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.

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The objective of this study was to determine whether a combination of crude glycerin (CG) and soyabean oil (SO) could be used to partially replace maize in the diet of Nellore steers while maintaining optimum feed utilisation. Eight castrated Nellore steers fitted with ruminal and duodenal cannulas were used in a double 4×4 Latin square design balanced for residual effects, in a factorial arrangement (A×B), when factor A corresponded to the provision of SO, and factor B to the provision of CG. Steers feed SO and CG showed similar DM intake, DM, organic matter and neutral-detergent fibre digestibility to that of steers fed diets without oil and without glycerine (P>0·05). Both diets with CG additions reduced the acetate:propionate ratio and increased the proportion of iso-butyrate, butyrate, iso-valerate and valerate (P<0·05). Steers fed diets containing SO had less total N excretion (P<0·001) and showed greater retained N expressed as % N intake (P=0·022). SO and CG diet generated a greater ruminal abundance of Prevotella, Succinivibrio, Ruminococcus, Syntrophococcus and Succiniclasticum. Archaea abundance (P=0·002) and total ciliate protozoa were less in steers fed diets containing SO (P=0·011). CG associated with lipids could be an energy source, which is a useful strategy for the partial replacement of maize in cattle diets, could result in reduced total N excretion and ruminal methanogens without affecting intake and digestibility.

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