4.1. Animals

All reagents were purchased from Sigma (Madrid, Spain) unless indicated otherwise. Animal experiments were conducted according to European legislation. All study protocols were approved by the Ethics Committee on Animal Experimentation of the INIA (Madrid, Spain) (21 September 2015) and were registered at the Direccion General de Agricultura y Ganaderia de la Comunidad de Madrid (Spain) (PROEX 261/15, 4 November 2015). For experiments we used CD-1 and B6 (C57BL/6NHsd) mice (Envigo, Envigo RMS Spain S.L.). All mice were kept in an animal facility with a controlled temperature and photoperiod (23 ± 1 °C and a cycle of 14 h light:10 h darkness) and had access to water and food ad libitum. Adult mice (3–4 months of age, virgin female mice) were bred for 2 weeks and then transferred to individual cages. Eight males of both strains were used in all crosses. Dams delivered naturally, fostered their own pups, and only litters with 5 to 7 (for inbred B6, average litter size of 6.4 in the mouse facility) and 7 to 9 (for outbreed CD1, average litter size of 8.6 in the mouse facility) animals were used in the experiments.

Neonatal growth restriction or microsomia was defined as a gender-specific weight on postnatal Day 2 (D2) above the 10th percentile for the colony [38]. To lessen the risk of rejection, nests were undisturbed for 24 h after delivery, and initial pup weights were obtained on postnatal D2. Pups whose weight on D2 was under the 10th percentile (IUGR) or within the 50th percentile (control) were marked with indelible ink, and they were later tattooed to identify each animal on Day 10. For every microsomic mouse detected (18 B6 males and 17 B6 females and 17 CD1 males and 15 CD1 females), a corresponding littermate control mouse was identified (14 and 15 B6 males and females respectively, and 15 and 14 CD1 males and females, respectively). No more than two microsomic and control mice were obtained from a given litter. Pups were weaned to postnatal D21 and kept in groups of 5 animals for 3 months, when their weights were recorded. Day 2 and Day 10 mice were euthanized by decapitation with surgical scissors, and adult mice were euthanized with CO₂.