Granja-Salcedo Yury Tatiana, Ramirez-Uscategui Ricardo Andrés, Machado Elwi Guillermo, Duarte Messana Juliana, Takeshi Kishi Luciano, Lino Dias Ana Veronica, Berchielli Telma Teresinha
Department of Animal Science, Faculdade de Ciências Agrárias e Veterinárias (FCAV), UNESP - Univ Estadual Paulista, Jaboticabal, São Paulo, Brazil.
Department of Clinical and Veterinary Surgery, Faculdade de Ciências Agrárias e Veterinárias (FCAV), UNESP - Univ Estadual Paulista, Jaboticabal, São Paulo, Brazil.
PLoS One. 2017 Apr 28;12(4):e0176701. doi: 10.1371/journal.pone.0176701. eCollection 2017.
The objective of this study was to investigate three storage methods and four storage times for rumen sampling in terms of quality and yield of extracted metagenomic DNA as well as the composition of the rumen bacterial community. One Nellore steer fitted with a ruminal silicone-type cannula was used as a donor of ruminal contents. The experiment comprised 11 experimental groups: pellet control (PC), lyophilized control (LC), P-20: pellet stored frozen at -20°C for a period of 3, 6, and 12 months, P-80: pellet stored frozen at -80°C for a period of 3, 6, and 12 months, and L-20: lyophilized sample stored frozen at -20°C for a period of 3, 6, and 12 months. Metagenomic DNA concentrations were measured spectrophotometrically and fluorometrically and ion torrent sequencing was used to assess the bacterial community composition. The L-20 method could not maintain the yield of DNA during storage. In addition, the P-80 group showed a greater yield of metagenomic DNA than the other groups after 6 months of storage. Rumen samples stored as pellets (P-20 and P-80) resulted in lower richness Chao 1, ACE, and Shannon Wiener indices when compared to PC, while LC and PC were only different in richness ACE. The storage method and storage time influenced the proportions of 14 of 17 phyla identified by sequencing. In the P-20 group, the proportion of Cyanobacteria, Elusimicrobia, Fibrobacteres, Lentisphaerae, Proteobacteria, and Spirochaetes phyla identified was lower than 1%. In the P-80 group, there was an increase in the proportion of the Bacteroidetes phylum (p = 0.010); however, the proportion of Actinobacteria, Chloroflexi, SR1, Synergistetes, TM7, and WPS.2 phyla were unchanged compared to the PC group (p > 0.05). The class Clostridium was the most abundant in all stored groups and increased in its proportion, especially in the L-20 group. The rumen sample storage time significantly reduced the yield of metagenomic DNA extracted. Therefore, the storage method can influence the abundance of phyla, classes, and bacterial families studied in rumen samples and affect the richness and diversity index.
本研究的目的是从提取的宏基因组DNA的质量和产量以及瘤胃细菌群落的组成方面，研究瘤胃采样的三种储存方法和四个储存时间。使用一头装有瘤胃硅胶型套管的内洛尔阉牛作为瘤胃内容物的供体。实验包括11个实验组：颗粒对照组（PC）、冻干对照组（LC）、P - 20：颗粒在-20°C冷冻储存3、6和12个月，P - 80：颗粒在-80°C冷冻储存3、6和12个月，以及L - 20：冻干样品在-20°C冷冻储存3、6和12个月。通过分光光度法和荧光法测量宏基因组DNA浓度，并使用离子激流测序法评估细菌群落组成。L - 20方法在储存期间无法维持DNA产量。此外，储存6个月后，P - 80组的宏基因组DNA产量高于其他组。与PC相比，以颗粒形式储存的瘤胃样品（P - 20和P - 80）导致较低的Chao 1、ACE和香农 - 维纳丰富度指数，而LC和PC仅在ACE丰富度上有所不同。储存方法和储存时间影响了测序鉴定的17个门中的14个门的比例。在P - 20组中，鉴定出的蓝细菌门、隐秘杆菌门、纤维杆菌门、 Lentisphaerae门、变形菌门和螺旋体门比例低于1%。在P - 80组中，拟杆菌门的比例增加（p = 0.010）；然而，与PC组相比，放线菌门、绿弯菌门、SR1、互养菌门、TM7和WPS.2门的比例没有变化（p > 0.05）。梭菌属在所有储存组中最为丰富，其比例增加，尤其是在L - 20组中。瘤胃样品储存时间显著降低了提取的宏基因组DNA产量。因此，储存方法可影响瘤胃样品中所研究的门、纲和细菌科的丰度，并影响丰富度和多样性指数。
