approved by the University Committee of Animal Resources at the University of Rochester, New York. Hematocrit and serum iron level measurement The animals were anesthetized with ketamine (100 mg/kg i.p.) and blood samples for hematocrit measurement were collected in heparinized microcapillary tubes (VWR, West Chester, PA) from the right cardiac atrium before the rats were perfused. Tubes were centrifuged for 15 minutes at room temperature in a high speed hematocrit centrifuge (IEC MB Microhematocrit Centrifuge). The Hematocrit value was calculated as the length of red blood cell column per total blood sample column length and expressed as a percentage. For a more detailed hematological analysis including hematocrit (Ht) values, hemoglobin (Hb) concentrations, red blood cell (RBC) counts, and mean corpuscular volumes (MCV), we analyzed the fresh blood samples, collected in EDTA-coated tubes, using HESKA Hema True Veterinary Hematology System. Whole blood aliquots for serum iron determination were allowed to clot, and then were centrifuged at 10,000 x g at 4uC for 15 minutes to separate cells from sera. Serum samples were isolated then frozen and stored at 280uC until analysis for iron concentrations by atomic absorption spectrophotometry (AAS). Tissue iron measurement using Atomic Absorption Spectroscopy (AAS) After anesthesia with ketamine (100 mg/kg i.p.) and blood collection for hematocrit and serum iron determination, the animals were transcardially perfused with11.2 mg/ml heparin in phosphate-buffered saline via the left ventricle to remove blood from organs. The embryos or CNS tissues were immediately dissected, washed in distilled deionized water, vacuum dried, weighed, and stored in iron-free teflon vials at 280uC until analyzed as previously described. Frozen embryonic and postnatal CNS tissues were wet-digested in ultra-pure nitric acid and analyzed for iron concentration using the graphite furnace atomic absorption spectrophotometer. Standards, blanks, and standard curves were used as previously specified [104,105,106]. Core body temperature measurement All experimental ID and control rats were subjected to core body temperature measurement by using a RET-2 rectal probe and TH-5 Thermalert Monitoring Thermometer (Physitemp). The core body temperature was consistently recorded for all groups of animals at six o’clock in the evening of the day of auditory testing. Auditory Brainstem Responses (ABR) recording and analysis Control (CTL) and iron deficient (ID) dietary groups of rats (ID - 2wks, ID E0, ID E7, and ID E14 groups) were ABR tested on postnatal day 40–45. For the test, rats were anesthetized with References 1. Stoltzfus RJ (2003) Iron deficiency: global prevalence and consequences. Food Nutr Bull 24: S99–103. 2. Baynes RD, Cook JD (1996) Current issues in iron deficiency. Curr Opin Hematol 3: 145–149. 3. Beard J (2003) Iron deficiency alters brain development and functioning. J Nutr 133: 1468S–1472S. 4. Conrad ME, Umbreit JN (2001) Iron absorption: relative importance of iron transport pathways. Am J Hematol 67: 215. 5. Conrad ME, Umbreit JN, Moore EG, Hainsworth LN, Porubcin M, et al. (2000) Separate pathways for cellular uptake of ferric and ferrous iron. Am J Physiol Gastrointest Liver Physiol 279: G767–774. Impact of Maternal Iron Restriction on Offspring acepromazine (1 mg/kg i.p.) and ketamine (80–100 mg/kg i.p.). Needle electrodes were inserted at vertex and pinna, with a ground near the tail. ABR potentials were evoked with click tone pips at 5 log-spaced frequencies from 5.6 to 24.4 kHz at 70 dB SPL. The response was amplified (10,000x) and 2,048 responses were averaged with using software and high-frequency speakers from Intelligent Hearing Systems (Smart EP, Miami, FL). To compare the change of latencies, the ABR peaks and troughs were identified by a semi-automated tool and verified by a trained observer by visual inspection of recorded wave forms. Latencies were calculated from the onset of the stimulus, and the interpeak latencies of wave II-I (P2-P1), wave III-I (P3-P1), and wave IV-I (P4-P1) were computed. The change of interpeak latencies was then calculated. Distortion Product of Otoacoustic Emissions (DPOAE) acquisition The function of the outer hair cells in the ear was analyzed by measuring DPOAEs with secondary tones (f2) at 5 log-spaced frequencies from 5.6 to 24.4 kHz. DPOAE testing was performed with f2/f1 = 1.2 and unequal primaries (L1 = 10dB+L2) at 70 dB and 60 dB SPL using high-frequency speakers and software from Intelligent Hearing Systems (SmartOAE) and high-frequency microphones (ER10B+) from Etymotic Research (Elk Grove Village, IL). Cochlear function was assessed for all animals at approximately 40–45 days of age and rats were sedated with ketamine (80–100 mg/kg i.p.) and acepromazine (1 mg/kg i.p.). Statistical analyses ‘‘n’’ represents the number of animals in any group analyzed and has a value $ 3. Regardless the sample size (n value), each control and experimental group was composed of animals from at least 3 separate litters. Statistical analyses were performed with the two-tailed Student’s t-test and conducted comparing nutritionally ID to control rats, under the same conditions. A p-value less than 0.05 was considered statistically significant. Comparison of mean values of ABR interpeak latencies between different dietary groups and controls was performed with analysis of variance (ANOVA) using a Bonferroni posttest analysis and significance was defined as a p value less than 0.05. Acknowledgments We thank Ollivier Hyrien for input regarding statistical analysis and other laboratory members for valuable assistance in this project. We also extend gratitude towards Mark Noble and Christopher Pro¨schel for helpful comments on this manuscript. Author Contributions Conceived and designed the experiments: MMP AEL CM. Performed the experiments: CM FGS DLL RMG. Analyzed the data: MMP AEL CM JS. Contributed reagents/materials/analysis tools: MMP AEL RMG. Wrote the paper: MMP CM. 6. Casanueva E, Viteri FE (2003) Iron and oxidative stress in pregnancy. J Nutr 133: 1700S–1708S. 7. Melamed N, Ben-Haroush A, Kaplan B, Yogev Y (2007) Iron supplementation in pregnancy–does the preparation matter? Arch Gynecol Obstet 276: 601–604. 8. Nguyen P, Nava-Ocampo A, Levy A, O’Connor DL, Einarson TR, et al. (2008) Effect of iron content on the tolerability of prenatal multivitamins in pregnancy. BMC Pregnancy Childbirth 8: 17. 9. Siega-Riz AM, Hartzema AG, Turnbull C, Thorp J, McDonald T, et al. (2006) The effects of prophylactic iron given in prenatal supplements on iron status and birth outcomes: a randomized controlled trial. Am J Obstet Gynecol 194: 512–519. PLoS ONE | www.plosone.org 11 March 2011 | Volume 6 | Issue 3 | e17483