the mechanisms of AA which improve the quality of WCCS during the aeration phase.

1. Introduction

Whole crop corn (WCC), which is a high-yield forage crop, is often used for silage production. Silage production is a microbiologically driven process, and a way to preserve nutrients. A moist anaerobic fermentation environment (60–70% moisture content) improves the palatability and storage time of the forage while minimizing nutrient losses [1]. Whole crop corn silage (WCCS) has a high nutritional value and high overall economic efficiency, and it has always been an important source of roughage for ruminants, especially in confined ruminant production [2]. However, at the end of the silage feed-out phase, the environmental temperature and humidity promote the extensive growth of molds and yeast in the silage, which consume sugars and organic acids, raising the temperature and pH of the silage and leading to aerobic deterioration [3], which results in a loss of up to 30% of the dry matter (DM) nutrients and the production of mycotoxins in silage [4,5]. Especially in WCCS, a highly water-soluble carbohydrate (WSC) content can induce the activity of none wanted microorganisms, including yeast and mold, to grow after opening, leading to secondary fermentation and the aerobic spoilage of the silage [6,7]. Therefore, additives are commonly used to improve the fermentation quality and aerobic stability of WCCS during production.
Lactic acid bacteria (LAB) have the ability to ferment WSC [8]. In LAB, heterofermentationis, commonly used as an additive to improve silage quality [9] as it can convert lactic acid into acetic acid and 1,2-propanediol under anaerobic conditions, along with acetic acid, improves the aerobic stability of microorganisms associated with aerobic deterioration—particularly yeasts and molds; this conclusion has been confirmed by many researchers [10]. The most famous of these is Lentilactobacillus (L.) buchneri [11]. However, L. buchneri can cause DM loss in silage and fermented feed, and even the mixed addition of L. buchneri and homofermentative LAB does not improve the aerobic stability and fermentation quality of silage [10,12]. Therefore, it is crucial to find and develop new additives to improve the aerobic stability and fermentation quality of silage.
Artemisia argyi (AA) is a medicinal plant of great economic value, containing a wide range of secondary metabolites including volatile oils, flavonoids, terpenoids, phenolic acids and other compounds, and AA exhibits broad-spectrum antibacterial properties, effectively inhibiting bacteria including Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Listeria monocytogenes, as well as fungi such as Saccharomyces cerevisiae and Candida albicans [13,14,15,16,17]. Our research group has conducted applied studies on ensiled corn [2], wheat [3], wheat straw [18] and fermented soybean meal [19]. The results showed that the addition of AA not only improved the quality of silage, but reduced the mycotoxin levels in the fermented feed. Therefore, it is hypothesized that Artemisia argyi would inhibit none wanted microorganisms, including coliform bacteria, aerobic bacteria, clostridia, molds and yeast during the ensiling process, thereby maintaining good fermentation quality and aerobic stability.
In our previous study, we conducted preliminary research on the mixed storage of WCC and AA, and have explored the phenomenon of AA affecting the quality of WCCS [2]. Nevertheless, in order to completely solve the aerobic fermentation problem of silage after opening, the mechanism of how additives improve the stability of the aerobic phase still needs further exploration. Currently, the development of extensive sequencing methods, that include metagenomics and metatranscriptomics, has greatly increased the understanding of the composition and activity of microbial communities in silage, revealing the role of additives in regulating silage quality [20,21,22,23]. Based on this, the present study aimed to further elucidate the mechanism of aerobic stability, combined with the fermentation characteristics, chemical composition and microbial communities of WCCS during the aerobic exposure phase, based on high-throughput sequencing and metagenomic technologies.

2. Materials and Methods

2.1. Materials Collection and Silage Preparation

In September 2022, whole crop corn (variety: Zhengdan 958) harvested in Xingyang, Henan Province (34.37° N, 113.28° E) and whole crop AA (variety: Tangyin Beiai) harvested in Anyang, Henan Province (35.92° N, 114.35° E) were cut into lengths of 1–2 cm for later use. L. buchneri was provided by China Agricultural University and cultured in Man Rogosa Sharpe (MRS) medium at 37 °C for 12 h (hours), followed by centrifugation at 4 °C and 12,000× g for 10 min. The precipitate was mixed with distilled water to achieve an OD600 of 0.80.
The experimental treatments were divided into 5 groups: (1) WCC, no additive; (2) LB, 2% L. buchneri (1.00 × 106 colony forming unit per milliliter (cfu/mL)); (3) 30% AA (whole crop corn + 30% AA, next same); (4) 60% AA, and (5) 90% AA. The moisture content of each group was adjusted to about 65%. The mixture (300 g) was placed in fermentation plastic bag (25 cm × 35 cm) and sealed with vacuum sealer (P-290, Shineye, Dongguan, China). Each treatment was prepared in 3 replicates and ensiled for 90 days (d), and then aerobically exposed for 1, 3, 5, and 7 d after each opening. Both ensiling and aerobic were conducted at room temperature (16–35 °C). Fermentation quality, chemical composition and microbial counts were analyzed during the fermentation and aeration phases.

2.2. Fermentation Quality, Chemical Composition, and Microbial Counts

Mix 10 g fresh material and each opened sample with 90 mL sterile water, filter the mixture through a four-layered cheesecloth to determine the pH value, ammonia nitrogen (NH3-N) after thorough shaking; for the determination of organic acids, the filtration process with four layers of gauze should be followed by another filtration with a 0.22 μm filter membrane [24]. The content of organic acids was determined using high-performance liquid chromatography (HPLC) [25]. Microbial counts were analyzed by following the method of Pang et al. [26], which involves consecutive dilutions (10−1 to 10−5) and spread-plate method.
Dry matter (DM) was determined using the drying method with 100 g sample, and WSC, neutral detergent fiber (NDF), acid detergent fiber (ADF) and crude protein (CP) content was measured in accordance with AOAC [3,27,28].

2.3. Aerobic Stability

According to Hu et al. [12] and He et al. [29], the improved method involved placing 12 bags of silage samples (300 g per bag, with 3 replicates for each of the 4 aeration phases), from the same treatment group of silage samples, into a 5 kg plastic drum and exposing them to air at room temperature (18–22 °C). A temperature logger, which automatically recorded the temperature, was placed in the center of the silage every 30 min, and the values were averaged every 2 h. Each sample was covered with a double-layer sterile coarse cotton cloth to prevent the contamination and drying of the silage while allowing air to penetrate the silage block.

2.4. Toxin Contents Determination

The enzyme-linked immunosorbent assay (ELISA) kits, provided by Ameko Biotech Co., Ltd. (AMEKO, Shanghai, China), were used to detect the levels of mycotoxins, including aflatoxin B1 (AFB1), zearalenone (ZEN) and fumonisin (FB).

