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.