Programming effects of intrauterine hyperthermia on adrenal gland development

Animals and Adrenal Gland Collection Procedures. Multiparous pregnant Holstein cows, in their dry period (preceding lactation), were exposed to environmental heat stress (Temperature-Humidity Index > 68) with access to shade and natural ventilation (HS, n = 41), or provided active cooling (CL) via access to shade plus fans, and water soakers (n = 41) to maintain thermoneutrality. The treatments of heifers born to these cows were reflected by their treatment during the dry period. Thus, female calves (heifers) born to HS dams were considered HS, and heifers born to CL dams were considered CL. Unless assigned to euthanasia at birth (d 0), heifers were fed 3.78 L if high-quality colostrum (protein concentration > 21%) within 2 h of birth (0.8 ± 0.5 h). Prophylactic interventions and vaccinations were performed according to the farm standard operating protocols and are available at Dado-Senn et al. (2021). Six heifers from each treatment were euthanized at birth (day 0). The remaining heifers were raised as a cohort, receiving the same postnatal management and nutrition, to isolate the in utero effects. A second subset of six heifers per group was euthanized one week after weaning (day 63). Adrenal glands were located above the kidneys and carefully dissected and washed in sterile PBS. The right adrenal was fixed in 10% NBF, and the left adrenal was bisected and snap-frozen in liquid nitrogen for RNA-sequencing.动物与肾上腺组织采集流程。 本研究的实验对象为处于干奶期（泌乳前期）的经产妊娠荷斯坦奶牛（Holstein cows），将其分为两组：一组暴露于环境热应激环境，温度湿度指数（Temperature-Humidity Index）>68，仅可获得遮阴与自然通风条件，记为HS组（n=41）；另一组通过遮阴结合风扇与浸水式饮水装置（water soakers）实施主动降温（CL组，n=41），以维持个体处于热中性状态（thermoneutrality）。 这些母牛所产的青年母牛（heifers，即母犊）的分组与其母亲在干奶期的处理分组保持一致：HS组母牛所产的青年母牛归为HS组，CL组母牛所产的青年母牛归为CL组。除在出生当天（第0天）被安排安乐死的个体外，所有青年母牛均在出生后2小时内（平均0.8±0.5小时）饲喂3.78 L优质初乳（蛋白质浓度>21%）。预防性干预与疫苗接种均按照养殖场标准操作流程实施，详细方案可参见Dado-Senn等人（2021）的研究。 每组各6头青年母牛在出生当天（第0天）实施安乐死。剩余青年母牛作为同一队列进行饲养，产后管理与营养供给完全一致，以分离宫内环境的效应影响。每组另有6头青年母牛在断奶后一周（第63天）实施安乐死。 肾上腺位于肾脏上方，需小心剥离并经无菌磷酸盐缓冲液（Phosphate Buffered Saline，PBS）冲洗。右侧肾上腺经10%中性福尔马林缓冲液（Neutral Buffered Formalin，NBF）固定，左侧肾上腺对半剖开后置于液氮中快速速冻，用于RNA测序（RNA-sequencing）。