Effects of diets for three growing stages by rumen inocula donors on in vitro rumen fermentation and microbiome

Ryukseok Kang1, Huseong Lee2,3, Hyeonsu Seon2, Cheolju Park2, Jaeyong Song4, Joong Kook Park4, Yong Kwan Kim5, Minseok Kim2,*, Tansol Park1,**
Author Information & Copyright
1Department of Animal Science and Technology, Chung-Ang University, Anseong-si, Gyeonggi-do 17546, Korea.
2Division of Animal Science, Chonnam National University, Gwangju 61186, Korea.
3Graduate School of Agricultural Science, Tohoku University, Sendai 980-0845, Japan.
4Nonghyup Feed Co., LTD., Seoul 05398, Korea.
5Seogwiposi Chuckhyup, Jeju-do 63585, Korea.
*Corresponding Author: Minseok Kim, Division of Animal Science, Chonnam National University, Gwangju 61186, Korea, Republic of. E-mail:
**Corresponding Author: Tansol Park, Department of Animal Science and Technology, Chung-Ang University, Anseong-si, Gyeonggi-do 17546, Korea, Republic of. E-mail:

© Copyright 2023 Korean Society of Animal Science and Technology. This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.


Hanwoo and Jeju Black cattle (Jeju Black) are native breeds of Korean cattle. Jeju Black cattle are recognized as natural monuments and are known to exhibit slower growth rates compared to Hanwoo. While several studies have analyzed the genetic characteristics of these cattle, there has been limited research on the differences in their microbiome. In this study, rumen fluid was obtained from three Hanwoo steers and three Jeju Black steers, and three different diets (total mixed rations [TMRs] for growing, early fattening, and late fattening periods) were used as substrates for <italic>in vitro</italic> fermentation. The <italic>in vitro</italic> incubation was conducted for 3 h and 24 h following a 2 × 3 factorial arrangement. After both incubation periods, fermentation characteristics were analyzed, and ruminal microbiome analysis was performed using 16S rRNA gene sequencing, employing both QIIME2 and PICRUSt2. The results revealed significant differences in the ruminal microbiota due to the inoculum effect. At the phylum level, Patescibacteria and Synergistota were found to be enriched in the Jeju Black inoculum-treated group. Additionally, using different inocula also affected the relative abundance of major taxa, including Ruminococcus, Pseudoramibacter, Ruminococcaceae CAG-352, and the [Eubacterium] ruminantium group. These microbial differences induced by the inoculum may have originated from varying levels of domestication between the two subspecies of donor animals, which mainly influenced the fermentation and microbiome features in the early incubation stages, although this was only partially offset afterward.  Furthermore, predicted commission numbers of microbial enzymes, some of which are involved in the biosynthesis of secondary metabolites, fatty acids, and alpha amylase, differed based on the inoculum effect. However, these differences may account for only a small proportion of the overall metabolic pathway. Conversely, diets were found to affect protein biosynthesis and its related metabolism, which showed differential abundance in the growing diet and were potentially linked to the growth-promoting effects in beef cattle during the growing period. In conclusion, this study demonstrated that using different inocula significantly affected <italic>in vitro</italic> fermentation characteristics and microbiome features, mainly in the early stages of incubation, with some effects persisting up to 24 h of incubation.

Keywords: in vitro fermentation; Korean native cattle; growing stages; rumen microbiome; functional prediction