Aspergillus oryzae and Aspergillus niger Co-Cultivation Extract Affects In Vitro Degradation, Fermentation Characteristics, and Bacterial Composition in a Diet-Specific Manner
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| 4. Discussion
Our objective was to evaluate the effects of adding AOAN on nutrient digestibility of a TMR and three roughages (corn silage, oat hay, and alfalfa hay) typical of those given to dairy cows in China by analyzing fermentation parameters and bacterial composition (Supplemental Figure S1). NDF digestibility after 30 h of incubation and starch digestibility after 7 h of incubation were related to the value and quality of feed [ 37 , 38 ]. AOAN significantly improved nutrient utilization and digestion. Several studies have examined the A. oryzae product, Amaferm, and have indicated that Amaferm increased NDF digestibility of switchgrass and bromegrass at 12 h [ 39 ], alfalfa hay at 48 h [ 40 ], and DM digestibility of a TMR at 24 h in vitro [ 41 ]. Positive results from the animal experiment also indicated that DM and CP digestibility were increased by Amaferm supplementation [ 42 ], and fiber digestibility of the rumen and total tract were improved [ 43 ]. Our results are consistent with these studies. However, the inconsistent results from Sievert et al. [ 17 ], who indicated that total tract digestibility was not changed and milk fat was depressed by Amaferm. Lactation cows are different from dry cows, which might partly explain the divergent results. Furthermore, a recent study reported that the percentage and types of roughage used could influence this response [ 13 ]. Many reasons may contribute to this response, including different strains used in fermentation or unique chemical linkages in the roughage [ 10 , 44 ]. Therefore, we used an extract from A. oryzae combined with A. niger to improve the use of fungal extracts. Although the results obtained by in vitro techniques cannot completely reflect or replace Animals 2021, 11, 1248 15 of 19
those obtained from in vivo studies. The results of the present study provide important insights into the efficacy of AOAN. GP and rate are indicators of fermentability and digestibility. Khazaal et al. [ 45 ] reported that GP increased with in vitro digestibility. In our study, the GP and ideal maximum GP were also increased with nutrient digestibility. The single most striking observation was that the HT of TMR was minimum and AGPR was maximum in these feeds, which may be attributed to the balance between energy generation from fermentable carbohydrates and nitrogen generation from fermentable nitrogen in the TMR. Overall, these results show that AOAN was effective to improve the nutritional value of different feeds and was characterized by its broad-spectrum. The acetate in the rumen, fat precursors, is associated with milk fat production. In our study, the higher molar proportion of acetate and higher acetate to propionate ratio were observed in oat hay and alfalfa hay by adding AOAN, and then this change may contribute to milk fat production by supplying acetate. In animal experiments, the milk fat depression was obtained from Sievert and Shaver [ 17 ] by A. oryzae extract, and Zicarelli et al. [ 46 ] by Saccharomyces cerevisiae plus A. oryzae extract. According to Campanile et al. [ 47 ], milk
fat depression could be due to the increased organic matter digestibility, which allowed higher energy availability for milk yield and reduced the fat mobilization during the first phase of lactation. The propionate in the rumen, glucose precursors, is associated with energy supply for maintenance requirement and milking. In our study, the higher starch digestibility and unchanged acetate to propionate ratio of corn silage remind us that acetate and propionate might be increased at the same time. Hence, we suggest that the AOAN not only increases acetate for milk fat production but also increases propionate for energy. However, Higginbotham et al. [ 48 ] found no significant differences in milk yield in dairy cows fed a diet supplemented with A. oryzae extract. Thus, an animal experiment is essential to confirm these reflections of milk performance in the future. The AOAN or other commercial fungal extracts contain inactivated microorganisms, their products, and the medium. The comprehensive enzymes in their products could stim- ulate the growth of microorganisms by converting macromolecular nutrients to available nutrients for microorganisms, which increase the nutrient digestibility of the substrate and produce a large quantity of VFA to decrease pH in the medium [ 39 , 49 ]. Alternatively, the in- activated microorganisms and medium also provide limited nutrients for microorganisms and then enhance the nutrient digestibility, as evidenced in other fungi [ 50 ]. Regardless of the pathway, the intention is to improve the diversity and function of microorganisms through AOAN supplementation. In the current study, AOAN improved the bacterial diversity in total, which was consistent with our digestibility data. Further analysis indi- cated that the differences were mainly derived from TMR and oat hay rather than corn silage and alfalfa hay. In addition, the increment was mainly driven by an increment in richness (number of OTUs, Chao1, and ACE), rather than evenness. In general, oat hay has the same contents of NDF and CP as corn silage, whereas corn silage contains a higher level of starch as readily fermentable carbohydrates, which are fermented within 7 h and long before 48 h [ 38 ]. Further work on bacterial composition at 7 h would be meaningful to investigate the effects of AOAN on corn silage fermentation. In summary, these results suggest that AOAN is effective in improving bacterial diversity and is more significant for TMR and oat hay. The increasing digestibility of feeds suggested that more substrate was provided for bacteria in this study. Many other studies have reported that the NH 3 -N concentration is not changed by A. oryzae extract [ 40 , 43 , 49 , 51 , 52 ]. The NH 3 -N concentration depends on the balance between generation and consumption. The significant increase in bacterial diversity in the TMR indicates that bacterial communities may grow and utilize NH 3 -N
