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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 in vitro fermentation. The in vitro 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 in vitro fermentation characteristics and microbiome features, mainly in the early stages of incubation, with some effects persisting up to 24 h of incubation.

Rumen cannulation is a surgical technique used to collect rumen contents from ruminants. However, rumen cannulation surgery may potentially impact the composition of the rumen microbiota. This study aimed to examine the longitudinal alterations in the rumen microbiota composition of Hanwoo steers after cannulation surgery. In this study, eight Hanwoo steers were used; four steers underwent rumen cannulation surgery (cannulation group), while the remaining four were left intact (control group). Rumen samples were collected from all eight steers using the stomach tubing method on the day before surgery (day 0) and on postoperative days 1, 4, 7, 10, 14, 17, 21, 24, and 28, resulting in 80 samples (10 timepoints × 8 animals). The microbiota of all 80 samples were analyzed using 16S rRNA gene amplicon sequencing with Quantitative Insights into Microbial Ecology version 2 (QIIME2). There were no significant differences (p > 0.05) in all major phyla and most major genera representing at least 0.5% of total sequences across all 80 samples between the control and cannulation groups on the preoperative and postoperative days. However, while the alpha diversity indices did not differ (p > 0.05) between the two groups on the preoperative day, they significantly differed (p < 0.05) between the two groups on the postoperative days. Further, the overall microbial distribution based on both unweighted and weighted principal coordinate analysis plots significantly differed (p < 0.05) between the two groups on both the preoperative and postoperative days. Orthogonal polynomial contrasts indicated that major genera and microbial diversity in the cannulation group decreased following surgery but returned to their initial states by postoperative day 28. In conclusion, this study demonstrates that rumen cannulation surgery affects some major taxa and microbial diversity, suggesting that the rumen cannulation method can alter the composition of rumen microbiota in Hanwoo steers.

Bovine fecal microbiota is important for host health and its composition can be affected by various factors, such as diet, age, species, breed, regions, and environments. The objective of this study was to evaluate the impact of diet and gender on fecal microbiota in Korean native Hanwoo cattle. The 16S rRNA gene amplicon sequencing of fecal microbiota was conducted from 44 Hanwoo cattle divided into four groups: (1) 11 heifers fed an oat hay plus total mixed ration (TMR) diet for breeding (HOTB), (2) 11 heifers fed an early fattening TMR diet (HEFT), (3) 11 steers fed the early fattening TMR diet (SEFT), and (4) 11 steers fed the late fattening TMR diet (SLFT). Firmicutes and Bacteroidota were the first and second most dominant phyla in all the samples, respectively. The Firmicutes/Bacteroidota (F/B) ratio associated with feed efficiency was significantly greater in the SLFT group than in the other groups. At the genus level, Romboutsia, Paeniclostridium, and Turicibacterwere the most abundant in the SLFT while Akkermansia, Bacteroides, and Monoglobus were the most abundant in the HOTB group. Although the same early fattening TMR diet was fed to Hanwoo heifers and steers, Marvinbryantia and Coprococcus were the most abundant in the HEFT group while Alistipes and Ruminococcus were the most abundant in the SEFT group. Shannon and Simpson diversity indices were significantly lower in the SLFT group than in the other groups. Distribution of fecal microbiota and functional genetic profiles were significantly different among the four treatment groups. The present study demonstrates that different diets and genders can affect fecal microbiota and the F/B ratio may be associated with feed efficiency in Hanwoo cattle. Our results may help develop strategies to improve gut health and productivity through manipulation of fecal microbiota using the appropriate diet considering Hanwoo cattle gender.