| The alfalfa is perennial legume forage with high protein and low fiber, and is planted throughout China. However, its nutritional value varies with area, growth period and stage. This study was conducted to compare the in vitro fermentation characteristics of alfalfa from different sources and reveal the bacterial community on the surface of their leaves. Batch culture system was used for in vitro fermentation, which contained 90 mL media and 10 mL rumen fluid, collected from 3 Holstein dairy cows with permanent rumen fistula, with 1 g alfalfa as substrate. The bottles were incubated at 39℃ for 96 h and samples were collected at 24,48,72 and 96 h for the analysis of in vitro degradability, pH, volatile fatty acids (VFA), ammonia nitrogen (NH3-N) and microbial protein production (MCP). Total gas production (GP) and methane production (MP) were measured during the fermentation. PCR and denaturing gradient gel electrophoresis (DGGE) were used for the analysis of bacterial community. Results were shown as follows:1. Comparative Study on in vitro Fermentation Characteristics of Alfalfa Collected at Different Growth StagesThe first stubble of alfalfa, collected at squaring stage, early flowering stage and bloom stage in Xinjiang, was used to compare the in vitro fermentation characteristics of alfalfa from different growth stages. Statistical analysis showed that the in vitro degradability of alfalfa were significantly (P<0.05) affected by the phenology. At the end of the fermentation, the GP, MP and VFA of alfalfa collected at squaring stage were significantly (P<0.05) higher than that of alfalfa collected at the early flowering stage. The DMD, OMD, NDFD, ADFD, NH3-N and MCP of alfalfa collected at squaring stage were significantly (P<0.05) higher than that of the alfalfa collected at bloom stage. The DMD, OMD, NDFD and ADFD of alfalfa collected at early flowering stage were significantly (P <0.05) higher than that of alfalfa collected at bloom stage. The nutritional value of alfalfa collected at squaring stage was the best.2. Comparative Study on in vitro Fermentation Characteristics of Alfalfa Grown with Different PeriodsThree stubbles of alfalfa, grown in 2008,2009, and 2011 and collected in 2012, were used to compare their in vitro fermentation characteristics with different growth periods. Statistical analysis showed that the in vitro degradability of alfalfa were significantly (P< 0.05) affected by growth periods. For the first stubble alfalfa, the GP, DMD, OMD, NDFD, ADFD of alfalfa grown in 2011 were significantly (P< 0.05) higher than that of alfalfa grown in 2009. Meanwhile, the MP, NH3-N and MCP of alfalfa grown in 2011 were significantly (P<0.05) higher than that of alfalfa grown in 2008. Futhermore, the butyrate of alfalfa grown in 2008 were significantly (P<0.05) higher than that of alfalfa grown in 2009. For the second stubble alfalfa, the MP, DMD, OMD, NDFD, ADFD, propionate, NH3-N and MCP of alfalfa grown in 2011 were significantly (P<0.05) higher than that of alfalfa grown in 2009, and the GP, butyrate and total VFA of alfalfa grown in 2008 were significantly (P< 0.05) higher than that of alfalfa grown in 2009. For the third stubble alfalfa, the GP, MP, propionate and MCP of alfalfa grown in 2011 were significantly (P< 0.05) higher than that of alfalfa grown in 2009, and the DMD, OMD, NDFD, ADFD, NH3-N of alfalfa grown in 2008 were significantly(P<0.05) higher than that of alfalfa grown in 2009. The nutritional value of alfalfa grown in 2011 and 2008 were higher than that grown in 2009.3. Comparative Study on in vitro Fermentation Characteristics of Alfalfa from Different Area and StubblesThe first, second, and third stubbles of alfalfa collected from Ningxia, Gansu, Xinjiang and Jiangsu were collected to compare their in vitro fermentation characteristics from different area and stubbles. Statistical analysis showed that the in vitro degradability of alfalfa were significantly (P<0.05) affected by area, stubble and their interaction. At the end of the fermentation, the GP, MP and concentrations of VFA of alfalfa from Xinjiang were significantly (P< 0.05) higher than those from the other three area. The DMD, OMD, NDFD, ADFD and concentrations of NH3-N of alfalfa from Ningxia were significantly(P< 0.05) higher than those from the other three area. The in vitro degradability of alfalfa from the third stubble was the highest and it was the lowest for the second stubble alfalfa (P< 0.05). The in vitro degradability of the third stubble alfalfa growing in Xinjiang and Gansu were significantly (P<0.05) higher than the others and the second stubble alfalfa growing in Gansu and Jiangsu showed the lowest in vitro degradability.4. Molecular Analysis of Bacterial Community on the Surface of Alfalfa Leaves at Different Growth Stages and StubblesThe first stubble of alfalfa growing in Jiangsu were collected in 2013 at the squaring stage, early flowering stage, and bloom stage, and the second stubble alfalfa were collected at the early flowering stage. PCR and denaturing gradient gel electrophoresis (DGGE) were used to analyze the bacterial community on the surface of alfalfa leaves and reveal the effects of growth stages and stubbles on the bacterial community on the surface of leaves. The regions of V6 to V8 of the 16S rRNA gene of phyllospere bacteria were amplified and the subsequently DGGE profiles from the PCR amplicons were analyzed by GelCompar. Results showed that the bacterial community from three growth stages differed from each other. The diversity indices of the squaring stage and bloom stage alfalfa were significantly (P< 0.05) higher than that of the early flowering stage alfalfa. Meanwhile the diversity indices of the first stubble alfalfa were significantly (P<0.05) higher than that of the second stubble alfalfa. The results suggested that the phyllospere microbial community were affected by the growth stages and stubbles. |
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