Interactive effect of inoculant and dried jujube powder on the fermentation quality and nitrogen fraction of alfalfa silage
Abstract
The interactive effect of inoculants and dried jujube powder (DJP) on the fermentation and nitrogen fraction (PA, PB1, PB2, PB3 and PC fractions) of alfalfa silage was investigated. Three of the Lactobacillus plantarum inoculants (LP1, LP2 or LP3) were used. The DJP was added at rates of 0, 3, 6, 9, 12 or 15% of the whole fresh forage. The combination of DJP and inoculants decreased the pH value and ammonia nitrogen content and increased the PC portion. As the DJP ratio increased, there was a peak in lactic acid : acetic acid ratio (12% of DJP ratio) and PB2 fraction (9% of DJP ratio) while the PA content decreased linearly. The LP1 and LP2 had the highest lactic acid content. Inoculants decreased the PB1 portion of true protein. The LP1 treated silage had the highest acetic acid content with the lowest lactic acid : acetic acid ratio and had lower PB3 and PC and higher PB2 than LP2 or LP3 treated silages. The result showed that the application of DJP or inoculants have positive effect on the fermentation, nutrition and N fraction value in the high moisture alfalfa silages, and the combination of DJP and inoculants preserves best.
Introduction
Harvesting alfalfa as hay has a high risk in southern China or in the summer of northern China. The moister climates cause famers to make ensiled alfalfa. However, making good alfalfa silage without wilting or any additive has long been known as a difficult issue for its high buffering capacity and low water soluble carbohydrate (WSC) content (McDonald et al. 1991). Differing from the herbage, large numbers of protein may be degraded into soluble non-protein nitrogen during ensiling (Fijalkowska et al. 2015), which would provide extra protein supplements to high-producing dairy cattle.
In recent years, microbial inoculant has been widely used in alfalfa silage which has positive effects on alfalfa silage fermentation (Filya et al. 2007). The homofermentative lactic acid bacteria (LAB) such as Lactobacillus plantarum is usually produces more lactic acid, rapidly decreased pH value, minimized dry matter (DM) losses and similar nutrition values to that of the crop at ensiling (Muck 2013). Dried jujube powder (DJP) which has high sugar and DM contents has a rich yield in north China and is often added as sugar supplement for animals. Little research has been done about the addition of DJP on silages. However, studies have shown that similar dry sugar sources were fermentation stimulants for alfalfa silage. Citrus pulp (Rodrigues et al. 2004), grape pomace (Canbolat et al. 2010), honey locust pods (Canbolat et al. 2013) and molasses (Khorvash et al. 2010) which have high sugar content have been reported to have positive effects on the fermentation of alfalfa silage, although they may decrease the crude protein.
The combination of DJP and inoculants could provide more LAB, enough sugars to consume and lower buffering capacity beneficial to rapid decrease of pH value. And then it could improve the fermentation quality of the alfalfa silages. However, when it comes to one certain inoculant, new problems should be considered. The effectiveness of the inoculant could be influenced by the characteristics of the crop at harvest, the epiphytic LAB population, additional amounts of inoculant in fresh material, sugar availability, and plant DM concentration (Ohmoto et al. 2002; Filya et al. 2007). When the ratio of DJP increases, the DM content and the sugar content increases, while the buffer capacity and the epiphytic LAB population decreases. The comparison of inoculants in alfalfa mixed with DJP would be necessary.
The protein quality of alfalfa silage is the most important determinant of silage nutritive value from an economic standpoint (Charmley 2001). The Cornell net carbohydrate and protein system (CNCPS system) which divided the nitrogen (N) fraction into five fractions (Sniffen et al. 1992) would be a more efficient method than just evaluating the crude protein or non-protein nitrogen. A study by Contreras-Govea et al. (2013) has shown that L. plantarum inoculant had positive effects on the preservation of true protein in alfalfa silage. However, the study by Hashemzadeh-Cigari et al. (2014) showed that the intermediate degraded true protein could be increased by the combination of molasses and inoculants, and inoculants used alone could not have this ability. Although the dry sugar sources could decrease the crude protein content of the silage, their effect on the true protein of alfalfa silage has not been considered. Choosing a suitable sugar source would be necessary.
Although many studies have indicated that inclusion of inoculants or dry sugar sources can significantly improve the fermentation quality of silages, little has been done to investigate the interactive effect between inoculants and dry sugar sources, especially for the nitrogen fraction. Therefore, the objective of the present study was to evaluate the effects of LAB inoculant, jujube powder and their combination on the fermentation, nitrogen fraction and nutrition value of high-moisture alfalfa silages.
Materials and Methods
Preparation of additives
Three of the inoculants (LP1, LP2 and LP3) applied in the silage were all L. plantarum strains and isolated in the forage processing laboratory of China Agricultural University from high-quality alfalfa silage samples (without addition of inoculants). The three inoculants with high acidification activity were isolated from different areas of China. More information about the isolation method and characteristics of the three L. plantarum strains can be found in Zhang et al. (2014). The addition level of the three inoculants and control were as follows: (i) distilled water and no inoculants; (ii) LP1, 106 colony forming units (cfu)/g of fresh forage; (iii) LP2, 106 cfu/g of fresh forage; or (iv) LP3, 106 cfu/g of fresh forage. The inoculants were dissolved in distilled water before application to the alfalfa; the same amount of distilled water was added when no inoculants were used.
