The quantification of targeted microbes, including cellulolytic bacteria such as Fibrobacter succinogenes, Ruminococcus albus, Ruminococcus flavefaciens, general bacteria, general anaerobic fungi, total protozoa, total methanogens and total archaea, was conducted as described in detail in our previous studies [24,27]. Briefly, the DNA was extracted from 300 µL of fermented rumen content (fluid and digesta) by QIAGEN DNA Mini Stool Kit (QIAGEN, Valencia, CA, USA) according to the manufacturer’s recommendations. The 16S rRNA of bacteria and 18S rRNA of protozoa and fungi were amplified by PCR (Bio-Rad, Hercules, CA, USA). The primer sets used in the current study are shown in Table 2. The PCR product was purified using a QIA quick PCR purification kit (QIAGEN, Inc., Valencia, CA, USA) and cloned in pCR®2.1-TOPO® TA cloning vector (Invitrogen, Carlsbad, CA, USA) and transformed into chemically competent E. coli TOP10 cells (Invitrogen, Carlsbad, CA, USA).The plasmid was extracted from all white colonies (ranged from 9–24 colonies). The extracted plasmids (ranged from 9–24 plasmids) were analyzed by EcoR1 restriction enzyme and separated on a 1% (w/v) agarose gel to confirm the presence and correct orientation of the inserts. Plasmids with the right cloned inserts were sequenced using an Applied Biosystems 3730xl DNA analyzer (Applied Biosystems, Foster City, CA, USA) in order to assert their identity. Bellerophon software was used to check the chimeric rDNA [16], and the sequences were blast-identified using basic alignment tool with those available in the GenBank [28]. Plasmids possessing sequences with more than 99% similarity to previously published sequences of target microorganisms were applied for standard curve construction by real-time PCR. The concentration and purity of the plasmids were evaluated using Nanodrop (Nanodrop Technologies, Wilmington, DE, USA) and the copy number was determined using below formula [29].

Microorganisms and characteristics of the primers used in this study [24,27].

F: forward; R: reverse.

A Bio-Rad CFX 96 real-time PCR thermocycler (Bio-Rad, Hercules, CA, USA) and SYBR Green (iQ Supermix, Bio-Rad Laboratories, Inc., Hercules, CA, USA) were used in this study. All the amplifications were conducted in triplicate and the data obtained from real-time PCR amplification were analyzed using CFX manager version 3 (Bio-Rad Laboratories, Alfred Nobel Drive, Hercules, CA, USA).

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