ROLE OF MELATONIN DURING MID-GESTATION ON FETAL GROWTH IN SHEEP

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 0226099
• Grant No.: 2011-67012-30683
• Cumulative Award Amt.: $130,000.00
• Proposal No.: 2010-05261
• Project Start Date: Sep 1, 2011
• Project End Date: Aug 31, 2013
• Grant Year: 2011
• Program Code: [A7201]- AFRI Post Doctoral Fellowships

Recipient Organization
NORTH DAKOTA STATE UNIV
1310 BOLLEY DR
FARGO,ND 58105-5750

Performing Department
Animal Sciences

Non Technical Summary
Mortality is increased in offspring of low birth weight, while surviving low birth weight offspring are associated with poor growth performance. Therefore, increasing fetal growth during gestation can decrease lamb mortality rates and improve offspring performance. The relationship between birth weight and offspring performance has a substantial impact on food production not only in the U.S. but other developing countries which rely heavily on sheep and goat production. The placenta is involved in transporting wastes and nutrients to the growing fetus; therefore, therapeutic supplements designed to improve blood flow to the placenta can have a direct impact on fetal growth and development. This project will examine the effect of melatonin supplementation on uterine and umbilical blood flow, both of which play an important role in supplying oxygen and nutrients to the growing fetus. We anticipate that melatonin will increase uterine and umbilical blood flow and improve fetal growth and development. In addition, we will evaluate the mechanism behind these melatonin mediated pathways, by chronically infusing pregnant sheep with a compound that blocks melatonin receptors. We anticipate that blocking melatonin receptors will result in decreased uterine and umbilical blood flow and fetal growth. The findings from this project will allow for the design and implementation of possible interventions that could be used to improve birth weights. In addition, novel research strategies could be developed to study placental and fetal development.

Animal Health Component

25%

Research Effort Categories

Basic

75%

Applied

25%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
301	3610	1020	80%
301	3610	1050	20%

Knowledge Area
301 - Reproductive Performance of Animals;

Subject Of Investigation
3610 - Sheep, live animal;

Field Of Science
1020 - Physiology; 1050 - Developmental biology;

Keywords

fetal growth

melatonin

placenta

pregnancy

sheep

umbilical cord

uterine artery

Goals / Objectives
The long-term goal is to determine the possible mechanisms that dietary supplements, particularly melatonin, may modify placental function and fetal development. This proposal is innovative because dietary melatonin supplementation during mid-gestation in sheep and the resultant fetal growth has not been determined. Recent evidence in nutrient restricted rats supplemented with melatonin showed an increase in birth weights compared to their respective controls. Moreover, low melatonin concentrations have been associated with an increased incidence of spontaneous abortions, which further exemplifies the need for research in this field. Interestingly, we identified melatonin receptor protein expression in the sheep placenta; however, a paucity of information exists on the role of melatonin in placental and fetal development. Therefore, the central hypothesis is that melatonin supplementation during mid-gestation will improve uteroplacental blood flow, placental efficiency and fetal growth. In addition, we believe these melatonin responses are receptor mediated. The hypothesis will be addressed through the specific objectives, which are to: Objective 1: Determine uteroplacental blood flow and fetal growth during mid-gestation in undernourished and control ewes supplemented with or without melatonin. Objective 2: Determine uteroplacental blood flow and fetal growth following chronic in vivo uterine artery infusions of vehicle, melatonin or melatonin receptor antagonist during mid-gestation. The specific outputs for the project include: conducting the animal experiments, analyzing data and disseminating research findings at national meetings and through peer-review publications. Moreover, as a USDA Fellow, the PI will seek strong research collaborations as well as assisting with the training of undergraduate and graduate students in Animal Science. Therefore, these outputs will be focused on establishing research independence and teaching credentials, which are main priorities of the Fellowship Program.