The DJP was bought from a market in Huanghu, (Jujube powder, Shunde, Zhuozhou, Hebei, China). The DJP was added with 0, 3, 6, 9, 12, or 15% of the whole fresh forage (alfalfa and DJP mixtures), respectively.
Preparation of silages
A fourth-cut, vegetative growing alfalfa crop was harvested from an experimental station in Huanghua (Hebei, China). The harvest was ahead of time to avoid the risk of rainfall, and the approximate DM content was between 200 g/kg to 300 g/kg. The materials were randomly collected and completely mixed from fields (about 1 hectare) at the time they were chopped for ensiling in conventional farm silos. Immediately after harvest the fresh alfalfa was chopped into particles with an average length of 2.5 cm. The alfalfa was ensiled in triplicate for each treatment in bag silos (45 cm × 26 cm) and each silo weighed approximately 500 g. Upon filling, the bag silos were sealed with a vacuum sealing machine (DZ-280/2SD; Jinqiao Co. Dongguan, Guangdong, China). All bag silos were transferred to the forage processing laboratory (China Agricultural University, Beijing, China) and preserved for 60 days at room temperature (20-23°C) until opened for sampling.
Chemical analysis
Immediately after the bag silos were opened, 20 g of the silage from each bag was diluted with 180 mL of sterilized distilled water, homogenized for 1 min at 30 pulses of 2 s by using an organization stamp mill (WaringTM-8010S; Waring Laboratory Science, Kendall, TX, USA) and then filtered through four layers of cheesecloth and a qualitative filter paper. The pH was measured using filtrate with a glass-electrode pH meter (PHS-3C; Shanghai Precision & Scientific Instrument Co. Ltd., Shanghai, China). The filtrate was further processed with dialyzer of 0.22 µm and then kept at −20°C for analyzing the organic acid and ammonia-N. The lactic acid, acetic acid, propionic acid and butyric acid contents were determined by high-performance liquid chromatography (HPLC; Shimadzu, Tokyo, Japan) (Xu et al. 2007). The HPLC conditions were as follows: column, Shodex RSpak KC-811S-DVB gel C (8.0 mm × 30 cm; Shimadzu, Tokyo, Japan); oven temperature, 50°C; mobile phase, 3 mmol/L HClO4; flow rate, 1.0 mL/min; injection volume, 5 μL; and detector, SPD-M10AVP. The ammonia nitrogen was determined according to the method of Broderick and Kang (1980).
The DM content was determined after being oven dried for 48 h at 65°C. The dried samples were ground to 40 mesh using microplant grinding machine (FZ102; Taisite Co. LTD, Tianjin, China). By using an automatic fiber analyzer (Ankom 2000i full; Ankom Tech Co., Macedon, NY, USA), the neutral detergent fiber (aNDF) was determined with a heat stable amylase and was expressed inclusive of residual ash (Van Soest et al. 1991). The acid detergent fiber (ADF) was determined after the aNDF by using the automatic fiber analyzer and expressed exclusive of residual ash (Van Soest 1973). The acid detergent lignin (ADL) was also determined according to Van Soest (1973) following the analysis of ADF. Ether extract (EE) was measured with an extractor (Ankom XT15i; Ankom Tech Co., Macedon, NY, USA) according to the method of the Amreican Oil Chemists’ Society (AOCS) Official Procedure Am 5–04 (AOCS 2009). WSC was determined by anthrone-sulphuric acid method (McDonald & Henderson 1964). Kjeldahl N (i.e. TN) was analyzed using the method 954.01 of the Association of Official Analytical Chemists (AOAC 1990). Crude protein (CP) was calculated as Kjeldahl N × 6.25. The non-protein nitrogen (NPN), soluble nitrogen, neutral detergent insoluble nitrogen and acid detergent insoluble nitrogen (ADIN) were determined as described by Licitra et al. (1996). The CNCPS fraction was calculated according to method from Sniffen et al. (1992). Briefly, fraction A contains NPN (PA). The B fraction includes true protein, and based on the rates of protein degradation in the rumen, it subdivides into rapidly (PB1), intermediate (PB2) and slowly (PB3) degradable fractions. Fraction C contains the ADIN (PC). The buffering capacity of alfalfa material and DJP was determined according to Playne and McDonald (1966).
Statistical analyses
Analysis of variance was used to test statistically significance differences among the effects of DJP, the L. plantarum inoculants (LP) and the interaction effect of DJP × LP. When the F test indicated significance (i.e. P < 0.05) in the two main effects (DJP or LP), means separations were conducted using a least significant difference test. A simple effects test was conducted when the interaction was significant. Polynomial contrasts were used to test the effects of DJP on fermentation quality, nutritive value and CNCPS fractions. Significance was declared at P < 0.05. All of the above statistical analyses used the GLM procedure of SPSS 20.0.
Results
Chemical composition of alfalfa material and DJP
The alfalfa forage had a relatively low DM (262.83 g/kg) concentration, compared with DJP which was 896.40 g/kg (Table 1). The WSC content of DJP was almost seven times as high as that of alfalfa and the buffering capacity of DJP was just one-quarter of the alfalfa material. The CP content of alfalfa was four times as high as that of DJP.
| DM (g/kg) | aNDF (g/kg DM) | ADF (g/kg DM) | WSC (g/kg DM) | CP (g/kg DM) | Buffering capacity (mE/kg DM) | |
|---|---|---|---|---|---|---|
| Alfalfa | 263 | 297 | 190 | 44 | 246 | 412 |
| DJP | 896 | 342 | 236 | 296 | 60 | 114 |
- DM, dry matter; aNDF, neutral detergent fiber assayed with a heat-stable amylase and expressed inclusive of residual ash; ADF, acid detergent fiber; WSC, water soluble carbohydrate; CP, crude protein; DJP, dried jujube powder.
