在这项研究中,我们旨在比较产后奶牛的子宫微生物概况,通过细菌学培养和下一代测序确定,使用三种子宫取样技术(拭子,细胞刷,和灌洗)和发情周期的诱导阶段(发情期和发情期)。在产后53±5天的15头健康产后奶牛被纳入研究。在固定时间的人工授精方案期间收集子宫样本。从每个样品的一部分需氧培养活细菌,通过16SrRNA基因的Sanger测序鉴定细菌分离株。从剩余的未稀释样品中提取总基因组DNA,以使用16SrRNAqPCR定量细菌负荷,并通过16SrRNA基因V1-V3区的宏基因组测序表征微生物组。使用Shannon-Weaver多样性指数和主成分分析分析了微生物的概况和组成,分别。87个样本中,88%(77/87)为培养阳性。培养阳性子宫样本的比例在采样技术(P=0.39)或发情周期阶段(P=0.99)之间没有差异。然而,拭子,细胞刷,灌洗技术产生1.5、9和9倍以上的细菌负荷(P<0.01),分别,在发情期比发情期。此外,在动情期,细胞刷法产生的细菌是灌洗法和拭子法的3和6倍(P<0.01)。从细菌学培养和宏基因组测序中鉴定出的最丰富的细菌属是芽孢杆菌和肠球菌,无论采样技术或发情周期的阶段。通过宏基因组测序,中至低丰度的细菌属包括链球菌,螺旋藻科,和落叶松科。值得注意的是,子宫微生物的概况和组成,通过宏基因组测序确定,采样技术(分别为P=0.55和P=0.60)或发情周期阶段(分别为P=0.34和P=0.17)没有差异。总之,我们的研究结果表明,任何一种采样技术都可以可靠地用于研究健康奶牛在发情周期随机阶段的子宫微生物组.然而,重要的是要考虑细菌产量的潜在差异作为混杂因素。
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.