2.5. Bacterial and Fungi Community Analyses

A total of 10 g of frozen sample was taken and 40 mL of sterile water was added. Homogenize the mixture and filter it through a four-layered sterile cheesecloth. Rinse the residue with sterile water three times to recover any remaining microorganisms. Centrifuge the filtered solution at 4 °C for 15 min (12,000× g). Qualified genomic DNA samples were obtained for PCR amplification. Illumina MiSeq with paired-end sequencing was performed on the V4 amplicons using barcodes. The raw data undergo data filtering to remove low-quality reads, and the remaining high-quality clean data were used for subsequent analysis. The reads were assembled into tags based on the overlap relationship between them. The tags were then clustered into Operational Taxonomic Units (OTUs) at a given similarity threshold. The OTUs were further annotated against a database for species identification. Species complexity and inter-group species difference analysis were performed based on the OTUs and species annotation results.

2.6. Metagenomic Sequencing

The treatment method was the same as mentioned above. Qualified genomic DNA samples were randomly fragmented using the ultrasonic high-performance sample processing system (Covaris, Woburn, MA, USA), and fragment sizes of approximately 300–400 bp were obtained after fragment selection. The DNA fragments were then subjected to library preparation, including adapter ligation. Finally, sequencing was performed on the MGISEQ 2000 platform using paired-end 150-bp sequencing. The raw data undergo quality control and data filtering, followed by metagenomic assembly, gene prediction, the construction of a reference gene set, and the subsequent analysis of species, genes, and functions (BGI Genomics Co., Ltd., Wuhan, China).

2.7. Statistical Analysis

The IBM Statistical Package for the Social Sciences, version 22.0 (SPSS 22.0, SPSS Inc., Chicago, IL, USA), was employed for analyzing the data related to fermentation quality, chemical composition, and microbial population. Duncan and Tukey tests were employed to evaluate the multiple comparison differences among treatment groups, with significance set at p < 0.05, and p < 0.01 indicating high significance. Additionally, paired t-tests were conducted to compare the association analysis between fermentation quality and microbial communities, with significance denoted as * p < 0.05, and ** p < 0.01.

3. Results

3.1. Characteristics of Whole Crop Corn and Artemisia Argyi before Ensiling

The characteristics of the fresh materials are shown in Table 1. The pH of both WCC and AA was above 5.00. The content of lactic acid (18.29 g/kg DM) and acetic acid (5.75 g/kg DM) in AA was higher than that in WCC, while propionic acid and butyric acid were not detected in either material. The counts of associated microorganisms in both fresh materials were approximately similar, with the quantity of LAB at around 5.73 lg cfu/g FM. However, the count of undesirable microorganisms in WCC was higher than that in AA, with the yeast count reaching 8.49 lg cfu/g FM. The CP content in AA was significantly higher than that in WCC, at 109.62 g/kg DM. There were significant differences in the levels of AFB1, ZEN, and FB between WCC and AA, but the levels of these three toxins were lower in AA than that WCC.

3.2. Fermentation Quality, Chemical Composition, and Microbial Population of Silage

3.2.1. Fermentation Quality

According to Table 2, the pH increased with prolonged aerobic exposure time. No presence of propionic acid and butyric acid was detected throughout the entire fermentation process. At 90 d of fermentation, the pH of LB was significantly lower than that of WCC, and NH3-N levels were significantly lower compared to those in 60% AA and 90% AA (p < 0.05). After 7 d of aerobic exposure, 60% AA showed a significantly lower pH than that of the other treatment groups (p < 0.01), at 3.90. Furthermore, at 90 d of fermentation, 60% AA demonstrated no significantly higher lactic acid content than that of the other treatment groups, at 84.78 g/kg DM; after 5 d of aerobic exposure, it reached peak content at 122.30 g/kg DM; and the lactic acid content on 7 d of aeration was statistically similar at 60% AA and 90% AA, and the acetic acid contents were statistically similar for all silages tested. After 90 d of fermentation, the WSC content of 60% AA and 90% AA treatments was significantly higher than all other treatment groups (p < 0.05). At the end of aeration, the WSC of 60% AA was only significantly higher than that of the 30% AA, LB and WCC silages (p < 0.05). The 90% AA silages had similar WSC compared to 60% AA (p > 0.05).

3.2.2. Chemical Composition

According to Table 3, it was observed that the DM increased with the increase in the amount of AA addition and aerobic exposure time. The CP of all treatment groups was higher than that of the WCC, and the WCC exhibits the fastest decline in CP.ADF and NDF, increased with increasing levels of AA supplementation. During the 90 d fermentation and aerobic exposure process, the NDF and ADF of each group show a decreasing trend, reaching their lowest values on the 7 d of aerobic exposure; and the AA addition group values were not significantly lower than those of the WCC and LB groups.

3.2.3. Microbial Population

According to Table 4, it was evident that with the increase in fermentation and aerobic exposure time, the number of none wanted bacteria with different proportions of AA was significantly lower than that in the WCC and LB groups (p < 0.01). After 90 d of fermentation, the number of bacilli in LB was significantly lower than that in WCC (p < 0.05), while the number of clostridium was not significantly lower than all other treatment groups. The quantity of coliform bacteria and yeast in the 60% AA was lower than that in the other treatment groups, at 4.43 and 3.90 g/kg DM, respectively; meanwhile, the quantity of LAB in the 60% AA treatment group was the highest, reaching 9.57 g/kg DM. After 7 d of aerobic exposure, the quantity of none wanted bacteria in the 60% AA reached the lowest level, and was significantly lower than that in the other treatment groups (p < 0.05); the quantity of LAB was higher than that in the other treatment groups, reaching 7.37 g/kg DM.

3.3. Aerobic Stability

The changes in temperature and aerobic stability during the 90 d fermentation and aerobic exposure stages were depicted in Figure 1. After 90 d of fermentation, the temperatures of all treatment groups were at similar levels. With prolonged aerobic exposure time, the WCC, LB, and 30% AA groups all exhibited temperature increases, with temperatures exceeding the ambient temperature (AT) by 2 °C, and the aerobic stability times were 17, 40, and 92 h, respectively. At the end of aerobic exposure, the temperatures of both the 60% AA and 90% AA groups remained at relatively low levels, with an aerobic stability time of 168 h. However, the temperature of the 90% AA group began to approach the ambient temperature.

3.4. Toxin Contents

The changes in toxin content during the 90 d fermentation and aerobic exposure stages are illustrated in Figure 2. After 90 d of ensiling, the levels of AFB1, ZEN, and FB toxins in all treatment groups were within the limits specified by the “Feed Hygiene Standards”. Additionally, the content of these three toxins in the AA-treated group was lower than that in the WCC group. With prolonged aerobic exposure time, there was an increasing trend in the toxin content across all treatment groups. Throughout the entire aerobic exposure phase, the WCC group exhibited the highest toxin content and the fastest rate of increase, while the 60% AA shows the lowest toxin content and the slowest rate of increase. After 7 d of aerobic exposure, the AFB1, ZEN, and FB toxin levels in the WCC group reached their peak values at 7.06 μg/kg, 5.47 mg/kg, and 6.23 mg/kg, respectively; meanwhile, the AFB1, ZEN, and FB toxin levels in the 60% AA group were the lowest, and were below those of the other treatment groups at 2.06 μg/kg, 0.44 mg/kg, and 3.42 mg/kg, respectively.

