Table_3_Oral swabs as a proxy for direct ruminal microbiome sampling in Holstein dairy cows is correlated with sample color.XLSX

Using oral swabs to collect the remnants of stomach content regurgitation during rumination in dairy cows can replicate up to 70% of the ruminal bacterial community, offering potential for broad-scale population-based studies on the rumen microbiome. The swabs collected from dairy cows often vary widely with respect to sample quality, likely due to several factors such as time of sample collection and cow rumination behavior, which may limit the ability of a given swab to accurately represent the ruminal microbiome. One such factor is the color of the swab, which can vary significantly across different cows. Here, we hypothesize that darker-colored swabs contain more rumen contents, thereby better representing the ruminal bacterial community than lighter-colored swabs. To address this, we collected oral swabs from 402 dairy cows and rumen samples from 13 cannulated cows on a research farm in Wisconsin, United States and subjected them to 16S rRNA sequencing. In addition, given that little is known about the ability of oral swabs to recapitulate the ruminal fungal community, we also conducted ITS sequencing of these samples. To correlate swab color to the microbiota we developed and utilized a novel imaging approach to colorimetrically quantify each swab from a range of light to dark. We found that swabs with increasing darkness scores were significantly associated with increased bacterial alpha diversity (p < 0.05). Lighter swabs exhibited greater variation in their community structure, with many identified amplicon sequence variants (ASVs) categorized as belonging to known bovine oral and environmental taxa. Our analysis of the fungal microbiome found that swabs with increasing darkness scores were associated with decreased alpha diversity (p < 0.05) and were also significantly associated with the ruminal solids fungal community, but not with the ruminal liquid community. Our study refines the utility of oral swabs as a useful proxy for capturing the ruminal microbiome and demonstrates that swab color is an important factor to consider when using this approach for documenting both the bacterial and fungal communities.

通过口腔拭子（oral swab）采集奶牛反刍时反流的胃内容物残留物，可复现高达70%的瘤胃细菌群落，为开展瘤胃微生物组（rumen microbiome）的大规模奶牛种群研究提供了可行路径。但从奶牛群体采集的口腔拭子样本质量往往差异悬殊，这可能受样本采集时间、奶牛反刍行为等多种因素影响，进而限制了单一样本准确反映瘤胃微生物组的能力。其中，拭子颜色便是此类影响因素之一，不同奶牛的拭子颜色差异显著。本研究提出假说：颜色更深的拭子所含瘤胃内容物更多，因此相比浅色拭子，能更准确地反映瘤胃细菌群落。为此，我们在美国威斯康星州的一处研究农场，从402头奶牛采集了口腔拭子，并从13头瘤胃瘘管奶牛采集了瘤胃样本，随后对所有样本开展16S核糖体RNA（16S rRNA）测序。此外，由于目前对口腔拭子能否复现瘤胃真菌群落的认知尚浅，我们还对所有样本开展了内转录间隔区（Internal Transcribed Spacer, ITS）测序。为探究拭子颜色与微生物组的关联，我们开发并应用了一种新型成像方法，可对颜色由浅至深的各类拭子进行比色定量分析。研究发现，颜色越深（灰度评分越高）的拭子，其细菌α多样性显著更高（p < 0.05）。浅色拭子的群落结构变异程度更高，其中大量已鉴定的扩增子序列变体（Amplicon Sequence Variant, ASV）被归类为已知的奶牛口腔及环境类群。我们对真菌微生物组的分析结果显示，颜色越深的拭子，其真菌α多样性显著更低（p < 0.05）；同时，这类拭子与瘤胃固相真菌群落显著相关，但与瘤胃液相真菌群落无显著关联。本研究优化了口腔拭子作为瘤胃微生物组替代采样手段的应用价值，并证实：在利用该方法开展细菌与真菌群落研究时，拭子颜色是一项需要重点考量的关键因素。