Fig 1. Overview of experimental design, timeline, and sampling schedule.

(A) Dairy calves were gestated by dams exposed to heat stress (access to the shade of a free-stall barn, indicated by the red rectangle) or heat stress abatement (access to shade plus fans and water soakers, indicated by the blue rectangle) during late-gestation (~last 46 d, June-August 2018). After birth, calves were raised under similar environmental conditions to their dams until weaning (56 d of age), resulting in two treatments: heat stress (HS, n = 6) or cooling (CL, n = 6) across the pre- and postnatal periods (~ 102 d). (B) Blood was collected weekly from d 1 to d 49 to assess serotonin and IgG concentrations. On d 1, 21, and 42, blood was collected using Tempus RNA tubes for gene expression analysis and tubes with K₂EDTA anticoagulant for blood hematology analysis. On d 21 and 47, blood was collected using sodium heparin tubes for flow cytometric analyses, and an on d 47 to perform an in vitro LPS-stimulation assay.