1. Introduction
Forage oat (
Avena sativa) silage is considered a palatable, highly digestible and beneficial forage for ruminants in most parts of the world [
1,
2]. Large amounts of forage are consumed by ruminants, and the global trade of oat has been restricted due to the COVID-19 pandemic [
3]. Meanwhile, oats have been widely cultivated in China, both as silage and hay, especially in northern China. In recent years, the research on oat silage has received continuous attention. In previous studies, low-temperature-tolerant lactic acid bacteria (LAB) and bacterial diversity in oat silage have been systematically studied [
4,
5]. However, different bacterial species in oats still need further exploration.
LAB plays an active role in silage processing and is widely used in various grass-based silages to reduce nutrient loss and extend the storage period. In addition, exogenous probiotics can normally change the type of fermentation in oat silage [
6]. Therefore, adding exogenous bacteria or microbiota to silage has become an important way to regulate its anaerobic fermentation, and it also has a profound impact on the organic acid content and dry matter loss of oat silage [
7].
Propionibacterium has been used as a microbial additive for forage oat to improve the silage quality [
8]. In some cases,
Propionibacterium provides better aerobic stability [
9]. Silage inoculated with
Propionibacterium can produce propionic acid in the anaerobic fermentation period to improve the aerobic stability [
9]. However, there are still few reported studies on fermentation quality and in vitro gas production of oat silage treated with
Propionibacterium.
Silage treated with lactic acid bacterial inoculants has been reported to increase ruminal microbial biomass when tested in vitro [
10]. The rumen is one of the most powerful fiber-degrading fermentation systems known to date [
11]. However, the degradation of fibers by a large number of microorganisms in the rumen produces organic acids, along with a large amount of methane and hydrogen. Methane (CH
4) is a potent greenhouse gas and, along with carbon dioxide and nitrous oxide, CH
4 emission from livestock production is a major contributor to global warming [
12]. It is well-established that factors such as nutrient composition and degradability of ruminant diets greatly affect CH
4 production [
13]. In the in vitro experiment,
Propionibacterium freudenreichii 53-W was found to exhibit the ability to reduce methane, but the relevant mechanism is not yet clear [
14]. We hypothesize that silage treated with lactic acid bacteria or
Propionibacterium can not only improve fermentation quality but also reduce methane emissions.
Therefore, this study aimed to investigate the effects of three different strains of LAB and one Propionibacterium strain on the fermentation quality of oat silage and in vitro gas production.
4. Discussion
The forage oat industry has been well-developed domestically, and the research on oat silage and lactic acid bacteria has attracted widespread attention. Propionibacterium widely exists in dairy products and silage. However, studies on the application of propionic acid bacteria in oat silage are relatively rare. Therefore, this study investigated the different effects of four additives including propionibacterium and common lactic acid bacteria on the fermentation quality and microbial composition of oat silages.
Higher dry matter was detected in the LR group compared to the other additives and CK, suggesting that LR has the potential to reduce silage dry matter losses. This supports the hypothesis that the substance metabolism of three lactic acid bacteria and
propionibacterium in oat silages is different. WSC also plays a key role in the energy metabolism of microorganisms during the ensiling process. This study found that LR, LP and LC all had a better lactate synthesis capacity compared to PP and CK, which is consist with the result of the previous study [
18].
The ensiling process reduces the pH through microbial anaerobic fermentation and provides a stable environment that could inhibit harmful microorganisms. As the DM content increases, the bacterial activity is restricted by low moisture. Usually, due to the lactic acid synthesis by lactic acid bacteria, the final pH of well-fermented and preserved silages could be reduced to 4.0 or lower [
19,
20]. In this study, the LP group had the lowest pH, which is consistent with the results of the previous studies [
21,
22]. LR could also reduce pH, which is consistent with Chen’s report [
23]. However, unlike lactic acid, propionic acid contributed less to pH reduction [
24], which may explain the higher pH in the PP group. Ammonia N is another component which is important for assessing fermentation quality. Palatability, intake and N utilization decline as the NH
3-N content increases in silages. Fast acidification in the initial days of ensiling is essential to controlling the reproduction of clostridium bacteria, which may lead to hydrolysis of proteins and amino acids and produce large amounts of ammonia nitrogen [
25]. Unlike PP, LC and CK, LP and LR could reduce the NH
3-N content in oat, which is consist with the results of the previous studies [
21,
26]. Therefore, the comparison between different treatments clearly showed the benefits of inoculation under favorable ensiling conditions. This is likely to exacerbate the differences among treatments under the high DM content of the oat silage. Lactic acid is an important product in silage fermentation that reduces pH to achieve a stable state conducive to storage. Compared to PP, LC and CK, LR significantly increased the LA content, which might be attributed to the high amount of lactic acid bacteria and the WSC content, resulting in rapid production of lactic acid. Britt reported that the lower LA content in silages might be attributed to the addition of propionic acid [
27]. In contrast, in this study, the PP-treated silage did not reduce the LA content probably due to the amount of lactic acid bacteria. Thus, propionic acid and
P. acidipropionici may have different functions in oat silage. Flieg’s point, which is based on pH and DM content, has been widely used to assess the fermentation quality of silages [
28]. Although additive treatments improved the fermentation quality of oat silage, only LP had a significantly increased Flieg’s point. Flieg’s point was above 50 for the treatments, which represented a moderate quality [
29].
