Phase 1 of a Transgenerational Study Effects of Endocrine Disrupting Chemicals (EDCs) on Reproductive Success and Offspring Viability in Adult Zebra Finches, Phase 1 of a Transgenerational Study D Christensen1, A Lambach1, K Dahlem1, C Hoover1, A Tucker1, R Reynolds1, A DenHartog1, R Rabideaux2 Biology1, Biochemistry, Cell and Molecular Biology2, Drake University, Des Moines, Iowa USA #339 ICN/SBN 2014 Introduction Image from: Matzuk and Lamb, 2008, The biology of infertility: research advances and clinical challenges. Nature Medicine (14)1197-1213 Endocrine-Disrupting Chemicals (EDCs) are released into the environment by degrading plastics. This includes food-grade plastic food and beverage containers. Most EDCs mimic estrogen, exerting the same influence that endogenous estrogens have on development of the nervous and reproductive systems. There is a complex relationship between body weight (acting via leptin in mammals), estrogen, and reproductive hormones. Acting on hypothalamic nuclei, estrogens can regulate reproductive timing & maturity. Estrogen can also affect prolactin release from the anterior pituitary and affect avian parental care. Results The average number of eggs laid per clutch did not differ between treatments (p = 0.9414). Egg viability per clutch was greatest in the NEG control and BPA- treatment sand lowest in the E2+ and BPA+ treatments (p < 0.0001). Even when viable, only 5% of the chicks that hatched from the +E2 treatment group survived more than 24 hours.   15.87% 68.81% 94.29% 83.33% Viability was determined by candling eggs 5 days after lay. Circulating levels of prolactin (PRL) during the basal and end incubation periods were not significantly different among the E2+, BPA+, and BPA- treatment groups. However, prolactin increased significantly more for the negative controls at the end of incubation (p=0.05). Other aberrant parental behaviors included chick and egg stealing among the +E2 parents. Summary Egg Viability Treatment did not affect egg production, but egg viability was markedly reduced in the E2+and BPA+ groups, suggesting estrogen is adversely affecting males, likely either by reducing sperm count or reducing copulation attempts, and that BPA is having a similar affect. Hatch Weight Offspring in the E2+, BPA+, and BPA- groups had significantly lower hatch weights than those in the NEG control group. Hatchlings rely on body stores for the first days of life before parental feeding begins, and we found most low-birth weight chicks were unable to survive this period (95% in the E2 treatment). Thus, hatch weight is tightly linked to survival. Age and Weight at Fledge At fledge, finches acquire ambulatory and nutritional independence. This coincides with development of adult plumage and a functional HPG axis. Timing of these events is precisely tuned to environmental conditions. Offspring in the +E2, BPA+, and BPA- groups weighed less at fledge than those in NEG control group despite no difference in age at fledge across treatment groups. Within treatment groups, +E2 females fledged earlier than +E2 males and BPA- males fledged earlier than BPA- females. Age and Weight at Reproductive Maturity Offspring in the BPA+ group reached reproductive maturity earlier than those in the +E2 group. Within treatment groups, there was no significant difference in age at reproductive maturity between males and females. Offspring in the BPA+ group weighed less at reproductive maturity than those in +E2, BPA-, and NEG control groups. Parental Care Plasma PRL was lower in all treatments when compared to the NEG control group and some abnormal parental behaviors were observed in the +E2 group. Pre- and post-fledge growth curves suggest that parents in the +E2 and BPA+ group may have fed offspring less, leading to nutritional stress. Following fledging, +E2 chicks were able to make up the lost growth but BPA+ chicks did not. Hatch weight was significantly lower for chicks in the +E2, BPA+, and BPA- groups than the NEG control (p=8.6E-10; p=0.028; p=0.00075, respectively). n=53 n=33 n=39 n=31 a b Fat Cells Study Objectives To determine if breeding zebra finches exposed to estrogen or EDCs differ in their ability to produce viable offspring To determine if body weight or growth differs between offspring of parent zebra finches exposed to EDCs and those not exposed to EDCs To determine if adult exposure to EDCs alters the timing of reproductive maturity in offspring (from onset of fledge to complete acquisition of adult plumage) To determine if EDC exposure has an effect on plasma prolactin (PRL) within the parent generation There was no significant difference in age at fledge across treatment groups. Within treatments, +E2 females fledged significantly earlier than +E2 males (p=0.00719). BPA- males fledged significantly earlier than BPA- females (p=0.0449). Offspring in the BPA+ group reached reproductive maturity significantly earlier than those in the +E2 group (p=0.0215). Within treatment groups, there was no significant difference in age at repro between males and females. Methods n=1 n=11 n=9 n=14 n=1 n=11 n=9 n=14 4 pairs of breeding zebra finches were randomly assigned to receive their daily drinking water from one of four containers: 1. Glass bottle (NEG) 2. BPA-positive (BPA+) plastic bottle 3. BPA-negative (BPA-) plastic bottle 4. Glass bottle, supplemented with estrogen (+E2) n=1 n=14 n=9 n=11 Offspring in the +E2, BPA+, and BPA- groups weighed significantly less at fledge than those in the NEG control (p=5.85E-7; p=0.00001; p=0.00215, respectively). Offspring in the BPA+ group remained significantly lighter at reproductive maturity than those in +E2, BPA-, and NEG control groups (p=0.00011; p=0.00036; and p=0.00066, respectively). Parents (P) breed Chicks Hatch 14 days Chicks Fledge 18-20 days Offspring Reach Reproductive Maturity 70-80 days ½ Offspring Breed (F1) We measured egg viability, chick hatch weight, daily chick weight and tarsus length (TL), and age and weight at fledge and reproductive maturity. Following fledging, juveniles were weighed and measured every 3 days, and adult plumage development was assessed using a spectrometer (Ocean Optics). Parental blood samples were taken before the onset of breeding and again during the last 2 days of incubation and assayed via ELISA for PRL . Estrogen & BPA+ chicks gained weight at a slower rate pre-fledge (p=0.06727) but Estrogen and BPA- chicks gained at a faster rate post-fledge (p=0.169760). Acknowledgements Research was funded, in part, by a DUSCI summer grant to AVL. All procedures were approved by Drake’s IACUC. Many thanks to Liz Stucker, Drake vivarian, for assistance with animal care. Thank you to Kaitie Parsons, Drew Harkins, Kaila Wechsler and Taylor Floyd for assistance with daily measurements, construction of nest boxes and data entry.