Fermentation characteristics of ensiled silages
The effects of DJP and inoculants on silage fermentation are shown in Table 2. The pH value and ammonia nitrogen content were significantly influenced by the DJP ratio (P < 0.001), the inoculants (P < 0.001) and the interaction between DJP and inoculants (P < 0.001). As the DJP ratio increased, the pH value and ammonia nitrogen content decreased linearly (P < 0.05). However, according to the simple effect analysis, there was no significant difference (P > 0.05) on pH value between 12% and 15% of DJP ratios in the LP2 or LP3 treated silages. In the control or the LP1 treated silages, there was no significant difference (P > 0.05) on pH value among 9%, 12% and 15% DJP ratios. For the ammonia nitrogen content, there was no significant difference (P > 0.05) among the DJP ratios of 6%, 9%, 12% and 15% in the control or LP3 treated silages. Except the silages without DJP added, there was no significant difference (P > 0.05) among the other five DJP ratios on the ammonia nitrogen content in LP1 treated silages. There was no significant difference (P > 0.05) on ammonia nitrogen content between 12% and 15% DJP ratios in the LP2 treated silages.
| Item | Ratio of DJP | Inoculants | SEM | Significance | ||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Control | LP1 | LP2 | LP3 | Mean | DJP | LP | DJP × LP | |||
| pH | 0 | 4.67aA | 4.39aD | 4.46aC | 4.62aB | 4.54a | 0.02 | ***, L | *** | *** |
| 3 | 4.50bA | 4.21bC | 4.26bB | 4.25bBC | 4.31b | |||||
| 6 | 4.42cA | 4.13cB | 4.17cB | 4.16cB | 4.22c | |||||
| 9 | 4.36dA | 4.04dC | 4.10dB | 4.06dBC | 4.14d | |||||
| 12 | 4.37dA | 4.03dB | 4.04eB | 4.03deB | 4.12e | |||||
| 15 | 4.34dA | 3.99dB | 4.04eB | 4.01eB | 4.10e | |||||
| Mean | 4.44A | 4.13C | 4.18B | 4.19B | ||||||
| Lactic acid (g/kg DM) | 0 | 63.4 | 65.2 | 66.9 | 46.4 | 60.5 | 1.67 | NS | *** | NS |
| 3 | 56.8 | 78.8 | 81.0 | 63.0 | 69.9 | |||||
| 6 | 54.1 | 86.5 | 69.0 | 56.8 | 66.6 | |||||
| 9 | 54.4 | 75.3 | 80.3 | 58.3 | 67.1 | |||||
| 12 | 54.2 | 76.3 | 65.4 | 53.1 | 62.3 | |||||
| 15 | 50.7 | 78.7 | 63.5 | 53.9 | 61.7 | |||||
| Mean | 55.6B | 76.8A | 71.0A | 55.3B | ||||||
| Acetic acid (g/kg DM) | 0 | 16.4 | 11.0 | 10.4 | 9.8 | 11.9 | 0.67 | NS | *** | *** |
| 3 | 10.1 | 15.0 | 9.8 | 6.2 | 10.3 | |||||
| 6 | 6.0 | 19.1 | 5.6 | 5.6 | 9.1 | |||||
| 9 | 6.6 | 18.0 | 6.3 | 13.5 | 11.1 | |||||
| 12 | 5.5 | 19.2 | 12.6 | 2.9 | 10.1 | |||||
| 15 | 5.1 | 20.1 | 11.1 | 6.5 | 10.7 | |||||
| Mean | 8.3B | 17.1A | 9.3B | 7.4B | ||||||
| Propionic acid (g/kg DM) | 0 | 0.00 | 0.91 | 0.24 | 0.00 | 0.29 | 0.08 | NS | NS | NS |
| 3 | 0.00 | 0.79 | 0.48 | 0.69 | 0.49 | |||||
| 6 | 0.00 | 0.30 | 0.33 | 0.00 | 0.16 | |||||
| 9 | 0.00 | 0.18 | 0.50 | 1.70 | 0.60 | |||||
| 12 | 0.00 | 0.25 | 0.49 | 0.62 | 0.34 | |||||
| 15 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | |||||
| Mean | 0.00 | 0.41 | 0.34 | 0.50 | ||||||
| Butyric acid (g/kg DM) | 0 | 0.00 | 0.00 | 6.03 | 4.18 | 2.55 | 0.30 | NS | NS | NS |
| 3 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | |||||
| 6 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | |||||
| 9 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | |||||
| 12 | 0.58 | 0.00 | 0.00 | 0.00 | 0.15 | |||||
| 15 | 0.21 | 0.18 | 0.00 | 0.00 | 0.10 | |||||
| Mean | 0.13 | 0.03 | 1.01 | 0.70 | ||||||
| Lactic acid : acetic acid ratio | 0 | 3.90dB | 5.93bA | 6.46cA | 4.75cdB | 5.26e | 0.42 | ***, Q | *** | *** |
| 3 | 5.77cC | 7.46aB | 8.31bB | 10.15bA | 7.94b | |||||
| 6 | 9.07bC | 3.61cD | 12.49aA | 10.15bB | 8.85a | |||||
| 9 | 8.26bB | 4.31cC | 12.73aA | 4.55dC | 7.46c | |||||
| 12 | 9.94abB | 3.99cD | 5.29dC | 18.23aA | 9.36a | |||||
| 15 | 10.04aA | 3.92cC | 5.76cdB | 5.68cB | 6.34d | |||||
| Mean | 7.82C | 4.87D | 8.50B | 8.93A | ||||||