3 -N concentration in TMR, and further suggests that AOAN may be given to modify microorganisms more effectively than A. oryzae extract alone. However, the rumen undegraded protein in the feed may be decreased and the effects of AOAN on metabolizable protein including rumen
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undegraded protein and microprotein is still not clear. Zicarelli et al. [ 46 ] observed no differences in terms of milk yield using A. oryzae (20 g/head/d) but a lower percentage of protein content in milk, while the milk protein yield was not calculated according to the numerical increase of milk production. Hence, further investigation is needed to investigate the effects of AOAN supplementation on the metabolizable protein of feedstuff with different protein quality. Similar to previous studies using 16S rRNA sequencing in vitro, the most abundant bacteria at the phylum level that were shared across all culture solutions were Firmi- cutes, Bacteroidetes, and Proteobacteria [ 53 ,
]. Previously, an in vitro experiment using bromegrass and switchgrass fermented with A. oryzae extract reported that cellulolytic organisms were 3.5 times higher than that of the control [ 39 ]. The dairy cows fed A. oryzae extract also had higher counts of cellulolytic bacteria and proteolytic bacteria [ 55 ]. With the development of bacterial culture technology, Beharka and Nagaraja [ 56 ] provided the effects in detail and found A. oryzae extract increased the growth rate of cellulolytic bacteria (Ruminococcus albus and Fibrobacter succinogenes), and lactate-utilizing bacteria (Megasphaera elsdenii, Selenomonas lactilytica, and Selenomonas ruminantium), which were consistent with the results from Sun et al. [ 13 ,
], and the increased relative abundance of Selenomonas_1 in our study. However, those studies used classical, culture-based, microbiology methods, which describe only a small fraction of the total bacterial population [ 57 ]. In our study, the results were obtained from Prevotella, which would previously have been described as both cellulolytic bacteria and proteolytic bacteria because at least a third of glycoside, hydrolases, and other carbohydrate enzymes are affiliated with Prevotella [ 58 ]. This genus utilizes starch, protein, and fiber to produce succinate and acetate, and is one of the most abundant core genera in the rumen of dairy cows. Thus, it was not surprising to identify the positive correlation between the relative abundance of Prevotella and molar proportion of acetate, and Prevotella might play a key role in DM digestibility and TVFA production. In addition, Anaerovibrio has commonly been reported to be involved in lipolysis, and the lipase from A. niger extract may release fatty acids in the substrate and contribute to the increased relative abundance [ 59 , 60 ]. The positive correlation between the relative abundance of Anaerovibrio and the molar proportion of acetate implied that lipolysis in this bacteria could produce acetate. Following the addition of AOAN, a significant decrease in the relative abundance of Ruminococcus and the negative correlation between this genus and DM digestibility were not expected, in disagreement with previous studies using A. oryzae extract [ 13 , 15 , 56 ]. The abundance of Ruminococcus albus that increased in previous studies is just one of the species in this genus. Furthermore, other results showed that the counts of microorganisms may increase with total VFA production and increased α diversity. Therefore, the actual amount of Ruminococcus rather than relative abundance may increase. Furthermore, the fermentation system is a complex ecosystem composed of anaerobic, bacterial, fungi, protozoa, methanogenic archaea, and phages. Other microorganisms besides bacteria may influence the relative abundance of Ruminococcus. Like Ruminococcus, most of the decreased genera, including Syntrophococcus, Desul- fovibrio, and Lachnospira were carbohydrate-degrading bacteria and acetogenic bacteria. Butyrivibrio_2 is responsible for producing butyrate from glucose fermentation or acetate. The negative correlation between relative abundance of Butyrivibrio_2 and molar propor- tion of acetate suggested that this genus might be considered as acetate-utilization bacteria. However, a pure culture study indicated that Prevotella grew more abundantly in the presence of water-soluble cellulose acetate, yielding enhanced levels of acetate [ 61 ]. These increased Prevotella spp. might be the main contributors to the higher molar proportion of acetate. In summary, our sequencing results indicate that AOAN is beneficial to bacterial diversity and manipulated acetogenic bacteria of TMR, oat hay, and alfalfa hay fermen- tation rather than corn silage to change the fermentation pattern, and Prevotella is more sensitive to AOAN.
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