3.5. Dynamics Changes in Microbial Communities in Fresh Materials and Silage

3.5.1. Microbial Community Abundance

The genus level of bacteria is shown in Figure 3. As can be seen, Cronobacter was the predominant microorganism in fresh WCC, while Erwinia and Pseudomonas were the main microorganisms in fresh AA. After 90 d of fermentation, the primary microorganisms in LB and WCC were Lactobacillus and Acetobacter, with Lactobacillus comprising the highest relative abundance in LB (60.24%), which was significantly higher than that of all other treatment groups (p < 0.05). Lactobacillus and Weissella were the main microorganisms in all AA treatment groups. With prolonged aerobic exposure, the dominant microbial communities in the WCC, LB, and 30% AA groups gradually shifted to Acetobacter, with a relatively high abundance of Xanthomonas in WCC. It is worth noting that throughout the entire aerobic exposure process, Lactobacillus and Weissella were always the dominant microbial communities in 60% AA and 90% AA, and had a higher relative abundance in 90% AA.
Figure 4, which concerns fungi at the genus level, shows that Candida was the predominant microorganism in the fresh samples of WCC, while Filobasidium was predominant in the fresh samples of AA. During the 90 d of anaerobic fermentation, WCC exhibited a relatively rich species diversity, with Candida accounting for the highest proportion at 26.88%, followed by Scedosporium. In contrast, Candida was predominant in all AA-added groups and LB. Throughout the aerobic exposure phase, WCC and LB showed a relatively rich species diversity. Upon 5 d of aerobic exposure, Wickerhamomyces was the predominant microorganism in WCC, while Candida was predominant in LB. The dominant genus in 30% AA changed to Kazachstania, while in 60% AA and 90% AA, Candida remained dominant, but the relative abundance of Issatchenkia gradually increased in 90% AA. These results did not significantly differ from those after 7 d of aerobic exposure.

3.5.2. Correlation Analyses of Microbial Communities and Fermentation Products

At the bacterial genus level (Figure 5a), Lactobacillus, Weissella, and Cronobacter were negatively correlated with pH (p < 0.01). Lactobacillus was positively correlated with lactic acid and acetic acid (p < 0.01), while Weissella exhibited a highly significant positive correlation with lactic acid (p < 0.01) and a non-significant positive correlation with acetic acid, while it was negatively correlated with ZEN and FB (p < 0.01). Acetobacter and Xanthomonas were positively correlated with pH (p < 0.01), and negatively correlated with lactic acid (p < 0.01), while they were positively correlated with AFB1 (p < 0.01); Xanthomonas was positively correlated with ZEN (p < 0.05).
At the fungal genus level (Figure 5b), Candida was negatively correlated with pH (p < 0.01), and positively correlated with lactic acid and acetic acid (p < 0.01), while it was negatively correlated with AFB1, ZEN and FB (p < 0.01). Kazachstania was positively correlated with pH and AFB1 (p < 0.01) and showed non-significant positive correlations with ZEN and FB, as well as non-significant negative correlations with lactic acid and acetic acid.

3.6. Metagenomic Analyses of Microorganisms

According to the KEGG level 1 annotation results (Figure 6a), all the treatment groups for metabolic pathways, and WCC, had a relatively higher gene abundance.
The KEGG level 2 annotation results (Figure 6b) show that WCC had the highest gene abundance in the global and overview maps, and in the carbohydrate metabolism pathway. The relative abundance of membrane transport in 60% AA and 90% AA was higher than that in other treatment groups.
Furthermore, according to the KEGG level 3 annotation results (Figure 6c), WCC had the highest gene abundance in metabolic pathways, with the biosynthesis of secondary metabolites, and microbial metabolic pathways in diverse environments. The relative abundance of ABC transporters in 60% AA and 90% AA was higher than that in the other treatment groups, and WCC showed a significantly higher relative abundance of two-component systems compared with the other treatment groups.
Compared with that in WCC, the relative abundance of genes involved in the porphyrin metabolism pathway was higher in both 60% AA and 90% AA. The biosynthesis pathways of phenylalanine, tyrosine, and tryptophan played important roles in amino acid metabolism in 60% AA. In lipid metabolism, the relative abundance of genes involved in fatty acid biosynthesis, and the biosynthesis of unsaturated fatty acids pathways, was higher. In xenobiotic biodegradation and metabolism in 90% AA, the gene abundance of the benzoate degradation pathway is relatively higher (Figure 7).
Furthermore, the relative abundance of genes involved in lysine biosynthesis in the amino acid metabolism pathway, the carbapenem biosynthesis in the biosynthesis of secondary metabolites pathway, N-glycan biosynthesis, and various types of N-glycan biosynthesis in the glycan biosynthesis and metabolism pathway was higher in 60% AA. On the other hand, the relative abundance of genes involved in valine, leucine, and isoleucine degradation in the amino acid metabolism pathway was higher in 90% AA. The gene quantity related to lipid metabolism was significantly higher in 60% AA than in 90% AA, while the relative abundance of genes involved in benzoate degradation in xenobiotic biodegradation and metabolism was still higher in 90% AA.
The functional annotation of microbial CAZy level 1 in the aerobic exposure of whole-plant corn silage for 7 d is shown in Figure 8. Carbohydrate metabolism primarily involves glycoside hydrolases (GHs), glycosyl transferases (GTs) and carbohydrate- binding modules (CBMs). Among them, GHs accounted for the highest proportion, especially in the 60% AA and 90% AA groups. Additionally, the relative abundances of GTs in the LB and 30% AA treatment groups were higher than those in the other groups.
The annotation of microbial antibiotic resistance genes is shown in Figure 9. After 7 d of aerobic exposure, the predominant antibiotic resistance genes in the WCC group were fluoroquinolone, tetracycline, aminoglycoside and elfamycin antibiotics. Furthermore, in LB and all AA treatment groups, there was a decrease in the relative abundances of these four antibiotic resistance genes compared with WCC, while the abundance of cephalosporin and penam antibiotics increased.

