Dnase rnase free

At days 1, 5, 15, 30, 45, and 60 after birth, 10 g of fecal samples was obtained from each calf. Samples were obtained aseptically before morning feeding, placed into sterile cryogenic vials (DNase-RNase free; Corning^®, Glendale, AZ, USA), and brought to the laboratory. Samples corresponding to the same treatment, the same sampling day, and the same dairy farm were pooled, and 0.2 g of each pooled sample was used for DNA extraction. Metagenomic DNA was extracted and purified using the Quick-DNA™ Fecal/soil Microbe Miniprep system (Zymo Research, Irvine, CA, USA) according to the manufacturer´s instructions. DNA integrity was verified by electrophoresis in 1% agarose gel. DNA was stored at −20 °C until sequencing procedures.

Fresh fecal samples were collected on different days (day 0, day 7, day 15, and day 30) aseptically to investigate intestinal bacterial composition using sterile gloves and lubricant from individual calves into sterile cryogenic vials (DNase-RNase free; Corning^®, USA). According to the manufacturer's protocol, bacterial genomic DNA was extracted from feces (0.2 g) using the commercial kit (QIAamp^® Fast DNA Stool Mini Kit, QIAGEN Inc., Valencia, CA, USA). After extraction, we purified the genomic DNA using the QIAamp 96 PowerFecal QIAcube HT Kit (QIAGEN Inc., Valencia, CA, USA) to obtain higher yields, better purity, and a more accurate representation of the microbial diversity. DNA purity and quantity were checked on agarose gel electrophoresis (2.0%) and using a NanoDrop 1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA), and the DNA samples were ready for high-throughput sequencing.