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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| Ingredient
Level (%) Corn
4.95 Soybean hull 2.78 Soybean meal 7.61 Molasses beet 2.23 Whole cottonseed 2.88 NaHCO
3 0.28
Yeast powder 0.06
Steam flaked corn 9.74
Alfalfa hay 7.93
Whole corn silage 50.07
Rumen-pass fatty acid 0.42
Corn bran 2.70
Distillers dried grains and soluble 4.17
Oat hay 2.31
Mycotoxin remover agent 0.06
Premix 1 1.81 1 One kg premix contained the following: VA, 130,000 IU; VE, 465 IU; Cu, 2600 mg; Mn, 6000 mg; Zn, 2600 mg; Se, 70 mg; I, 120 mg; Co, 70 mg. 2.2. Experiment Design and In Vitro Batch Culture The in vitro experiments in anaerobic glass bottles (volume capacity of 120 mL) com- bined with the Automated Trace Gas Recording System for Microbial Fermentation (AGRS III, Beijing, China), incubated with a substrate, buffer solution, and rumen fluid were per- formed according to the description of Zhang and Yang [ 24 ]. The fermentation system in each bottle included 500 mg substrate, 50 mL rumen fluid, and 25 mL buffer solution. The rumen fluid from each cow was collected 2 h after morning feeding, filtered through four layers of cheesecloth, transported in a pre-warmed thermos bottle at 39 ◦ C, and then mixed in the laboratory with CO 2 at 39 ◦ C. The rumen fluid sampling process was conducted according to Yáñez-Ruiz et al. [ 25 ]. The buffer solution was prepared following the method of Menke [ 26 ]. After mixing, the buffer solution was bubbled with CO 2 until the pH value was 6.8. The substrates consisted of a TMR, corn silage, oat hay, and alfalfa hay obtained from the same commercial farm where the rumen-cannulated dairy cows resided. The fresh TMR sample was collected after mixing (MVS-16-H self-propelled vertical double auger, TATOMA, Monzón, Spain). The ingredients of TMR are shown in Table 1 . The corn silage, oat hay, and alfalfa hay samples were taken from the silage pit or feed warehouse. All the samples were taken from five points and mixed. After 2 h transportation from the farm to the laboratory, TMR and corn silage were dried at 60 ◦ C for 48 h, and then all feeds were ground to pass through a 2 mm screen (FNF-400, Qingdao Jimo, and Pulverizer Plant, Shandong, China). The chemical compositions are shown in Table 2 .
fermentation substrates were TMR, corn silage, oat hay, and alfalfa hay, respectively. Finally, according to the substrate amount (500 mg each bottle), 30 mg AOAN was weighted into each bottle. The AOAN product was supplied by Bioscreen Technologies SRL (Bologna, Italy). The additive amount of AOAN was referred to Sun et al. [ 13 ]. The main ingredient of the commercial product was corncob power with A. oryzae and A. niger fermentation (94.5% dry matter, 26% crude protein, 4.2% crude fiber, 3.8% crude fat). The TMR and corn silage fermentation included four-time points (7 h, 24 h, 30 h, 48 h), and hay or alfalfa hay fermentation included three-time points (24 h, 30 h, 48 h). Each time point included six replicates. These bottles were immediately connected to the Automated Trace Gas Recording System. Three bottles without substrate were used as the blank. |