4. Discussion

4.1. Analysis of Fermentation Quality Changes during the Fermentation and Aerobic Exposure Phases

The characteristics of fresh raw materials and epiphytic microorganisms have a significant impact on fermentation quality. Previous studies have reported that the optimal DM content of high-quality silage is between 30 and 35%, and a WSC content above 50 g/kg DM indicates successful ensiling [30,31]. In this study, the DM of WCC and AA was 362.11 and 478.74 g/kg (36.21% and 47.87%), respectively, with a WSC content of 140.73 g/kg DM and 127.58 g/kg DM, which indicated good fermentation characteristics. CP is one of the main nutrients in silage, and protein degradation can affect the nutritional value of forage [32]. AA had a higher CP content (109.62 g/kg DM), indicating that it could improve the nutritional value of feed as an additive. Previous studies have shown that silage with an LAB count greater than 5.00 lg cfu/g FM can produce high-quality silage [33]. In this study, the LAB counts of both raw materials were above 5.73 lg cfu/g FM and, thus, were sufficient to initiate lactic acid fermentation under anaerobic conditions. LAB can convert WSC into organic acids and provide a lower pH [2]. However, the number of none wanted microorganisms, such as coliform bacteria, aerobic bacteria and yeast, was also relatively high in both raw materials, presenting a severe challenge for ensiling without additives. To make matters worse, the proliferation of undesirable aerobic microorganisms, including yeast and mold, is inevitable when silage makes contact with air after opening for feeding, leading to the aerobic deterioration of the silage. Therefore, it is necessary to use additives that can effectively inhibit none wanted aerobic microorganisms and reduce aerobic deterioration during the exposure process, to promote the fermentation process and enhance the aerobic stability of silage.
The pH of silage is considered a key indicator of fermentation [34]. A lower pH ensures better anaerobic fermentation and further suppresses the growth of none wanted microorganisms [35]. In this study, after 90 d of fermentation, the pH values of the LB, 30% AA and 60% AA groups were all lower, and the LAB counts and lactic acid content were higher, while unwanted bacteria, including coliform bacteria, aerobic bacteria, and bacilli were significantly decreased. During the fermentation process, L. buchneri can produce a large amount of acetic acid and even decompose lactic acid into substances such as acetic acid, ethanol, and propylene glycol, which can further inhibit the reproduction of none wanted bacteria in the feed and improve the quality of silage. Meanwhile, the active substances in AA are activated after fermentation, promoting the proliferation of LAB and inhibiting the growth of undesirable microorganisms during the fermentation process [18]. With the prolongation of the aerobic exposure time, the pH of all treatment groups increased, while the acetic acid contents decreased. This might be because, upon opening the fermented feed, it came into contact with air, and carbohydrates, organic acids, proteins, and amino acids were decomposed by aerobic microorganisms, generating heat and raising the pH value [3]. As for lactic acid content, the content of WCC and LB showed a downward trend with the extension of aerobic exposure time, while the content of 30% AA reached its peak on the 3 d of aerobic exposure, and the contents of 60% AA and 90% AA reached their peaks on the 5 d of aerobic exposure. However, the lactic acid content of 60% AA was not significantly higher than that of 90% AA. This indicates that the addition of AA can increase the lactic acid content of silage during aerobic exposure, thus improving the aerobic stability of silage. However, at 7 d of aerobic exposure, the pH of 60% AA remained stable at 3.90, which was significantly lower than that of WCC, LB and 30% AA; moreover, both 60% AA and 90% AA had the highest lactic acid contents (p < 0.05), and lowest yeast count. This could be due to the presence of active ingredients in AA, such as essential oils, flavonoids and polyphenols, which possess antimicrobial properties, inhibiting the growth of undesirable microorganisms during fermentation [36,37]. The active components present in AA suppressed the activity of yeast, thereby reducing the consumption of lactic acid.
During the 90 d fermentation period, the DM of 60% AA was within the range of high-quality silage feed (25–35%) [38], and it increased with the increase in the amount of AA added. This might have been due to the initially higher DM content of AA and the reduction in DM loss caused by microbial metabolism and decomposition, similarly to the results reported by Wang et al. [2,3,17]. The degradation of plant and microbial proteins leads to changes in nitrogen compounds in silage, and fermentation increases soluble N and NH3-N [35]. The NH3-N contents in the LB and 30% AA treatment groups was the lowest after 90 d of ensiling and aerobic exposure, respectively, which may have been related to the lower pH and higher levels of organic acid in these two groups inhibiting the hydrolysis of protein by none wanted microorganisms. The CP content increased with the increase in the amount of AA added, indicating that adding AA helped improve the CP content of the silage feed, as AA had a higher initial CP content. ADF and NDF in all treatment groups decreased after ensiling in the present study, which could help improve intake and digestibility by animals. The content of ADF and NDF determines the quality of roughage feed because they are difficult to digest and absorb in feed, and intake decreases by animals with the increase in feed NDF, while digestibility decreases with the increase in ADF [39].
Temperature is an important indicator for assessing the aerobic stability of silage, and aerobic stability was defined as the time (in hours) when the temperature of the silage after opening was 2 °C higher than the ambient temperature [40]. When silage is exposed to air, aerobic fungi—particularly yeast—rapidly proliferate. These fungi are considered catalysts for aerobic deterioration, as they release a significant amount of heat during metabolism and nutrient consumption, leading to an increase in the temperature of the silage feed [29]. As the aerobic exposure time increased in the present study, only the 60% AA group maintained a lower temperature level, with an aerobic stability time of 168 h, which was also consistent with previous results, showing that various bioactive components in AA, and low pH, inhibit the growth and proliferation of aerobic microorganisms [41].
Mycotoxins, including AFB1, ZEN, and FB [42], are toxic secondary metabolites produced by different fungi, such as Aspergillus, Fusarium, Penicillium, and Claviceps. They harm animal health through various mechanisms, causing hepatotoxicity, nephrotoxicity, immune modulation, genotoxicity, and neurotoxic effects, as well as reproductive and developmental disorders [43]. It is gratifying that the contents of these three mycotoxins in the 60% AA group was the lowest after 90 d of fermentation. Up to 7 days of aerobic exposure, the toxin levels remained lower than in the other treatment groups. This could also be attributed to the inhibition of mycotoxin-producing fungi by bioactive components, and the higher count of lactic acid bacteria, thus eliminating mycotoxin production at the source [44]. This is also in line with the findings of Wang et al. [18]. As for the specific inhibition mechanism, our team is still in the process of further research.

