Long evans female rats

We used our established model of dietary iron administration in rats (Huebner et al., 2018 (link), 2016 (link)). Nulliparous, 8-week old Long-Evans female rats (Envigo, Richmond IN) were randomly assigned to consume one of three diets ad libitum: the iron-sufficient (IS) diet (containing 100 ppm iron; TD.06016); the iron-deficient (ID) diet (containing 100 ppm iron from GD0.5–5 [TD.06016], 20 ppm iron from GD5–13.5 [TD.06013], and 2–6 ppm iron from GD13.5–20.5 [TD.80396]); or the iron-fortified (IF) diet (containing 500 ppm iron; TD.110880). Diet composition was based on AIN-76A (Huebner et al., 2018 (link), 2016 (link)), and diets were produced by Teklad-Envigo (Madison, WI). Rats consumed the assigned diet at least 2 weeks prior to and throughout pregnancy. Gestational day (GD) 0.5 was defined as the morning a vaginal plug was discovered. Litters containing <9 or >15 pups were excluded from analyses to eliminate extremes in iron distribution across litters within a treatment group. On GD20.5, following isoflurane overdose and maternal perfusion, maternal and fetal tissues were collected, flash frozen in liquid nitrogen, and stored at −80°C until further analysis. The Institutional Animal Care and Use Committee approved all protocols.

We used our established model of dietary iron intervention (Rufer, et al., 2012 (link); Huebner et al., 2015 (link), 2016 (link)). Nulliparous, 8-week old Long Evans female rats (Envigo, Richmond IN) were randomly assigned to consume ad libitum either an iron-sufficient (IS, 100ppm Fe; TD.06016) or iron-fortified (IF, 500ppm; TD.110880) diet at least 2 wks prior to and throughout pregnancy. The diet composition is based on AIN-93G (Supplemental Table 1), and food intake did not differ by treatment. Gestational day (GD) 0.5 was defined as the morning a vaginal plug was discovered. Litters containing < 9 or >15 pups were excluded from all analyses to minimize differences in iron distribution across litters within a treatment group. Fetuses were analyzed on GD20.5, which is the day prior to birth and is when placental iron transport is largely complete; it is the equivalent of human fetus at 39-40 weeks with respect to maternal-fetal iron transfer (Rao and Georgieff, 2002 ). On GD20.5, following isoflurane overdose and maternal perfusion, maternal and fetal blood and tissues were collected, flash frozen in liquid nitrogen, and stored at −80°C until further analysis. A subset of liver specimens was formalin-fixed and paraffin-blocked for histological analysis. All protocols were approved by the Institutional Animal Care and Use Committee.