Tissue collection: Multiparous ewes were fed an NR (50% NRC recommendations; n= 8) or C (100% NRC; n= 6) diet from 28-78 days of gestation. Two placentomes with similar size were collected from each placenta. A cross section of each placentome was embedded in paraffin. The rest of the placentome was then separated into COT and CAR. COT tissue was collected and immediately snap frozen in liquid nitrogen.

Measurements: Three 5 µm cross sections of each placentome were stained with eosin and hematoxylin and evaluated for COT numbers and diameters via image analysis by an experienced individual blinded to the experimental groups, and the results averaged. Arteriole number from each COT was then corrected for the total COT area measured (per unit area as mm2). COT tissue was pulverized in liquid nitrogen and total RNA and protein were extracted for Realtime-PCR and Westernblot evaluation of VEGF, PLGF, FGF-2, ANG-1&2 and HIF-1α.