4.2. Analysis of Microbial Community Changes during Fermentation and Aerobic Exposure Stages

Studies have shown that the relative abundance of Acetobacter increases after aerobic exposure, and the aerobic stability of silage deteriorates [45]. The dominant bacteria at genus level in the WCC, LB and 30% AA were Acetobacter in this study, which also verified the conclusion. The relative abundance of Xanthomonas, a plant pathogen that can cause various diseases in crops [46], gradually increased in WCC. Moreover, Acetobacter and Xanthomonas were positively correlated with pH and negatively correlated with lactic acid, which corresponded to the poor fermentation quality and short aerobic stability time of these three groups. This also suggested that WCC alone cannot achieve good fermentation quality and aerobic stability. During fermentation and aerobic exposure, the dominant bacteria in 60% AA and 90% AA were Lactobacillus and Weissella, which were negatively correlated with pH and positively correlated with organic acid. Lactobacillus and Weissella are ideal microbial species, involved in acid production during ensiling [47]. Lactobacillus improves fermentation quality by consuming nutrients to produce lactic acid, lowering the pH and inhibiting the growth of other microorganisms [48], while Weissella is sensitive to low pH and is inhibited by Lactobacillus in acidic environments [49]. Thus, during the aerobic exposure phase, the 60% AA group had the lowest pH, and the relative abundance of Lactobacillus was higher than that of Weissella. Studies have shown that when adding AA to ferment whole crop corn and soybean meal together, the total flavonoid content in AA is positively correlated with the relative abundance of LAB [2,19]. This indicates that AA could increase the relative abundance of Lactobacillus and Weissella.
After 90 d of fermentation, the fungal microbiota of WCC were more diverse, while Candida was the dominant genus in all treatment groups, which was in keeping with findings that the yeast community is mainly composed of fermentative species belonging to the Candida and Pichia genera in silage under anaerobic conditions [50]. During the aerobic exposure phase, the fungal community of WCC became more diverse with prolonged aerobic exposure time. This might be one of the reasons for its poor aerobic stability. With the prolongation of aerobic exposure time, the dominant genus in the 30% AA treatment group gradually changed to Kazachstania, which was positively correlated with pH and AFB1. Kazachstania play an important role in the aerobic deterioration of silage, as the relative abundance of Kazachstania gradually increases after exposure to air [40,51].
When exposed to aerobic conditions for 7 d, Candida remained the dominant microbial group in 60% AA and 90% AA. Some studies have suggested that Candida is a lactic acid assimilating fungus, that can metabolize organic acids to increase pH and cause the aerobic deterioration of silage [50]. There are also studies indicating that Candida utilis in silage can produce various metabolites and proteins [52]; using a novel yeast microbial protein source—Candida utilis—in concentrated feed for cows can allow soybean meal to be replaced without affecting the quality of Norwegian Gouda cheese [53]; gut commensal Candida induces host antifungal immunoglobulin G (IgG) through CARD9-dependent production, producing systemic antibodies against specific fungal infections [54]. In our study, when exposed to aerobic conditions for 7 d, Candida remained the dominant microbial group in 60% AA and 90% AA, and it was negatively correlated with pH, AFB1, ZEN, and FB, while it was positively correlated with lactic acid and acetic acid in this study. It is no surprise that both groups had relatively lower pH (below 4.07) and higher contents of lactic acid (more than 89.77 g/kg DM); thus, the overall fermentation effect of these groups was good and did not cause aerobic decay, which was consistent with the results of Chen et al. [55]. Candida is a facultative anaerobic fungus that can survive under both aerobic and anaerobic conditions [56]. Research has shown that mixed fermentation of Lactobacillus and Candida can improve the sensory quality characteristics of feed color, aroma, taste, and quality [57,58], because, under the acidic conditions of fermentation, the vitamins and other nutrients released by yeast cell autolysis may even be beneficial for the growth of LAB [59]. This also explains why the relative abundance of Lactobacillus and Candida was high in the LB and AA added groups during the anaerobic phase in the present study.

4.3. Metagenomic Analysis at the End of Aerobic Exposure

In the functional difference analysis, compared with WCC, the relative abundance of genes involved in the porphyrin metabolism pathway in the metabolism of cofactors and vitamins was higher in both the 60% AA and 90% AA groups. Some intermediate metabolites in the porphyrin metabolism pathway, such as 5-aminolevulinic acid, heme, chlorophyll, and vitamin B12, have important physiological and metabolic functions [60]. Therefore, the identification of the porphyrin metabolism pathway in these two groups might make silage easy for animals to absorb, and has significant functions in preventing or treating anemia in young animals while improving their antioxidant and disease resistance abilities. The biosynthesis pathways of phenylalanine, tyrosine, and tryptophan in the amino acid metabolism of the 60% AA treatment group played an important role. These three amino acids are aromatic amino acids, which not only improve the sensory characteristics of feed and increase animal feed intake but also increase the protein content in synthesized protein [61]. Amino acids are essential for the growth and reproduction of LAB, and the hydrolysis of amino acids increases the lactate content [62], which explains the higher lactate and protein content in the 60% AA treatment group. As fatty acids and unsaturated fatty acids can improve the rumen microbial community and reduce methane (CH4) production in animals [63,64], the biosynthesis of fatty acids and unsaturated fatty acids in the lipid metabolism pathway in the 60% AA group might improve the quality of silage.
The higher relative abundance of carbapenem biosynthesis genes in the 60% AA group suggests the presence of microorganisms that are capable of producing carbapenems, which are broad-spectrum bactericidal antibiotics with antibacterial activity against many Gram-positive and Gram-negative pathogens [65]. As a member of the Poaceae family, corn has a cell wall composed of a large amount of phenolic cross-linked polysaccharides (cellulose, hemicellulose), which structurally hinder the enzymatic hydrolysis of polysaccharides [66]. GHs are hydrolytic enzymes that can degrade insoluble polysaccharides (chitin, cellulose) and convert polysaccharides into readily absorbable WSC (monosaccharides and oligosaccharides), including N-glycans in the intestine [67]. N-glycans are oligosaccharides that result from the N-glycosylation of plant proteins, and have various biological functions, such as promoting the colonization of beneficial bacteria in the intestine and immunoregulation [68,69,70]; they serve as substrates for the growth and proliferation of Lactobacillus, and can convert WSC into lactic acid to lower the pH of silage and maintain good fermentation quality [71]. The relatively higher abundance of GHs in the 60% AA treatment group could correspond to the higher WSC and lactic acid content, lower pH, and better fermentation quality observed in this group during aerobic exposure.
Additionally, it was unexpectedly found that AA could reduce the relative abundance of antibiotic resistance genes in the present research. The overuse and misuse of antibiotics, which are commonly used to treat or prevent bacterial infections, has led to a more rapid emergence of antibiotic-resistant bacteria (ARB) and antibiotic-resistant genes (ARGs), reducing antibiotics’ therapeutic potential against human and animal pathogens [72]. ARGs are genes that encode the ability of bacteria to develop resistance to antibiotics, and the resistance of bacteria is usually associated with the abundance of their resistance genes [73,74]. During the ensiling process of silage, if the bacteria involved carry ARGs, the number of these genes in the silage will increase in parallel with the bacterial count [75]. In the KEGG level 3 functional analysis, the relative abundance of two-component systems (TCSs) in the WCC group was significantly higher than that in the other treated groups. TCSs are the main mechanism by which bacteria perceive and respond to environmental changes, regulating antibiotic defense mechanisms and changing cellular physiological functions to enhance antibiotic resistance [76], and this might be the main reason for the higher relative abundance of annotated mainstream antibiotic resistance genes in WCC. In the CARD functional gene annotation, the relative abundance of four antibiotics—fluoroquinolone, tetracycline, aminoglycoside, and elfamycin—decreased in all AA-treated groups, further confirming that the addition of bioactives to silage can effectively reduce the abundance of antibiotic resistance genes [3,77,78,79]. Therefore, using AA and L. buchneri as feed additives can effectively reduce the relative abundance of antibiotic resistance genes in silage during aerobic exposure. Additionally, the study of antibiotic resistance genes in silage microorganisms is very important for mitigating or interrupting the spread and transmission of resistance genes from silage as an intermediate pathway.

