Marques J C S, Maciel J P O, Denis-Robichaud J, Conceicao R S, Moore S, Piau T, Bartolomeu C C, Cerri R L A
Faculty of Land and Food Systems, University of British Columbia, Vancouver V6T 1Z4, Canada.
Faculty of Land and Food Systems, University of British Columbia, Vancouver V6T 1Z4, Canada; Faculty of Veterinary Medicine, Rural Federal University of Pernambuco, Recife 52171-900, Brazil.
J Dairy Sci. 2025 Sep;108(9):10391-10409. doi: 10.3168/jds.2025-26522. Epub 2025 Jul 18.
The main objective of this study was to evaluate if different concentrations of progesterone (P4) during superovulation and different intensities of estrous expression affected endometrial gene expression 7 d after artificial insemination (AI) in Holstein heifers. Our secondary objective was to explore the potential role of estradiol (E2) concentrations before estrus in relation to these factors. Post-puberty Holstein heifers were randomly assigned into 2 treatment groups: high P4 or low P4. Animals received a presynchronization protocol followed by a protocol of superovulation that included the assigned P4 treatment. Activity was monitored by an automated wearable sensor, and estrous behavior (intensity and duration) was recorded. A total of 39 heifers (high P4: n = 19; low P4: n = 20) were submitted to uterine biopsy 7 d after AI. We conducted RNA extraction for each sample, and the count of mRNA molecules for 91 target genes were determined using the NanoString nCounter system. The intensity and duration of estrus were used to categorize heifers as high or low estrous expression using the median. Count outcome data were normalized to housekeeping genes and analyzed using a mixed linear regression model with heifer as random effect. Significance for differential expression was set at false discovery rate <0.1 and absolute fold change ≥1.5. The P4 treatment or estrous expression alone were not associated with changes in endometrial gene expression; however, their interaction significantly influenced the expression of 12 target genes. Most of these genes were related to the immune system signaling (CXCL8, IL1B, MUC1, PTX3), indicating an upregulation of this biological function in the endometrium of heifers treated with low P4 and having low estrous expression. Pathway analysis confirmed the involvement of the immune response with downstream effects linked to transmigration of leukocytes, cell movement of monocytes, accumulation of phagocytes and mobilization of neutrophils. Additional E2 analysis demonstrated that concentrations of E2 before estrus influenced the expression of genes related to extracellular matrix remodeling (MMP14, MMP19, POSTN), immune system (MUC1, IL1B, CSF1), growth factors (IGF1, IGF1R, FGF2), and eicosanoid metabolic processes (AKR1B1, AKR1C4, PTGIS, HPGD) regardless of the P4 treatment. In conclusion, heifers treated with low P4 during superovulation and exhibiting low intensity and duration of estrus had distinct endometrial gene expression profiles 7 d after AI compared with those treated with high P4. Additional E2 analysis demonstrated that concentrations of E2 2 d before estrus influenced post-estrus endometrial gene expression independently of the pre-estrus P4 profile.
本研究的主要目的是评估在超排期间不同浓度的孕酮（P4）以及不同强度的发情表现是否会影响荷斯坦小母牛人工授精（AI）后7天的子宫内膜基因表达。我们的次要目的是探讨发情前雌二醇（E2）浓度与这些因素相关的潜在作用。青春期后的荷斯坦小母牛被随机分为2个处理组：高P4组或低P4组。动物先接受预同步方案，然后是包含指定P4处理的超排方案。活动通过自动可穿戴传感器进行监测，发情行为（强度和持续时间）被记录下来。共有39头小母牛（高P4组：n = 19；低P4组：n = 20）在AI后7天接受子宫活检。我们对每个样本进行RNA提取，并使用NanoString nCounter系统测定91个靶基因的mRNA分子计数。发情的强度和持续时间用于将小母牛分为发情表达高或低的类别，以中位数为界。计数结果数据经管家基因标准化后，使用以小母牛为随机效应的混合线性回归模型进行分析。差异表达的显著性设定为错误发现率<0.1且绝对倍数变化≥1.5。单独的P4处理或发情表达与子宫内膜基因表达的变化无关；然而，它们的相互作用显著影响了12个靶基因的表达。这些基因中的大多数与免疫系统信号传导（CXCL8、IL1B、MUC1、PTX3）相关，表明在接受低P4处理且发情表达低的小母牛子宫内膜中该生物学功能上调。通路分析证实了免疫反应的参与，其下游效应与白细胞迁移、单核细胞的细胞运动、吞噬细胞的积累和中性粒细胞的动员有关。额外的E2分析表明，无论P4处理如何，发情前E2的浓度都会影响与细胞外基质重塑（MMP14、MMP19、POSTN）、免疫系统（MUC1、IL1B、CSF1）、生长因子（IGF1、IGF1R、FGF2）以及类花生酸代谢过程（AKR1B1、AKR1C4、PTGIS、HPGD）相关的基因表达。总之，在超排期间接受低P4处理且发情强度和持续时间短的小母牛，与接受高P4处理的小母牛相比，在AI后7天有明显不同的子宫内膜基因表达谱。额外的E2分析表明，发情前2天的E2浓度独立于发情前的P4情况影响发情后子宫内膜基因表达。