| Ammonia nitrogen (g/kg TN) | 0 | 98.1aA | 42.9aC | 57.1aB | 52.6aB | 62.7a | 2.30 | ***, L | *** | *** |
| 3 | 58.2bA | 29.9bB | 35.6bB | 26.0bB | 37.4b | |||||
| 6 | 43.7cA | 27.7bB | 25.5cB | 20.3bcB | 29.3c | |||||
| 9 | 41.5cA | 21.7bBC | 26.0bcB | 13.6cC | 25.7cd | |||||
| 12 | 34.6cA | 22.1bB | 21.7cdBC | 12.2cC | 22.7d | |||||
| 15 | 34.6cA | 23.3bB | 15.1dBC | 11.9cC | 21.2d | |||||
| Mean | 51.8A | 27.9B | 30.2B | 22.8C | ||||||
- LAB, lactic acid bacteria; DM, dry matter; TN, total nitrogen; DJP, effect of dried jujube powder; LP, effect of lactic acid bacteria inoculants, DJP × LP, interaction effects between dried jujube powder and lactic acid bacteria inoculants
- a-eMeans within a column with different superscripts differ (P < 0.05); A-DMeans within a row with different superscripts differ (P < 0.05)
- *** P < 0.001, NS, not significant; SEM, stand error of the mean; L, linear effect, P < 0.05; Q, quadratic effect, P < 0.05.
The inoculants used had lower pH value and ammonia nitrogen contents than the control (P < 0.05). The LP1 treated silages had the lowest (P < 0.05) pH value among the three inoculant-treated silages and control silages. However, according to the simple effect analysis, in the silages added with 0%, 3% or 9% of DJP, the LP1 treated silages had significantly lower (P < 0.05) pH values than LP2 treated silages. And in silages without DJP added, the LP1 treated silages had significant lower (P < 0.05) pH value than the LP3 treated silages. The LP3 treated silages had the lowest ammonia nitrogen content (P < 0.05). The ammonia nitrogen content of LP3 treated silages was lower (P < 0.05) than that of the LP2 treated silages in the silages treated with 9% DJP and lower (P < 0.05) than that of the LP1 treated silages in the silages treated with 12% or 15% DJP. The LP1 treated silages had lower ammonia nitrogen than the LP2 or LP3 treated silages in the silages without DJP added.
The inoculants could significantly influence the lactic acid (P < 0.001) content and the acetic acid (P < 0.001) content. The LP1 and LP2 treated silages had the highest (P < 0.05) lactic acid content while the LP1 treated silages had the highest (P < 0.05) acetic acid content. The inoculants and the DJP ratio had no significant (P > 0.05) effect on the propionic acid and butyric acid contents. However, butyric acid was detected in the control silages with 12% or 15% DJP added, LP1 treated silages with 15% DJP added or LP2 and LP3 treated silages without DJP added.
The DJP ratio (P < 0.001), the inoculants (P < 0.001) and the interaction between DJP and inoculants (P < 0.001) could significantly influence the lactic acid : acetic acid ratio. As the DJP ratio increased, the change of lactic acid : acetic acid ratio was quadratic (P < 0.05). In the control silages, the 15% DJP ratio reached the highest (P < 0.05) lactic acid : acetic acid ratio, while there was no significant difference (P > 0.05) between the DJP ratios of 12% and 15%. In the LP1 treated silages, 3% DJP ratio got the highest (P < 0.05) lactic acid : acetic acid ratio, while 12% DJP ratio got the highest (P < 0.05) in LP3 treated silages. In the LP2 treated silages, the 9% DJP ratio got the highest (P < 0.05) lactic acid : acetic acid ratio while there was no significant difference (P > 0.05) between the DJP ratios of 6% and 9%.
For the silages without DJP added, the LP1 and LP2 treated silages had higher (P < 0.05) lactic acid : acetic acid ratio than the control silages and LP3 treated silages. For the silages added with 3% or 12% DJP, the LP3 treated silages had higher (P < 0.05) lactic acid : acetic acid ratio than the control, LP1 treated or LP2 treated silages. For the silages added with 6% or 9% DJP, the LP2 treated silages had higher (P < 0.05) lactic acid : acetic acid ratio than the control, LP1 treated or LP3 treated silages.