5. Conclusions

In whole crop corn silage, although the addition of L. buchneri improved fermentation quality, its aerobic stability was poor. However, ensiling with the addition of 60% Artemisia argyi improved the fermentation quality and aerobic stability by reducing the relative abundance of none wanted microorganisms, decreasing the content of mycotoxins and lessening the relative abundance of antibiotic resistance genes. Thus, this research provides a theoretical basis for understanding the mechanism of how Artemisia argyi improves the quality of whole crop corn silage during the aerobic exposure stage.

Author Contributions

Designed experiments, H.P.; carried out experiments, P.Z.; analyzed experimental results, P.Z., Z.W., Z.Y., Z.T., Y.W., G.Q. and Y.C.; wrote and edited the manuscript, P.Z. and H.P. All authors have read and agreed to the published version of the manuscript.

Funding

This work were sponsored by National Natural Science Foundation of China (No. 32201468), Natural Science Foundation of Henan (No. 232300421153) and Qinghai Province Key R&D and Transformation Plan of China (Grant no. 2023-NK-137).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article.

Conflicts of Interest

The authors declare no conflicts of interest.

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Agriculture 14 01015 g001
Figure 1. Variation in temperature (a) and aerobic stability (b) during 90 d fermentation and aerobic exposure stage. Different letters (a–d) indicate significant differences according to Duncan’s test (p < 0.05). WCC, whole crop corn; LB, whole crop corn mixed with 2% L. buchneri; 30% AA, 60% AA and 90% AA, whole crop corn mixed with 30%, 60% and 90% Artemisia argyi, respectively; AE1 d, AE3 d, AE5 d and AE7 d, aerobic exposure for 3, 5 and 7 days after ensiling 90 days, respectively.
Figure 1. Variation in temperature (a) and aerobic stability (b) during 90 d fermentation and aerobic exposure stage. Different letters (a–d) indicate significant differences according to Duncan’s test (p < 0.05). WCC, whole crop corn; LB, whole crop corn mixed with 2% L. buchneri; 30% AA, 60% AA and 90% AA, whole crop corn mixed with 30%, 60% and 90% Artemisia argyi, respectively; AE1 d, AE3 d, AE5 d and AE7 d, aerobic exposure for 3, 5 and 7 days after ensiling 90 days, respectively.
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Figure 2. Content of mycotoxins after 90 d fermentation and through aerobic exposure stage. Aflatoxin B1 (µg/kg DM) (a), zearalenone (mg/kg DM) (b), fumonisin (mg/kg DM) (c); WCC, whole crop corn; LB, whole crop corn mixed with 2% L. buchneri; 30% AA, 60% AA and 90% AA, whole crop corn mixed with 30%, 60% and 90% Artemisia argyi, respectively; AE5 d and AE7 d, aerobic exposure for 5 and 7 days after ensiling 90 days, respectively.
Figure 2. Content of mycotoxins after 90 d fermentation and through aerobic exposure stage. Aflatoxin B1 (µg/kg DM) (a), zearalenone (mg/kg DM) (b), fumonisin (mg/kg DM) (c); WCC, whole crop corn; LB, whole crop corn mixed with 2% L. buchneri; 30% AA, 60% AA and 90% AA, whole crop corn mixed with 30%, 60% and 90% Artemisia argyi, respectively; AE5 d and AE7 d, aerobic exposure for 5 and 7 days after ensiling 90 days, respectively.
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Figure 3. The structure of the bacterial community at the genus levels. WCC, whole crop corn; LB, whole crop corn mixed with 2% L. buchneri; 30% AA, 60% AA and 90% AA, whole crop corn mixed with 30%, 60% and 90% Artemisia argyi, respectively; AE5 d and AE7 d, aerobic exposure for 5 and 7 days after ensiling 90 days, respectively.
Figure 3. The structure of the bacterial community at the genus levels. WCC, whole crop corn; LB, whole crop corn mixed with 2% L. buchneri; 30% AA, 60% AA and 90% AA, whole crop corn mixed with 30%, 60% and 90% Artemisia argyi, respectively; AE5 d and AE7 d, aerobic exposure for 5 and 7 days after ensiling 90 days, respectively.
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Figure 4. The structure of the fungal community at the genus levels. WCC, whole crop corn; LB, whole crop corn mixed with 2% L. buchneri; 30% AA, 60% AA and 90% AA, whole crop corn mixed with 30%, 60% and 90% Artemisia argyi, respectively; AE5 d and AE7 d, aerobic exposure for 5 and 7 days after ensiling 90 days, respectively.
Figure 4. The structure of the fungal community at the genus levels. WCC, whole crop corn; LB, whole crop corn mixed with 2% L. buchneri; 30% AA, 60% AA and 90% AA, whole crop corn mixed with 30%, 60% and 90% Artemisia argyi, respectively; AE5 d and AE7 d, aerobic exposure for 5 and 7 days after ensiling 90 days, respectively.
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Figure 5. Correlation analysis between bacterial (a) and fungal (b) communities, and fermentation characteristics and correlation at genus level. Positive correlations are shown in red, and negative correlations are shown in blue. * means p < 0.05, and ** means p < 0.01.
Figure 5. Correlation analysis between bacterial (a) and fungal (b) communities, and fermentation characteristics and correlation at genus level. Positive correlations are shown in red, and negative correlations are shown in blue. * means p < 0.05, and ** means p < 0.01.
