**2.4. Analysis of fermentation products**

To terminate fermentation at the end of incubation, 25 µL of formaldehyde solution (35%) were injected into serum bottles, which were immediately sealed and cooled at room temperature. Gas samples were collected by air syringe from the serum bottles and injected into a gas chromatograph (GC323, GL Sciences, Tokyo, Japan) equipped with a thermal conductivity detector and a stainless steel column (WG-100 SUS, 1.8 m × 6.35 mm OD), and the methane production in each serum bottle was measured. The analytical conditions were as follows: column oven temperature at 50°C, injector temperature at 50°C, and detector temperature at 50°C.

### **2.5.** *In vitro* **DM digestibility, and methane and VFA production**

Separate sub-samples of the supernatant were taken to determine the pH and VFA concentration. The bottles were rinsed with warm water to remove all solid residues, which were then oven-dried at 60°C and stored for further analyses. In total, 2 g of dried residue were oven-dried at 135°C and stored to determine DM digestibility.

### **2.6. Statistical analyses**

Analyses were performed using the general linear model procedure (SAS institute, Cary, NC, USA). Data on fermentative characteristics, in vitro DM digestibility, and ruminal methane and VFA production of TMR silages were subjected to one-way analysis of variance Tukey's test was used to identify differences (*P* < 0.05) between means. Data on the fermentative characteristics of each vegetable silage opened on d 30 were analyzed by oneway analysis of variance. Data on chemical compositions, fermentative characteristics, in vitro ruminal DM digestibility, and fermentation products after 6-h incubation of silages opened on d 60 were analyzed using a completely randomized design with a 4 × 4 (vegetable residues × additive treatment) factorial treatment structure. The general linear model procedure of SAS version 9.0 (SAS Institute, Inc., Cary, NC) was used for the analysis, and the model included the main effects of vegetable residues and additive treatment, and their interactions. Sealing time and ensiling duration were excluded from the model because the 60-d silages, wastes processed, and silos were made only one time. The Tukey test was used to identify differences (*P* < 0.05) between means.

Ruminal Digestibility and Quality of Silage Conserved via Fermentation by Lactobacilli 369

Treatment SEM3

B

Control M1 LAB2 M+LA

Item White cabbage Chinese cabbage Red cabbage Lettuce DM, % 3.6 ± 0.02 2.2 ± 0.01 5.2 ± 0.02 2.0 ± 0.01 OM, % of DM 81.6 ± 0.30 70.8 ± 0.08 87.3 ± 0.24 75.4 ± 0.09 CP, % of DM 21.9 ± 0.01 20.6 ± 0.21 22.8 ± 0.32 21.1 ± 0.41 Ether extract, % of DM 2.8 ± 0.07 1.5 ± 0.02 1.7 ± 0.14 4.9 ± 0.07 ADF, % of DM 31.0 ± 0.20 26.5 ± 1.49 31.6 ± 0.42 29.4 ± 0.14 ADL, % of DM 2.3 ± 0.19 5.8 ± 0.02 3.2 ± 0.03 5.9 ± 0.30 NDF, % of DM 32.8 ± 0.56 27.7 ± 0.24 33.6 ± 0.40 31.4 ± 0.01 Sucrose, % of DM 12.1 ± 0.15 1.1 ± 0.02 6.3 ± 0.10 3.1 ± 0.13 Glucose, % of DM 4.3 ± 0.04 3.6 ± 0.07 2.5 ± 0.01 1.3 ± 0.04 Fructose, % of DM 5.3 ± 0.12 3.8 ± 0.05 3.7 ± 0.10 4.0 ± 0.18

LAB (mg kg–1 FM4) - - 5 5 M (% FM) - 4 - 4 Whole crop rice (% DM5) 30 30 30 30 Concentrate6 (% DM) 25 25 25 25

Dried beet pulp (% DM) 13.5 13.5 13.5 13.5 Tofu cake (% DM) 30 30 30 30

1Molassess; 2Lactic acid bacteria (Lactobacillus plantarum); 3Standard error of means; 4Fresh matter; 5Dry matter; 6Formula feed ("Koushi Ikusei Special Mash" made by Zenno; total digestible nutrients, 70.0%; crude protein, 12.0% in fresh matter); 7Commercial vitamin-mineral supplement product (Snow brand seed, Iwate, Japan); 8Non-fibrous

**Table 3.** Ingredient and chemical composition of total mixed ration silages (Cao et al., 2010b)

carbohydrate (100 – crude protein – ether extract – neutral detergent fiber – ash).

DM (%) 35.9 36.2 36.4 37.0 1.98

Organic matter (% DM) 92.6 92.5 92.6 92.3 0.25 Crude protein (% DM) 15.3 14.6 15.4 15.1 0.38 Ether extract (% DM) 5.1 5.3 5.5 5.6 0.24 Nitrogen free extract (% DM) 57.1 58.4 58.4 57.5 0.40 Crude fiber (% DM) 15.7 14.9 14.2 14.5 0.88 NFC8 (% DM) 30.9 32.0 29.5 32.0 1.54 Crude ash (% DM) 7.4 7.5 7.4 7.7 0.25 Acid detergent fiber (% DM) 19.0 20.0 18.9 20.2 1.21 Neutral detergent fiber (% DM) 41.4 40.6 42.2 39.6 1.12

Vitamin-mineral supplement7 (% DM) 1.5 1.5 1.5 1.5

1Values are means ± SD.

**Table 2.** Chemical composition1 of vegetables residues (Cao et al., 2011)

Ingredient

Chemical composition