Chemical composition of ensiled silages
The concentrations of DM (P < 0.001), CP (P < 0.001), residue WSC (P < 0.001), aNDF (P < 0.01), ADF (P < 0.05), ADL (P < 0.001) and EE (P < 0.001) were significantly influenced by the DJP ratio (Table 3). There were linear increases in DM (P < 0.05), residue WSC (P < 0.05), aNDF (P < 0.05), ADF (P < 0.05) and ADL (P < 0.05) contents and linear decrease in CP (P < 0.05) and EE (P < 0.05) contents as the DJP ratio increased.
| Item | Ratio of DJP | Inoculants | SEM | Significance | ||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Control | LP1 | LP2 | LP3 | Mean | DJP | LP | DJP × LP | |||
| DM (g/kg) | 0 | 287 | 296 | 301 | 318 | 300e§ | 4.84 | ***, L | *** | NS |
| 3 | 307 | 306 | 309 | 364 | 322d | |||||
| 6 | 331 | 333 | 360 | 390 | 354c | |||||
| 9 | 355 | 350 | 373 | 381 | 365c | |||||
| 12 | 369 | 372 | 391 | 386 | 380b | |||||
| 15 | 397 | 389 | 407 | 422 | 404a | |||||
| Mean | 341C | 341C | 357B | 377A | ||||||
| CP (g/kg DM) | 0 | 233 | 242 | 240 | 238 | 238a | 3.85 | ***, L | *** | NS |
| 3 | 219 | 218 | 223 | 228 | 222b | |||||
| 6 | 209 | 206 | 218 | 210 | 211c | |||||
| 9 | 197 | 193 | 204 | 201 | 199d | |||||
| 12 | 183 | 184 | 193 | 194 | 189e | |||||
| 15 | 170 | 172 | 178 | 177 | 174f | |||||
| Mean | 202B | 203B | 209A | 208A | ||||||
| WSC (g/kg DM) | 0 | 4.6 | 4.1 | 5.5 | 4.5 | 4.7d | 1.69 | ***, L | * | NS |
| 3 | 6.7 | 6.0 | 7.6 | 6.8 | 6.8d | |||||
| 6 | 9.8 | 8.6 | 10.5 | 7.3 | 9.1cd | |||||
| 9 | 14.9 | 13.6 | 12.8 | 12.5 | 13.5c | |||||
| 12 | 28.8 | 31.8 | 15.9 | 16.1 | 23.2b | |||||
| 15 | 40.9 | 37.2 | 34.6 | 23.9 | 34.2a | |||||
| Mean | 17.6A | 16.9A | 14.5AB | 11.8B | ||||||
| aNDF (g/kg DM) | 0 | 293 | 295 | 299 | 312 | 300c | 2.60 | **, L | * | NS |
| 3 | 310 | 310 | 305 | 317 | 311bc | |||||
| 6 | 301 | 323 | 304 | 332 | 315bc | |||||
| 9 | 322 | 323 | 299 | 341 | 321ab | |||||
| 12 | 324 | 329 | 308 | 281 | 311bc | |||||
| 15 | 323 | 344 | 322 | 350 | 335a | |||||
| Mean | 312AB | 321A | 306B | 322A | ||||||
| ADF (g/kg DM) | 0 | 204 | 229 | 232 | 207 | 218c | 2.43 | *, L | *** | NS |
| 3 | 216 | 245 | 232 | 229 | 231abc | |||||
| 6 | 196 | 250 | 232 | 237 | 229bc | |||||
| 9 | 222 | 247 | 226 | 244 | 235ab | |||||
| 12 | 221 | 252 | 233 | 226 | 233ab | |||||
| 15 | 225 | 260 | 242 | 251 | 245a | |||||
| Mean | 214C | 247A | 233B | 232B | ||||||
| ADL (g/kg DM) | 0 | 41.7 | 48.6 | 46.7 | 42.0 | 44.8e | 0.99 | ***, L | *** | NS |
| 3 | 45.7 | 54.0 | 46.7 | 47.2 | 48.4de | |||||
| 6 | 39.7 | 60.8 | 48.1 | 48.7 | 49.3cd | |||||
| 9 | 44.9 | 60.2 | 51.6 | 54.6 | 52.8bc | |||||
| 12 | 47.5 | 63.0 | 55.7 | 50.1 | 54.1b | |||||
| 15 | 48.1 | 64.4 | 61.9 | 63.0 | 59.4a | |||||
| Mean | 44.6C | 58.5A | 51.8B | 50.9B | ||||||
| EE (g/kg DM) | 0 | 39.8 | 34.6 | 27.0 | 23.7 | 31.3a | 1.07 | ***, L | *** | NS |
| 3 | 36.0 | 23.5 | 22.7 | 21.4 | 25.9b | |||||
| 6 | 31.4 | 19.1 | 14.3 | 18.7 | 20.9c | |||||
| 9 | 29.4 | 18.6 | 11.6 | 14.5 | 18.5c | |||||
| 12 | 27.6 | 16.9 | 10.1 | 12.1 | 16.7d | |||||
| 15 | 27.4 | 14.9 | 8.4 | 10.4 | 15.3d | |||||
| Mean | 31.9A | 21.3B | 15.7C | 16.8C | ||||||
- DM: dry matter; EE: ether extract; WSC: water soluble carbohydrate; aNDF, neutral detergent fiber assayed with a heat stable amylase and expressed inclusive of residual ash; ADF, acid detergent fiber; ADL, acid detergent lignin; CP: Crude protein; TN, total nitrogen.