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Figure 6. Heat map of differential abundance of microbial KEGG level 1 (a), level 2 (b) and level 3 (c) function. WCC, whole crop corn; LB, whole crop corn mixed with 2% L. buchneri; 30% AA, 60% AA and 90% AA, whole crop corn mixed with 30%, 60% and 90% Artemisia argyi, respectively; AE7 d, aerobic exposure for 7 days after ensiling 90 days.
Figure 6. Heat map of differential abundance of microbial KEGG level 1 (a), level 2 (b) and level 3 (c) function. WCC, whole crop corn; LB, whole crop corn mixed with 2% L. buchneri; 30% AA, 60% AA and 90% AA, whole crop corn mixed with 30%, 60% and 90% Artemisia argyi, respectively; AE7 d, aerobic exposure for 7 days after ensiling 90 days.
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Figure 7. Functional differences in KEGG pathway enrichment: 30% AA, 60% AA and 90% AA, whole crop corn mixed with 30%, 60% and 90% Artemisia argyi, respectively; AE7 d, aerobic exposure for 7 days after ensiling 90 days.
Figure 7. Functional differences in KEGG pathway enrichment: 30% AA, 60% AA and 90% AA, whole crop corn mixed with 30%, 60% and 90% Artemisia argyi, respectively; AE7 d, aerobic exposure for 7 days after ensiling 90 days.
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Figure 8. Annotation of microbial CAZy level 1 function: 30% AA, 60% AA and 90% AA, whole crop corn mixed with 30%, 60% and 90% Artemisia argyi, respectively; AE7 d, aerobic exposure for 7 days after ensiling 90 days.
Figure 8. Annotation of microbial CAZy level 1 function: 30% AA, 60% AA and 90% AA, whole crop corn mixed with 30%, 60% and 90% Artemisia argyi, respectively; AE7 d, aerobic exposure for 7 days after ensiling 90 days.
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Figure 9. Annotation of microbial antibiotic resistance genes: 30% AA, 60% AA and 90% AA, whole crop corn mixed with 30%, 60% and 90% Artemisia argyi, respectively; AE7 d, aerobic exposure for 7 days after ensiling 90 days.
Figure 9. Annotation of microbial antibiotic resistance genes: 30% AA, 60% AA and 90% AA, whole crop corn mixed with 30%, 60% and 90% Artemisia argyi, respectively; AE7 d, aerobic exposure for 7 days after ensiling 90 days.
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Table 1. Characteristics of fresh materials (Mean ± SD, n = 3).
Table 1. Characteristics of fresh materials (Mean ± SD, n = 3).
ItemWCCAA
Microbial population
(lg cfu/g FM)		Coliform bacteria9.75 ± 0.139.47 ± 0.16
Aerobic bacteria9.77 ± 0.189.47 ± 0.14
Bacilli6.08 ± 0.165.89 ± 0.05
Clostridia6.16 ± 0.186.19 ± 0.11
Yeast8.49 ± 0.055.43 ± 0.23
Lactic acid bacteria5.82 ± 0.025.73 ± 0.34
Fermentation qualitypH5.24 ± 0.025.62 ± 0.09
WSC, g/kg DM140.73 ± 0.87127.58 ± 1.61
NH3-N, g/kg DMND0.32 ± 0.03
Lactic acid, g/kg DM13.36 ± 0.3618.29 ± 0.55
Acetic acid, g/kg DMND5.75 ± 0.08
Propionic acid, g/kg DMNDND
Butyric acid, g/kg DMNDND
Chemical compositionDM, g/kg36.21 ± 0.4847.87 ± 0.41
NDF, g/kg DM543.88 ± 3.58565.84 ± 1.02
ADF, g/kg DM430.23 ± 2.07418.48 ± 1.76
CP, g/kg DM86.89 ± 0.32109.62 ± 0.17
MycotoxinsAFB1, μg/kg1.15 ± 0.110.77 ± 0.03
ZEN, mg/kg4.84 ± 0.544.03 ± 0.08
FB, mg/kg5.63 ± 1.284.77 ± 0.05
WCC, whole crop corn; AA, Artemisia argyi; DM, dry matter; WSC, water-soluble carbohydrates; CP, crude protein; NDF, neutral detergent fiber; ADF, acid detergent fiber; NH3-N, ammonia nitrogen; cfu, colony forming unit; FM, fresh material; AFB1, aflatoxin B1; ZEN, zearalenone; FB, fumonisin; ND, not detected.
Table 2. Fermentation quality of whole crop corn after ensiling and through aerobic exposure.
Table 2. Fermentation quality of whole crop corn after ensiling and through aerobic exposure.
ItemTreatmentDays of Ensiling (Day, d)SEMp Value
90AE1AE3AE5AE7TET × E
pHWCC4.20 Ad3.76 Ad5.71 Ac6.43 Ab7.00 Aa0.165<0.01<0.01<0.01
LB3.63 Bc3.71 Ac5.57 Ab6.48 Aa6.48 Ba
30% AA3.75 ABc3.75 Ac3.79 Bc5.51 Bb6.53 ABa
60% AA3.83 ABa3.88 Aa3.88 Ba3.89 Ca3.90 Ca
90% AA4.02 ABa4.04 Aa4.05 Ba4.05 Ca4.07 Ca
NH3-N
(g/kg DM)		WCC0.14 B0.17 BND0.15 B0.33 B0.164<0.010.62<0.01
LB0.13 BNDNDND0.23 B
30% AA0.43 AB0.40 AB0.59 ANDND
60% AA0.71 A0.73 AB0.74 A0.82 A0.86 A
90% AA0.68 A0.72 A0.63 A0.91 A1.00 A
WSC
(g/kg DM)		WCC48.79 Aa47.29 ABab46.08 Bb45.14 Bb44.57 Bb2.29<0.01<0.01<0.01
LB49.43 Bb53.76 ABa51.58 Bab45.93 Bb45.60 Bb
30% AA49.43 Ba50.85 Ba49.86 Ba46.06 Ba45.83 Ba
60% AA55.71 ABa55.01 ABa59.04 Aa55.71 Aa55.70 Aa
90% AA57.89 Aa59.47 Aa56.80 ABa55.44 Aa53.26 Aa
Organic acid (g/kg DM)Lactic acidWCC54.83 Aab77.03 Aa34.51 Bb11.78 Bb4.33 Bb14.34<0.01<0.01<0.01
LB71.70 Aa70.21 Aa36.82 Bab12.14 BbND
30% AA81.81 Aa80.96 Aa118.68 Aa35.02 BbND
60% AA84.78 Aab63.98 Ab95.39 ABab122.30 Aa98.21 Aab
90% AA56.74 Ab61.97 Ab68.82 Bab106.12 Aa89.77 Aab
Acetic acidWCC16.16 ABa17.72 Aa7.63 Ba11.99 Aa10.65 Aa3.60<0.01<0.01<0.01
LB18.56 Aa14.91 ABab7.23 Bb7.11 Ab4.21 Ab
30% AA17.19 ABa14.62 ABab18.46 AaND6.15Ab
60% AA14.37 ABa10.14 ABa15.95 ABa12.35 Aa11.58 Aa
90% AA7.79 Ba6.44 Ba6.14 Ba7.12 Aa6.45 Aa
WCC, whole crop corn; LB, whole crop corn mixed with 2% L. buchneri; 30% AA, 60% AA and 90% AA, whole crop corn mixed with 30%, 60% and 90% Artemisia argyi, respectively; NH3-N, ammonia nitrogen; DM, dry matter; AE1, AE3, AE5 and AE7, aerobic exposure for 1, 3, 5 and 7 days after ensiling 90 days, respectively; ND, not detected; means with different uppercase and lowercase letters in the same column or row indicate a significant difference according to Duncan test (p < 0.05); SEM, standard error of means; T, treatment; E, ensiling days; T × E, interaction between treatment and ensiling days.
