Sample type and time of collection of ruminal content for identification and quantification of archaeas INS cattle

• Grant number: 19/11484-5
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: August 01, 2019
• End date: July 31, 2020
• Field of knowledge: Agronomical Sciences - Animal Husbandry - Animal Nutrition and Feeding
• Principal Investigator: Telma Teresinha Berchielli
• Grantee: Yasmin Andrade Pinheiro
• Host Institution: Faculdade de Ciências Agrárias e Veterinárias (FCAV). Universidade Estadual Paulista (UNESP). Campus de Jaboticabal. Jaboticabal , SP, Brazil

Abstract

The aim of this study is to compare the use of three types of samples (NET fraction, solid fraction and net fraction + solid) and three schedules (0, 4 and 8h after feeding) of ruminal content collection, to identify and quantify the total population of Archaeans Methogenic in cattle using molecular methods. Will be used four Nellore cattle cannulated in the rumen (mean body weight of 445 kg and mean age of 24 months) receiving diet with 50% of corn silage as forage source and 50% of concentrate consisting of ground corn, dry grains by distillation From Corn processing (DDG), urea and 100 g/d of mineral supplement. After 21 days of adaptation of the animals to the diet, the ruminal cannula will be collected from samples of ruminal content (500 g/animal) in the ventral region of the rumen at 0, 4 and 8h after feeding. The samples will be immediately chilled and taken to the laboratory for processing. After homogenization, each sample will be divided into three fractions: 1) solid fraction + Liquida: 50 g of ruminal content. 2) Liquid fraction: 50 mL of ruminal fluid obtained from the filtration of ruminal content in a cloth of 100 microreicras. 3) Solid fraction: 50 g of dry ruminal content, corresponding to the remaining solid residue after filtration in a cloth of 100 microremicras. Each fraction will be added 50 mL of phosphate-saline buffer (PBS, pH 7.4) at 4 ° C, then the mixture will be homogenized by slight manual agitation for 3 minutes. Subsequently, the material will be filtered in fabric with 100 micras mesh. The filtrate will be subjected to centrifugation of 8000 X G for 30 minutes at a temperature of 4 º C and the supernatant will be discarded. The DNA extraction will be performed with 250 mg of the obtained pellet, and the qPCR method will be used for the identification and quantification of the total bacteria and the abundance of the Archaeans using the method 2-””Ct. The relative absorbance readings of the DNA, the apparent yield and the proportions of methogenic Archaeas will be compared between the collection methods (fractions) and the collection times (schedules) by the Dunns post-Test Friedman test. Thus, the project intends to contribute to the knowledge of appropriate collection methodologies for the molecular quantification of methogenic Archaeas in ruminants.