Abstract 143 – First Trial of Semen Microbiome Analysis by Next-Generation Sequencing in Asiatic black bear.

Nari Kim, Kyung-Hyo Do, Ki-Yoon Kim, Kwang-Won Seo, Jeong-Jin Yang, Eui-Beom
Kyeong, Seung-Hyo Lim, Dong-Hyuk Jeong

Captive breeding, a global practice for endangered species conservation, faces challenges due
to reduced fertility among many captive individuals. While the reproductive microbiome has
been studied in animals like bulls and stallions to enhance fertility, no research has been
conducted on bears for conservation purposes. Therefore, this study aims to establish a
protocol for analyzing sperm microbiomes in Asiatic black bears (Ursus thibetanus) bred for
conservation in Korea and confirm their microbiome profiles through 16S Sequencing.
A total of 100µL semen was obtained via urethral catheter from an Asiatic black bear bred at
Korea National Park Research Institute. DNA extraction was performed individually using
aliquots of 5µL, 10µL, 15µL, and 50µL of semen. 16S V3–V4 amplicon libraries were prepared,
followed by sequencing on the Illumina MiSeq platform. The acquired sequences were
processed and analyzed using QIIME 2.
The 5µL, 10µL, and 15µL aliquots showed a total number of sequence reads exceeding 70,000,
which is sufficient for further analysis. The predominant phylum among semen bacteria was
Firmicutes (42.1%), followed by Bacteroidetes (35.0%). The most abundant genera were
Barnesiella, Eisenbergiella, Bacteroides, and Rothia, comprising approximately 45.7% of the
total semen microbiome.
Based on the results, we found that only a small amount of semen (5–15µL) is required for
microbiome analysis in Asiatic black bear. Given the challenge of extracting sufficient amounts
of semen in bears, the protocol has proven to be efficient for practical application. The bacterial
compositions of the three samples were aligned, demonstrating the protocol’s stability for
analysis.
This study represents the first attempt to establish a protocol for semen microbiome analysis in
bears. The application of this protocol to compare the reproductive microbiome of captive and
wild bears holds promise for enhancing Asiatic black bear fertility and species restoration.