Establishment and maintenance of pregnancy has a substantial economic impact within the dairy industry each year. Recent research has indicated a potential role of the reproductive microbiome on impacting pregnancy status as a result of hormone concentrations and breeding strategies. However, the association between alpha (within sample metric) and beta (between samples metric) bacterial diversity with pregnancy establishment has not been extensively evaluated in dairy cattle. Thereby, the objective of this study was to analyze vaginal bacterial diversity in lactating dairy cows 1) between day of artificial insemination (AI; day 0) and maternal recognition of pregnancy (MRP; d 18), and 2) between resulting Open and Pregnant females. Lactating Holstein dairy cows (n = 53) were sampled of day of AI and day of MRP. Pregnancy determination occurred on day 32 via transrectal ultrasonography. Sterile swabs were inserted into the vagina, rotated eight times against the vaginal wall, and stored in sterile microcentrifuge tubes at -80°C. Swab samples were utilized for subsequent bacterial diversity analyses targeting the V4 hypervariable region of the 16S rRNA gene. Cows underwent transrectal ultrasonography on gestational day 32 for pregnancy determination. Sample quality was determined by FastQC before being processed with the qiime2 pipeline for taxonomic classification. Alpha diversity metrics were analyzed with Kruskal-Wallis and beta diversity was measured utilizing PERMANOVA in the qiime2 pipeline. At day 32 of gestation, the pregnancy rate was 47.2%. According to Faith’s Phylogenetic Diversity metric for alpha diversity, there was no significant difference in diversity richness (i.e., number of different bacterial species within a sample) between day of AI and MRP or between cows diagnosed as Open or Pregnant (P >0.05). Pielou’s Evenness metric (i.e., distribution or balance of bacterial species within a sample) for alpha diversity indicated greater diversity within samples at day of AI compared with day of MRP (P = 0.02). Shannon’s diversity index metric (i.e., richness and evenness within a sample) for alpha diversity also indicated greater diversity at day of AI compared with day of MRP (P = 0.04). However, there was no difference between resulting Open and Pregnant females for Pielou’s or Shannon’s metrics (P > 0.05). Distinctions between samples (beta diversity) were compared utilizing pairwise comparisons of the unweighted and weighted Unifrac distance metrics. Specifically, day of AI indicated lower diversity compared with day of MRP for both metrics (P < 0.01). However, upon comparisons of Open and Pregnant status, there was no difference between samples for unweighted or weighted Unifrac distances (P > 0.05). Based on these results, bacterial diversity within and between vaginal samples appear to shift between day of AI and day of MRP. Additional studies are needed to elucidate the mechanisms altering bacterial diversity during early gestation in lactating dairy cows.

中文翻译：

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99 泌乳奶牛阴道细菌多样性对妊娠建立的影响

妊娠的建立和维持每年都会对乳制品行业产生重大的经济影响。最近的研究表明，由于激素浓度和繁殖策略，生殖微生物组对影响妊娠状态具有潜在作用。然而，α（样本指标内）和β（样本指标之间）细菌多样性与妊娠建立之间的关联尚未在奶牛中得到广泛评估。因此，本研究的目的是分析泌乳奶牛的阴道细菌多样性：1) 人工授精当天（AI；第 0 天）和母亲识别妊娠（MRP；第 18 天）之间，以及 2) 开产和怀孕之间女性。在 AI 日和 MRP 日对泌乳荷斯坦奶牛 (n = 53) 进行取样。第 32 天通过经直肠超声检查确定妊娠。将无菌拭子插入阴道，抵着阴道壁旋转八次，并在-80°C下储存在无菌微量离心管中。拭子样本用于随后针对 16S rRNA 基因 V4 高变区的细菌多样性分析。奶牛在妊娠第 32 天接受经直肠超声检查以确定妊娠。样本质量由 FastQC 确定，然后使用 qiime2 管道进行分类学分类。使用 Kruskal-Wallis 分析 Alpha 多样性指标，并使用 qiime2 管道中的 PERMANOVA 测量 Beta 多样性指标。妊娠第32天，妊娠率为47.2%。根据 Faith 的 α 多样性系统发育多样性指标，AI 和 MRP 之间或诊断为开放或怀孕的奶牛之间的多样性丰富度（即样本中不同细菌种类的数量）没有显着差异（P > 0.05） 。Pielou 的 α 多样性均匀度指标（即样本内细菌种类的分布或平衡）表明，与 MRP 日相比，AI 日样本内的多样性更高（P = 0.02）。香农的 α 多样性多样性指数指标（即样本内的丰富度和均匀度）也表明，与 MRP 日相比，AI 日的多样性更高（P = 0.04）。然而，对于 Pielou 或 Shannon 指标而言，结果开放和怀孕女性之间没有差异（P > 0.05）。利用未加权和加权 Unifrac 距离度量的成对比较来比较样本之间的差异（β 多样性）。具体而言，与 MRP 日相比，AI 日的两个指标的多样性较低（P < 0.01）。然而，在比较开放状态和怀孕状态时，样品之间的未加权或加权 Unifrac 距离没有差异（P > 0.05）。根据这些结果，阴道样本内和之间的细菌多样性似乎在 AI 日和 MRP 日之间发生了变化。