- DJP, effect of dried jujube powder; LP, effect of lactic acid bacteria inoculants, DJP × LP, interaction effects between dried jujube powder and lactic acid bacteria inoculants;
- a-fMeans within a column with different superscripts differ (P < 0.05). A-CMeans within a row with different superscripts differ (P < 0.05);
- * P < 0.05,
- ** P < 0.01,
- *** P < 0.001, NS, not significant; SEM, stand error of the mean; L, linear effect, P < 0.05; Q, quadratic effect, P < 0.05.
The inoculants also had significant influence on the DM (P < 0.001), CP (P < 0.001), residue WSC (P < 0.05), aNDF (P < 0.05), ADF (P < 0.001), ADL (P < 0.001) and EE (P < 0.001) contents. The LP1 treated silages had the highest ADF (P < 0.05) and ADL (P < 0.05) contents and had lower EE (P < 0.05) content than the control silages. The LP2 or LP3 treated silages had higher DM (P < 0.05), CP (P < 0.05), ADF (P < 0.05) and ADL (P < 0.05) contents and lower EE (P < 0.05) content than the control silages. Also the LP3 treated silages had a lower residue WSC (P < 0.05) content than the control silages.
N-fraction of ensiled silages
The DJP ratio could significantly influence the PA (P < 0.001), PB2 (P < 0.001), PB3 (P < 0.05) and PC (P < 0.001) contents (Table 4). As the DJP ratio increased, the PA content decreased linearly (P < 0.05) while the PC content increased linearly (P < 0.05). Silages added with 15% DJP had the lowest (P < 0.05) PA content and highest (P < 0.05) PC content. However, there was no significant difference (P < 0.05) on PA content between 12% and 15% DJP ratios in control or LP3 treated silages, among the DJP ratios of 9%, 12% or 15% in LP1 treated silages or among the five DJP ratios except 0% in LP2 treated silages. The highest (P < 0.05) PC content was reached by 12% in control silages and 15% DJP ratio in the three inoculant treated silages, while in control silages DJP ratios of 12% and 15% had no significant (P > 0.05) effect on the PC fraction. For the PB2 content, the DJP effect was quadratic. The highest (P < 0.05) PB2 content in LP2 or LP1 treated silages was achieved when the DJP ratio was 9%, while the highest (P < 0.05) PB2 content of LP3 treated silages was achieved when the DJP ratio was 6%. In the LP2 and LP3 treated silages, there was no significant (P > 0.05) difference among silages with 3%, 6% and 9% DJP added. In the LP1 treated silages, there was no significant (P > 0.05) difference among silages with 9%, 12% or 15% DJP added. For the PB3 fractions, the main change by DJP ratio was in the LP2 treated silages, and the silages without DJP added got the highest (P < 0.05) PB3 content in the LP2 treated silages.
| Item | Ratio of DJP | Inoculants | SEM | Significance | ||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Control | LP1 | LP2 | LP3 | Mean | DJP | LP | DJP × LP | |||
| PA (g/kg TN) | 0 | 657aB | 620aB | 704aA | 703aA | 671a | 5.82 | ***, L¶ | ** | * |
| 3 | 594bcB | 641aAB | 636bAB | 669aA | 635b | |||||
| 6 | 620ab | 617ab | 591b | 615b | 611b | |||||
| 9 | 585bc | 574c | 579b | 603b | 585c | |||||
| 12 | 547cd | 577bc | 597b | 595bc | 579c | |||||
| 15 | 505dB | 569cA | 590bA | 561cA | 556d | |||||
| Mean | 585C | 600BC | 616AB | 624A | ||||||
| PB1 (g/kg TN) | 0 | 77 | 81 | 9 | 46 | 53 | 5.18 | NS | *** | * |
| 3 | 121 | 4 | 16 | 31 | 43 | |||||
| 6 | 106 | 9 | 42 | 24 | 45 | |||||
| 9 | 109 | 25 | 31 | 37 | 51 | |||||
| 12 | 115 | 29 | 41 | 44 | 57 | |||||
| 15 | 153 | 4 | 35 | 53 | 61 | |||||
| Mean | 114A | 25B | 29B | 39B | ||||||
| PB2 (g/kg TN) | 0 | 190B | 222cA | 154dC | 146cC | 178c | 4.82 | ***, Q | *** | ** |
| 3 | 201C | 265abA | 236abB | 202abC | 226ab | |||||
| 6 | 190C | 262bA | 250aAB | 229aB | 233ab | |||||
| 9 | 205C | 293aA | 261aB | 210abC | 242a | |||||
| 12 | 217B | 275abA | 203cB | 200bB | 224b | |||||
| 15 | 220B | 282abA | 210bcB | 197bB | 227ab | |||||
| Mean | 204C | 267A | 219B | 197C | ||||||
| PB3 (g/kg TN) | 0 | 8B | 12B | 67aA | 24B | 28a | 1.92 | * | *** | *** |
| 3 | 10 | 21 | 19c | 14 | 16bc | |||||
| 6 | 9 | 26 | 17c | 28 | 20ab | |||||
| 9 | 14B | 7B | 17cB | 39A | 19abc | |||||
| 12 | 4C | 22B | 40bA | 29AB | 24a | |||||
| 15 | 10 | 8 | 8c | 18 | 11c | |||||
| Mean | 9B | 16B | 28A | 25A | ||||||
| PC (g/kg TN) | 0 | 69cB | 65dB | 66eB | 82dA | 70f | 3.01 | ***, L | *** | *** |
| 3 | 74cBC | 68dC | 93dA | 84dAB | 80e | |||||
| 6 | 75bcC | 87cBC | 99cdAB | 105cA | 92d | |||||
| 9 | 87bB | 102bA | 111bcA | 111cA | 103c | |||||
| 12 | 116aA | 98bcB | 119bA | 131bA | 116b | |||||
| 15 | 112aC | 137aB | 156aA | 171aA | 144a | |||||
| Mean | 89D | 93C | 107B | 114A | ||||||
- TN, total nitrogen. PA, fraction which contains non-protein Nitrogen; PB1, rapidly degradable fraction of true protein; PB2, intermediate degradable fraction of true protein; PB3, slowly degradable fraction of true protein; PC, Fraction which contains the acid-detergent insoluble nitrogen.
