Table_4_Evaluation of Reproductive Tract Cytokines in Post-partum Beef Cows Relating to Reproductive Microbiota and Fertility Outcomes.DOCX

The activity of the immune system in the reproductive tract has been proven to be crucial in the response to uterine diseases, normal reproductive functions, and tolerance to the allogeneic fetus during pregnancy. The objectives of the current study were to (1) evaluate uterine and vaginal cytokine concentrations in postpartum cows undergoing estrus synchronization followed by timed artificial insemination (TAI) and (2) correlate bacterial communities with cytokine concentrations. Postpartum Angus cows (n = 20) were subjected to a 7-Day Co-Synch protocol with pre-synchronization beginning 21 days prior (d −21) to TAI (d 0). Uterine and vaginal flushes were collected on d −21 and −2. Pregnancy was determined by transrectal ultrasound on d 30. Cytokines include interleukin (IL)-1b, IL-6, IL-10, transforming growth factor beta (TGF-β), and immunoglobin A (IgA) and concentrations were determined by commercial ELISA kits. No differences by day or pregnancy status in cytokine concentrations were detected in vaginal samples. No differences by day or pregnancy status in IgA, IL-10, or IL-1b concentrations were detected in uterine samples. Overall TGF-β concentrations in the uterus were greater in resulting pregnant than non-pregnant cows (44.0 ± 13.4 pg/mL vs. 14.7 ± 4.9 pg/mL; P = 0.047). Uterine TGF-β was correlated with the relative abundance of genera Treponema (r = −0.668; P = 0.049) in resulting non-pregnant cows on d −21 and with the relative abundance of genera Ureaplasma (r = 0.901; P = 0.0004) in resulting pregnant cows on d −2. In resulting pregnant animals, a tendency for a strong correlation was detected between d −2 progesterone concentrations and uterine TGF-β concentrations (r = 0.591, P = 0.07). Overall IL-6 concentrations in the uterus were greater in resulting non-pregnant than pregnant cows (198.7 ± 21.8 pg/mL vs. 144.3 ± 16.1 pg/mL; P = 0.045). A correlation was also detected between uterine IL-6 concentrations and the relative abundance of genera Butyrivibrio (r = 0.742; P = 0.022) in resulting non-pregnant cows on d −21. These results suggest possible relationships between different bacterial communities and cytokine concentrations within the uterus of beef cattle prior to TAI that may ultimately affect fertility outcomes.

生殖道免疫系统的活性，在子宫疾病应答、正常生殖功能维持以及妊娠期间对同种异体胎儿的免疫耐受过程中，已被证实发挥关键作用。
本研究旨在达成两个研究目标：（1）评估经同期发情处理后实施定时人工授精（timed artificial insemination, TAI）的产后奶牛的子宫与阴道细胞因子浓度；（2）分析细菌群落与细胞因子浓度之间的相关性。
本研究共纳入20头产后安格斯奶牛，采用7天同步方案，于定时人工授精当天（d 0）前21天（d -21）启动预同步流程。分别于d -21与d -2采集子宫及阴道冲洗液样本；于d 30通过直肠超声检查判定妊娠状态。
本研究检测的细胞因子包括白细胞介素（interleukin, IL）-1β、IL-6、IL-10、转化生长因子β（transforming growth factor beta, TGF-β）以及免疫球蛋白A（immunoglobin A, IgA），所有指标浓度均通过商业化ELISA试剂盒测定。
阴道样本的细胞因子浓度未观察到随采样日或妊娠状态产生的显著差异。子宫样本中，IgA、IL-10及IL-1β的浓度未发现随采样日或妊娠状态存在显著差异。
子宫整体TGF-β浓度在最终妊娠的奶牛中显著高于未妊娠奶牛（44.0±13.4 pg/mL vs. 14.7±4.9 pg/mL; P=0.047）。在d -21时，未妊娠奶牛的子宫TGF-β浓度与密螺旋体属（Treponema）的相对丰度呈显著负相关（r=-0.668; P=0.049）；而在d -2时，妊娠奶牛的子宫TGF-β浓度与脲原体属（Ureaplasma）的相对丰度呈显著正相关（r=0.901; P=0.0004）。在最终妊娠的动物中，d -2的孕酮浓度与子宫TGF-β浓度呈现较强的相关趋势（r=0.591, P=0.07）。
子宫整体IL-6浓度在最终未妊娠的奶牛中显著高于妊娠奶牛（198.7±21.8 pg/mL vs. 144.3±16.1 pg/mL; P=0.045）。在d -21时，未妊娠奶牛的子宫IL-6浓度与丁酸弧菌属（Butyrivibrio）的相对丰度呈显著正相关（r=0.742; P=0.022）。
本研究结果提示，定时人工授精前肉牛子宫内的不同细菌群落与细胞因子浓度之间可能存在关联，该关联或可最终影响繁殖结局。