Department of Large Animal Clinical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, S7N 5B4, Canada.
Department of Veterinary Microbiology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, S7N 5B4, Canada.
Theriogenology. 2024 Jan 15;214:298-306. doi: 10.1016/j.theriogenology.2023.11.004. Epub 2023 Nov 7.
In this study, we aimed to compare uterine microbial profiles in postpartum dairy cows, determined by bacteriological culture and next-generation sequencing, using three uterine sampling techniques (swab, cytobrush, and lavage) and induced phases of the estrous cycle (estrus and diestrus). Fifteen healthy postpartum dairy cows at 53 ± 5 days postpartum were enrolled in the study. Uterine samples were collected during a fixed-time artificial insemination protocol. Viable bacteria were aerobically cultured from part of each sample, and bacterial isolates were identified through Sanger sequencing of the 16S rRNA gene. Total genomic DNA was extracted from the remainder of undiluted samples to quantify bacterial load using 16S rRNA qPCR and characterize the microbiome by metagenomic sequencing of the V1-V3 region of the 16S rRNA gene. Microbial profiles and composition were analyzed using the Shannon-Weaver diversity index and principal component analysis, respectively. Out of 87 samples, 88 % (77/87) were culture positive. The proportion of culture-positive uterine samples did not differ between sampling techniques (P = 0.39) or estrous cycle phases (P = 0.99). However, swab, cytobrush, and lavage techniques yielded 1.5, 9 and 9 times greater bacterial loads (P < 0.01), respectively, during diestrus than estrus phase. Moreover, during diestrus phase, the cytobrush method yielded 3 and 6 times more bacteria (P < 0.01) than both the lavage and swab methods. The most abundant bacterial genera identified from both bacteriological culture and metagenomic sequencing were Bacillus and Enterococcus, regardless of sampling technique or phases of the estrous cycle. Bacterial genera in moderate to low abundance through metagenomic sequencing included Streptococcus, Oscillospiraceae, and Lachnospiraceae. Notably, the uterine microbial profiles and composition, determined by metagenomic sequencing, did not differ by sampling techniques (P = 0.55 and P = 0.60, respectively) or estrous cycle phases (P = 0.34 and P = 0.17, respectively). In conclusion, our results suggest that any of the sampling techniques can be reliably used to study the uterine microbiome of healthy cows at random phases of the estrous cycle. However, it is important to consider potential differences in bacterial yield as a confounding factor.
在这项研究中，我们旨在比较产后奶牛的子宫微生物谱，通过细菌培养和下一代测序来确定，使用三种子宫取样技术（拭子、细胞刷和冲洗）和发情周期的诱导阶段（发情和间情期）。15 头产后 53 ± 5 天的健康奶牛参与了这项研究。在固定时间人工授精方案期间收集子宫样本。从每个样本的一部分中需氧培养有活力的细菌，并通过 Sanger 测序 16S rRNA 基因鉴定细菌分离株。从未稀释样品的其余部分提取总基因组 DNA，使用 16S rRNA qPCR 定量细菌负荷，并通过 16S rRNA V1-V3 区的宏基因组测序来描述微生物组。使用 Shannon-Weaver 多样性指数和主成分分析分别分析微生物谱和组成。在 87 个样本中，88%（77/87）为培养阳性。采样技术（P=0.39）或发情周期阶段（P=0.99）之间的培养阳性子宫样本比例没有差异。然而，在间情期，拭子、细胞刷和冲洗技术分别产生 1.5、9 和 9 倍的细菌负荷（P<0.01）。此外，在间情期，细胞刷方法产生的细菌比冲洗和拭子方法分别多 3 倍和 6 倍（P<0.01）。无论是采样技术还是发情周期阶段，细菌培养和宏基因组测序都鉴定出最丰富的细菌属是芽孢杆菌属和肠球菌属。通过宏基因组测序鉴定出的丰度适中至较低的细菌属包括链球菌属、 Oscillospiraceae 和 Lachnospiraceae。值得注意的是，通过宏基因组测序确定的子宫微生物谱和组成不受采样技术（P=0.55 和 P=0.60）或发情周期阶段（P=0.34 和 P=0.17）的影响。总之，我们的研究结果表明，在发情周期的任意阶段，任何一种采样技术都可以可靠地用于研究健康奶牛的子宫微生物组。然而，需要考虑细菌产量的潜在差异作为混杂因素。