- DJP, effect of dried jujube powder; LP, effect of lactic acid bacteria inoculants, DJP × LP, interaction effects between dried jujube powder and lactic acid bacteria inoculants;
- a-fMeans within a column with different superscripts differ (P < 0.05). A-DMeans within a row with different superscripts differ (P < 0.05);
- * P < 0.05,
- ** P < 0.01,
- *** P < 0.001, NS, not significant; SEM, stand error of the mean; L, linear effect, P < 0.05; Q, quadratic effect, P < 0.05.
The inoculants also had significant influence on the PA (P < 0.01), PB1 (P < 0.001), PB2 (P < 0.001), PB3 (P < 0.001) and PC (P < 0.001) contents. All inoculant treated silages had lower (P < 0.05) PB1 content and higher (P < 0.05) PC content than the control silages. The LP2 and LP3 treated silages had higher (P < 0.05) PA content than the control. The LP3 treated silages had higher (P < 0.05) PA content than the control silages in the silages treated with 0%, 3% or 15% DJP. The three inoculant treated silages all had higher (P < 0.05) PA content than control silages in the silages added with 15% DJP. The LP3 treated silages had higher (P < 0.05) PC content than the control silages in the silages treated with 0%, 6%, 9% or 15% DJP. The LP2 and LP3 had a similar effect (P > 0.05) on the PC content except a lower (P < 0.05) PC content of LP2 than that of LP3 in silages without DJP added. The PC content of LP1 treated silages was lower (P > 0.05) than that of the control silages in the silages added with 12% of DJP and higher (P < 0.05) than that of the control silages in the silages added with 9% or 15% DJP. PB2 content of the LP1 treated silages was higher (P < 0.05) than that of the other two inoculant silages and control silages. The PB3 content of LP2 treated silages was the highest (P < 0.05) in the silages without DJP added and higher than that of the control or LP1 treated silages in the silages treated with 12% DJP. The LP3 treated silages got the highest (P < 0.05) PB3 content in the silages with 9% DJP added.
Discussion
For a satisfactory compromise among quantity, quality and the threat of rainfall, the fourth cut alfalfa was harvested at the vegetative stage. Chemical composition of alfalfa forage was similar to that in previous studies except for lower aNDF concentration which is similar to the report of Contreras-Govea et al. (2013). This is possibly because early cutting permitted very low NDF and ADF contents (Colombari et al. 2001). Good silages have a dominance of lactic acid bacteria and a rapid decline and final low pH value to reduce the activities of plant enzymes and undesirable microorganisms. In this experiment, all silages had relatively good fermentation with relatively low pH value (lower than 4.2), little or no detectable butyric acid, low ammonia nitrogen content (lower than 10%), low acetic acid content and high production of lactic acid (5-7%).
Jujube powder is usually used to replace corn meal as an energy source (Ma et al. 2014), which is considered to contain high contents of soluble sugars. Addition of dry sugar sources like DJP not only can increase the WSC concentration of alfalfa silages but also can decrease the moisture contents and the buffer capacity of the whole silages (Weiss & Underwood 2009). In this experiment, the pH value in DJP added silages without inoculants decreased. However, the lactic acid content decreased numerically. These results were similar to the molasses effect on alfalfa silages reported in the study of Hashemzadeh-Cigari et al. (2014). The decrease of pH value may mainly be due to the decrease of buffer capacity instead of more production of lactic acid. High addition of DJP had no more effect on the silage fermentation. This may be because the excess WSC from high levels of DJP was not changed to lactic acid. The high level of DM due to the increased ratio of DJP also could influence lactic acid. The study by Guo et al. (2013) showed that lactic acid in untreated and LP-treated silages could be decreased by the increase of silage DM. Butyric acid was detected in control silages with 12–15% DJP added in this experiment. On the other hand, the study by Jones et al. (1992) and Gül et al. (2008) believed that supplementing extra sugar to wilted forage caused heterofermentative fermentation or the conversion of lactic acid to acetic acid, and thereby an increased acetic acid concentration in silage.
Lower pH in the treated silages usually warrants homolactic fermentation. The inoculants LP1 or LP2 used had an obvious effect on lactic acid production, and then the pH value and ammonia nitrogen decreased. The L. plantarum strains are known as homofermentative lactic bacteria which would increase the lactic acid : acetic acid ratio of the silage (Muck 2013). But in this experiment, the three inoculants could not increase the lactic acid : acetic acid ratio in all conditions. The LP3 used could increase the lactic acid : acetic acid ratio only in silages added with 3%, 6% or 12% DJP. The LP2 used could increase the lactic acid : acetic acid ratio only in silages without DJP added or added with 3%, 6% or 9% DJP. The LP1 used could increase the lactic acid : acetic acid ratio in silages without DJP added or added with 3% DJP. A small amount of butyric acid contents in LP2 or LP3 treated alfalfa silages were detected in silages without DJP added or in LP1 treated silages combined with 15% DJP, which showed an undesirable fermentation quality. With the same amount of lactic acid, observing lower pH in LP3 silages may be attributed to the lower production of NH3-N, and thereby lowering its buffering effects (Hashemzadeh-Cigari et al. 2014).