Project Methods
Objective 1 Approach: On day 50 of gestation, singleton pregnant ewes will be assigned randomly to one of 4 dietary treatments in a 2 x 2 factorial design: nutrient restricted to 60% of requirements or receive 100% of their nutrient requirements and ewes will be supplemented with or without (controls) 5 mg of melatonin per day (absorbed onto a feed pellet). Maternal blood samples will be collected throughout gestation to determine melatonin concentrations. Umbilical blood flow will be determined at day 50, 60, 70 and 90 of gestation by color Doppler ultrasonography. On day 90 of gestation ewes will be anesthetized and uterine blood flow will be determined. Blood samples will be collected from the uterine artery and fetus for determination of melatonin concentrations. Following uterine blood flow measurements ewes will be euthanized for tissue collection. Placentome weight, fetal weight, and fetal liver, perirental fat, heart and right ventricle, left ventricle and septum weights will be determined. Maternal and fetal placental samples as well as perirenal adipose tissue will be snap frozen for later determination of melatonin receptor 1 and 2 protein expression. In addition, placental tissue will be fixed and used to localize melatonin receptors at day 90 of gestation using immunofluorescence. This experiment is intended to determine whether feeding melatonin during a short timeframe of gestation is an applicable approach to increasing fetal growth. Therefore, a primary output for evaluating the success of this project will be fetal weight and development. Objective 2 Approach: Beginning on day 62 of gestation, the uterine artery of adequate fed ewes will be fitted with Alzet mini osmotic pumps attached to a catheter and infused with vehicle (ethanol), melatonin, luzindole (melatonin receptor 1 and 2 antagonist) or a combination of both melatonin and luzindole (4 treatments). Osmotic pumps will deliver treatments chronically for duration of 4 weeks. Moreover, we are unaware of any chronic 4 week infusions of vasodilators or vasoconstrictors into the uterine artery with mini osmotic pumps. Thus, the current proposal utilizes a novel technique. Uterine and umbilical blood flow and fetal growth will be monitored weekly using color Doppler ultrasonography. Following blood flow measurements ewes will be euthanized with an overdose of sodium pentobarbital on day 90 of gestation. Placental weight, fetal weight and fetal organ weight will be recorded to assess intrauterine growth restriction following the 4 week chronic infusions. The following experiment will allow us to determine uteroplacental blood flow and placental and fetal development during chronic melatonin and/or melatonin receptor antagonist infusion. Therefore, direct effects of melatonin on gestational performance will be examined.

Progress 09/01/11 to 08/31/13

Outputs
OUTPUTS: To date the PI has completed the animal work and animal surgery portion of the experiments. For specific aim 2 the PI developed a novel surgical procedure to implant mini-osmotic infusion pumps directly into the pregnant uterus of sheep during mid-gestation. This allowed us to monitor fetal growth and placental blood flow via Doppler ultrasonography after chronic hormone infusions. In addition to the conduct and analysis of experiments, the PI gained valuable experience as the principal instructor for the undergraduate reproductive physiology course at NDSU during the spring semester of 2012. This research and teaching experience allowed the PI to apply for an Assistant Professor Faculty position (65% research, 35% teaching appointment) at Mississippi State University. Since then the PI has successfully negotiated and accepted an offer from Mississippi State University as an Assistant Professor of Reproductive Physiology. Over the last year the PI has attended 4 scientific conferences and given three invited research talks. These invited talks were at the IETS meeting in Phoenix, AZ (January 9, 2012), the ASAS Midwest meeting in Des Moines, IA (March 20, 2012), and the NDSU Summer Research Forum in Fargo, ND (May 15, 2012). In addition, the PI attended the Joint Annual Meeting for ASAS/ADSA at Phoenix, AZ (July 17, 2012; session titled, Physiology & Endocrinology: Pregnancy) and presented his latest research from the current USDA grant. Abstracts for these talks are listed under publications. During this project the PI has interacted with several undergraduate and graduate students. In addition, the PI advised an undergraduate student (Mattia Lein) on a research project examining glucose flux across the uteroplacenta unit. Ms. Lein presented her research at the Midwest Meeting in Des Moines, IA (March 20, 2012; abstract listed under publications). Several new collaborations were fostered during the PI's time at North Dakota State University. Currently the PI is working on several manuscripts with not only his mentor, Dr. Kim Vonnahme, but also Drs. Steve O'Rourke, Dale Redmer, Kendall Swanson, and Allison Meyer. Apart from these collaborations we have also successfully implemented a new surgical technique to examine uteroplacental blood flow following chronic hormone infusions. This new technique will hopefully foster more collaborations in the near future. PARTICIPANTS: Dr. Caleb Lemley is the principal investigator for this USDA postdoctoral fellowship grant. He conducted the experiments, analyzed data, and prepared abstracts and manuscripts for dissemination of said data. The PI is mentored by Dr. Kim Vonnahme and she was responsible for assisting with the day to day functions of the project including assistance with fostering outside collaborations. Several tissues were collected from the current project and used for collaborative projects. Dr. Steve O'Rourke served as a collaborator and assisted with placental vascular reactivity studies. Dr. Dale Redmer collected placentomes from the current project and is examining placental vascularity. Dr. Kendall Swanson collected maternal and fetal pancreas for examination of exocrine pancreatic function and Dr. Allison Meyer examined oxygen consumption from the liver and small intestines of both the dam and fetus. In addition to these collaborations, the PI assisted with the training of several graduate students and undergraduate student workers during these experiments. Graduate student L.E. Camacho and undergraduate student M.A. Lein presented abstracts at the Midwest ASAS meeting in Des Moines, IA in March of 2012. These projects examined either steroid or glucose flux across the uteroplacenta unit after supplementing dams with melatonin. TARGET AUDIENCES: Data from this grant proposal was presented at 4 separate scientific conferences in 2012. These presentations were given at the International Embryo Transfer Society meeting in Phoenix, AZ (January 2012), the American Society of Animal Science Midwest meeting in Des Moines, IA (March 2012), the NDSU Summer Research Forum in Fargo, ND (May 2012), and the Joint Annual Meeting for ASAS/ADSA at Phoenix, AZ (July 2012). PROJECT MODIFICATIONS: The major change to this project is that the PI (Dr. Lemley) has accepted an Assistant Professor position at Mississippi State University. Dr. Lemley is in the process of transferring this postdoctoral fellowship to Mississippi State University. This may delay a portion of the laboratory analysis as well as the time to publication (due to publication costs) until the transfer of funds are complete. Beyond this transfer no other project modifications have occurred.