Table 3. Chemical composition of whole crop corn after ensiling and through aerobic exposure.
Table 3. Chemical composition of whole crop corn after ensiling and through aerobic exposure.
ItemTreatmentDays of Ensiling (Day, d)SEMp Value
90AE1AE3AE5AE7TET × E
DM
(g/kg)		WCC235.43 Ca241.97 Da239.13 Da243.47 Da245.72 Da1.53<0.01<0.01<0.01
LB221.76 Da235.61 Da250.24 Da251.12 Da254.23 Da
30% AA279.98 BCa296.53 Ca310.24 Ca319.39 Ca324.81 Ca
60% AA314.08 Bb398.63 Ba405.95 Ba414.36 Ba416.84 Ba
90% AA485.58 Ab534.33 Aa578.81 Aa580.94 Aa582.03 Aa
NDF
(g/kg DM)		WCC536.25535.29532.68533.63533.042.25<0.010.671.00
LB528.15525.30517.51515.74514.34
30% AA498.83496.88490.18489.33487.28
60% AA488.19485.49489.09486.36477.89
90% AA484.98482.06480.30479.97470.56
ADF
(g/kg DM)		WCC418.65415.04409.88406.99400.353.720.280.491.00
LB415.41396.25392.84374.82370.04
30% AA396.33395.21383.05371.43363.98
60% AA388.05382.01374.59365.51361.53
90% AA384.35381.23371.58364.16354.39
CP
(g/kg DM)		WCC84.64 Ca82.54 Ca80.09 Ba78.52 Ba73.27 Ba3.95<0.01<0.010.97
LB87.63 Ca87.29 Ca85.77 Ba85.92 Ba85.33 Ba
30% AA103.92 Ba99.06 Ba95.50 Ba94.67 Ba94.43 Ba
60% AA110.02 Aa109.31 ABa108.37 Aa107.89 Aa107.12 Aa
90% AA118.40 Aa114.88 Aa112.33 Aa112.16 Aa108.08 Aa
WCC, whole crop corn; LB, whole crop corn mixed with 2% L. buchneri; 30% AA, 60% AA and 90% AA, whole crop corn mixed with 30%, 60% and 90% Artemisia argyi, respectively; DM, dry matter; WSC, water-soluble carbohydrates; CP, crude protein; NDF, neutral detergent fiber; ADF, acid detergent fiber; AE1, AE3, AE5 and AE7, aerobic exposure for 1, 3, 5 and 7 days after ensiling 90 days, respectively; ND, not detected; means with different uppercase and lowercase letters in the same column or row indicate a significant difference according to Duncan test (p < 0.05); SEM, standard error of means; T, treatment; E, ensiling days; T × E, interaction between treatment and ensiling days.
Table 4. Microbial population of whole crop corn after ensiling and through aerobic exposure (lg cfu/g FM).
Table 4. Microbial population of whole crop corn after ensiling and through aerobic exposure (lg cfu/g FM).
ItemTreatmentDays of Ensiling (Day)SEMp Value
90AE1AE3AE5AE7TET × E
Coliform bacteriaWCC7.43 Ad7.35 Ad8.23 Ac9.65 Ab10.17 Aa0.17<0.01<0.01<0.01
LB7.22 Ab7.61 Ab7.38 Bb9.69 Aa9.58 Ba
30% AA5.06 Bc5.15 BCc6.12 Cb9.44 Aa9.39 Ba
60% AA4.43 Cb4.68 Cb5.59 Da5.18 Ca5.32 Da
90% AA4.36 Cc5.64 Bb7.54 Ba7.24 Ba7.40 Ca
Aerobic bacteriaWCC9.54 Ab7.54 Ac9.71 Aab9.59 Ab10.08 Aa0.15<0.01<0.01<0.01
LB7.39 Bb7.64 Ab9.70 Aa9.34 Aa9.63 Ba
30% AA5.50 Cc5.58 Bc7.42 Bb9.32 Aa9.46 Ba
60% AA5.49 Ca5.54 Ba5.67 Ca5.60 Ca5.38 Da
90% AA5.32 Cb5.48 Bb7.61 Ba7.35 Ba7.24 Ca
BacilliWCC6.35 Ab6.54 Ab6.70 Aab6.69 Aab6.95 Aa0.12<0.01<0.01<0.01
LB5.18 Bc5.17 Cc5.66 Bb5.55 Bb6.75 Aa
30% AA5.33 Bb5.43 BCb5.38 BCb5.40 Bb5.81 Ba
60% AA5.38 Bab5.59 Ba5.23 Cb5.48 Bab5.32 Cab
90% AA5.32 Bb5.78 Ba5.25 Cb5.58 Bab5.23 Cb
ClostridiumWCC4.67 Ab5.56 Ab5.37 Aab5.36 Aab7.16 Aa0.16<0.01<0.01<0.01
LB4.48 Ac5.14 ABc5.20 ABb5.09 Ab6.77 ABa
30% AA4.52 Ab5.25 ABb4.85 Bb5.34 Ab6.61 Ba
60% AA4.60 Aab4.96 Ba5.02 ABb5.14 Aab5.31 Cab
90% AA4.57 Ab4.98 Ba4.93 ABb5.16 Aab5.41 Cb
YeastWCC7.75 Ab7.89 Ab9.75 Aa9.90 Aa9.45 Aa0.27<0.01<0.01<0.01
LB7.43 Ac8.05 Ab9.41 Aa9.64 Aa9.74 Aa
30% AA4.82 Bc5.06 Bc6.21 Cb9.90 Aa9.69 Aa
60% AA3.90 Cb4.14 Cb4.58 Db5.16 Cab5.49 Ca
90% AA3.80 Cc5.82 Bb7.42 Ba7.30 Ba7.34 Ba
Lactic acid bacteriaWCC9.04 Da8.13 Dc8.51 Bb6.75 Dd5.45 Ce0.14<0.01<0.01<0.01
LB9.24 Ba9.38 Aa8.54 Ba7.67 Ab6.65 Cb
30% AA9.32 Aa8.62 Cba8.38 Cb7.46 Ac6.83 Cc
60% AA9.57 Aa9.14 Ba8.84 Ab7.38 Bb7.37 Ab
90% AA9.19 Ca8.76 Bb8.50 Cc7.31 Cc6.93 Bc
FM, fresh material; cfu, colony forming unit. AE1, AE3, AE5 and AE7, aerobic exposure for 1, 3, 5 and 7 days after ensiling 90 days, respectively; ND, not detected; means with different uppercase and lowercase letters in the same column or row indicate a significant difference according to Duncan test (p < 0.05); SEM, standard error of means; T, treatment; E, ensiling days; T × E, interaction between treatment and ensiling days.
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Pang, H.; Zhou, P.; Yue, Z.; Wang, Z.; Qin, G.; Wang, Y.; Tan, Z.; Cai, Y. Fermentation Characteristics, Chemical Composition, and Aerobic Stability in Whole Crop Corn Silage Treated with Lactic Acid Bacteria or Artemisia argyi. Agriculture 2024, 14, 1015. https://doi.org/10.3390/agriculture14071015

AMA Style

Pang H, Zhou P, Yue Z, Wang Z, Qin G, Wang Y, Tan Z, Cai Y. Fermentation Characteristics, Chemical Composition, and Aerobic Stability in Whole Crop Corn Silage Treated with Lactic Acid Bacteria or Artemisia argyi. Agriculture. 2024; 14(7):1015. https://doi.org/10.3390/agriculture14071015

Chicago/Turabian Style

Pang, Huili, Pilong Zhou, Zishan Yue, Zhenyu Wang, Guangyong Qin, Yanping Wang, Zhongfang Tan, and Yimin Cai. 2024. "Fermentation Characteristics, Chemical Composition, and Aerobic Stability in Whole Crop Corn Silage Treated with Lactic Acid Bacteria or Artemisia argyi" Agriculture 14, no. 7: 1015. https://doi.org/10.3390/agriculture14071015

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