The studies by Jones et al. (1992) and Nadeau et al. (2000) showed that with a low WSC content, inoculants will not produce enough lactic acid due to a limitation of substrates. The combination of inoculants and DJP additively could decrease the pH value and ammonia nitrogen content and influence the lactic acid : acetic acid ratio. However, there was no interaction between the inoculants and DJP on the lactic acid. The numerically reduced butyric acid concentration was in agreement with the result that interaction of L. plantarum and sugar could inhibit the fermentation of clostridia (McDonald et al. 1991). Decreased ammonia nitrogen content due to combination of DJP and inoculants is in agreement with Umana et al. (1991) regarding wilted bermudagrass. The properties of a LAB bacterial strain vary even within the same species (Ohmoto et al. 2002). The three inoculants were all L. plantarum strains but had significant differences in this experiment. To enhance the fermentation quality of silage, the effectiveness of LP1 got the highest with only low ratio of DJP while the LP2 and LP3 needed higher ratios of DJP than LP1. The effectiveness of LP1 is higher than that of LP2 or LP3 on the decrease of pH value and ammonia nitrogen content in alfalfa silage without DJP added.
Because of the big differences of chemical composition between DJP and alfalfa, the DM, residue WSC and fiber fractions (aNDF, ADF and ADL) in silages increased while CP and EE contents decreased linearly as the rate of DJP increased. Addition of inoculants decreased the EE and residue WSC contents and increased the DM, CP, ADF and ADL concentrations. Kozelov et al. (2008) and Filya et al. (2007) observed lower content of aNDF in inoculated alfalfa silage. In addition, there are some researches showing reduced fiber concentration in silage that is mainly due to the degradation of cell walls (Wang et al. 2009). In this experiment, the higher ADF and ADL contents may be due to the large consumption of WSC by the three inoculants. The reason for the reduced EE contents may be attributed to lipid degradation during ensiling.
During ensiling, the nitrogen fraction changed a lot and most of the true protein was degraded to NPN (PA in this study), although the PA portion can also be utilized by the rumen, and a study by Licitra et al. (1996) showed that it is rapidly converted to ammonia in the rumen and metabolically closer to soluble protein. How to decrease the proteolysis of true protein is still one of the most important contents in alfalfa silage. The DJP used decreased the CP content of the silage linearly which is similar to the study by Canbolat et al. (2010) and the PA content was also decreased linearly by DJP. However, the inoculants LP2 and LP3 used could increase the PA content which is similar to the study by Rizk et al. (2005). The PC portion is nutritionally unavailable for ruminants. In this experiment, the PC portion could be increased by the DJP and the inoculants.
The effects of DJP ratio on the PB2 and PB3 fractions were limited. There was a quadratic cure for the change of PB2 fraction and 9% DJP ratio received the peak. The influence of DJP ratio on PB3 focused on the LP2 or LP3 treated silages. Inoculants were supposed to have no effect on the true protein and NPN except for the decrease in ammonia nitrogen (Muck 1989). However, in this experiment, the inoculants consumed large amounts of the PB1 portion. As a result, other portions (PA, PB2, PB3 and PC portions) of the CNCPS increased. The LP1 performed better than the LP2 and LP3 for the lower PB3 and PC content and higher PB2 content and lower PA content than the LP3. Combination of DJP ratio and inoculants also could influence the PB2 and PB3 fractions.
Addition of DJP had positive effects on the silage. As the DJP ratio increased, the pH value, ammonia nitrogen content and PA fraction decreased. There was a peak in lactic acid : acetic acid ratio (12% DJP ratio) and PB2 fraction (9% DJP ratio). The three inoculants also had positive effects on the silages. The LP1 or LP2 obtained the highest lactic acid content. The three inoculants decreased the PB1 portion of true protein. The LP1 obtained the highest acetic acid content with the lowest pH value and lactic acid : acetic acid ratio and had lower PB3 and PC and higher PB2 than LP2 or LP3. There were synergistic effects between DJP ratio and inoculants on the pH value, ammonia nitrogen and opposite effects on the PA or PC contents. On the other hand, 12% DJP ratio combined with LP3 obtained the highest lactic acid : acetic acid ratio while 9% DJP ratio combined with LP1 obtained the highest PB2 content.
Conclusion
The DJP and inoculants could benefit the fermentation quality, nutrition value and N fractions of high-moisture alfalfa silage, while 9% DJP ratio would be appropriate. The inoculant LP1 had better effects on fermentation quality and N fraction than the LP2 or LP3. Combination of DJP and inoculants is more effective than only an inoculant or DJP addition on fermentation quality and N fraction.
Acknowledgments
The funding of this research by the Special Fund for Agro-scientific Research in the Public Interest (201303061), the National Forage Industry System (CARS-35) and the Inner Mongolia science and technology project is gratefully acknowledged.