Coverage of all treatments exceeded 0.999, indicating that the sequencing data were sufficient to reflect the profile of bacterial community. In general, the stable state of aerobic and acidic environments inhibits the growth of most microorganisms, leading to low ecological diversity. PP and LC treatments led to the significant increase in the Shannon index of the oat silage, suggesting that PP and LC had a highly diverse bacterial community. Due to different treatments, index calculation methods and the number of observed OTUs, only the Shannon index showed differences. The Simpson index also indicated that PP had high bacterial community diversity. The ACE and Chao indices indicated that all treatments had the same richness of oat silage. In addition, the ACE and Chao indices may be less susceptible to inoculant treatment in oat silage, which is consist with the result of Jia’s previous study [
8].
To the best of our knowledge, forage sources affect the bacterial community of silages [
30]. Apparently,
Kosakonia has been observed by many researchers in barley silage [
31], stylo silage [
32], ricestraw silage [
33] and cornstalk silage [
34].
Kosakonia is a genus of
enterobacteria in the family of Enterobacteriaceae. To date, it has been identified in many studies, but no adverse effects on the ensiling process have been found. Most importantly,
Kosakonia is considered to have the ability to reduce ammonia nitrogen [
32]. Unlike LP, which may have a tendency to increase the abundance of
kosakonia in oat silage, the addition of vanillic acid could reduce the abundance of
kosakonia in stylo silage [
35]. In addition, tannic acid could lead to the higher abundance of
kosakonia in stylo silage [
36].
Kosakonia is positively related to many volatile chemicals in silage [
37]. In addition, the different abundances of
Clostridium sensu stricto 12 could be related to the inhibitory ability of different strain treatments and soil contamination of the plants during harvest [
38]. Despite the increasing use of high-throughput sequencing in the field of ensiling in recent years, efforts in analyzing certain unknown microorganisms, especially non-cultivable bacteria, are still expected [
39]. It is difficult to draw a definitive conclusion from the relative abundance of microbial communities, so we tried to obtain more intuitive characteristics through PCA analysis.
Lacticaseibacillus rhamnosus was thought to inhibit ethanol fermentation and
enterobacter activity in grass silage [
40]. In this study, we hypothesized that LR and other additives had slightly different metabolic patterns and expression of functional genes in the fermentation process. Moving forward, increasing studies are expected to consider not only how relative abundance of bacterial communities in the silage varieties, but also the functional genes, present.
In vitro gas production is not only applied to determine the nutritive values of silage, but is also used to predict the digestion status of ruminants [
41]. In vitro gas production is highly dependent on the availability of soluble fractions, which favors ruminal fermentation at an earlier fermentation stage [
42]. In this study, LP increased gas production within the first 48 h, and this might be because LP treatments could provide more available substrates for microbial degradation at the initial rumen liquid fermentation stages. However, PP and LC treatments may consume the key substrates in the ensiling process, which is good for gas production in in vitro fermentation. Intriguingly, gas production within 72 h had no significant difference, which may possibly be explained by different bacterial inoculants preferring slowly/rapidly degradable DM fractions in oat silages. Similar to Chen’s reports [
41], there was no significant difference in the gas production rate constant between oat silage treated with LP and PP, which is possibly due to the rumen liquid source (ruminant species) or forage characters. Therefore, further studies should pay more attention to those different bacterial inoculants, whereas their internal biological mechanisms need to be clarified. Methane (CH
4) is the third most important greenhouse gas, following water vapor and CO
2, contributing to climate change [
43]. Of the 16.5 billion tons of greenhouse gas emissions from global total agri-food systems in 2019, 7.2 billion tons came from the farm gate according to the new analysis [
44]. The emission of methane will also cause energy loss (from 2% to 12% of gross energy (GE)) [
45]. Therefore, significant research investments are needed to reduce the carbon footprint of ruminants, improve rumen fermentation and clarify the proton transfer strategy [
46]. Although the in vitro gas production (both methane and carbon dioxide) of oat silage fermented with PP and LC showed no significant difference from other groups, both reduced the emissions of methane and carbon dioxide. We hypothesized that both PP and LC might have positive effects on rumen methane proton transfer. Both oat grain and whole plants contain several types of antioxidants, such as α-tocopherol [
47] and polyphenols [
48].
Propionibacterium acidipropionici 1.1161. and
Lacticaseibacillus paracasei treatments may protect the antioxidants in oat ensiling processing [
49], thereby regulating the metabolism of rumen methane bacteria and reducing gas production. Moreover, those additives may provide a new prospect for oat silage fermentation and digestion and utilization of ruminants.