Impacts
The long-term goal is to determine the possible mechanisms that dietary supplements, particularly melatonin, may modify placental function and fetal development. Therefore, the central hypothesis is that melatonin supplementation during mid-gestation will improve uteroplacental blood flow, placental efficiency (fetal weight to placental weight ratio) and fetal growth. In addition, we are testing whether these responses are melatonin receptor mediated. Currently, we have completed the majority of animal work for the project and we are in the process of analyzing samples via bioassays and histological procedures. The objectives of the current project were to determine uteroplacental blood flow and fetal growth following chronic, 4 week, in vivo uterine infusions of vehicle, melatonin or melatonin receptor antagonist (luzindole) from day 62 to day 90 of gestation. We observed an increase in umbilical artery blood flow, fetal aorta blood flow, and fetal growth parameters following melatonin infusion during pregnancy. Blocking placental melatonin receptors during mid-gestation, by infusing a melatonin receptor antagonist (luzindole), decreased umbilical artery blood flow and fetal descending aorta blood flow (or fetal cardiac output). Placentome weight and dimensions (length, width, and depth) were similar across all infusion treatment groups. Therefore, the increased fetal abdominal girth and placental efficiency in melatonin infused versus luzindole infused ewes is more than likely due to altered placental blood perfusion and/or placental nutrient uptake. The melatonin mediated alterations in umbilical artery blood flow and fetal descending aorta blood flow may be dependent on increasing nitric oxide bioavailability. Total nitrites (an indicator of nitric oxide production) were increased in umbilical cord serum of both melatonin and luzindole infused animals compared to controls. Therefore, the alterations in umbilical artery blood flow may be independent of nitric oxide pathways. Infusing melatonin into the uterus increased Mn superoxide dismutase (SOD) activity in the caruncle (maternal portion of the placental) compared to both luzindole and control infused ewes. This alteration to the placental antioxidant enzyme (SOD) activity may potentially aid in improving placental oxidative status; however it does not fully explain the alterations to fetal blood flow during luzindole infusions. This relationship describes a novel pathway, whereby melatonin may partially modulate uteroplacental blood flow and fetal development through placental melatonin receptors. In addition to the results, we have described a new experimental technique which will allow researchers to design future experiments on identifying placental vasomediators during pregnancy. The findings from this project will allow for the design and implementation of possible interventions that could be used to improve birth weights and potentially offspring performance.

Publications

• Camacho, L.E., C.O. Lemley, A.M. Meyer, D.M. Hallford, and K.A. Vonnahme. 2012. Uteroplacental steroid flux in an ovine maternal nutrient restriction model during melatonin supplementation. Journal of Animal Science. 90 (supplement 2): abstract 89.
• Lemley, C.O., L.E. Camacho, and K.A. Vonnahme. 2012. Chronic uterine infusion of melatonin or melatonin receptor antagonist alters ovine placental efficiency and fetal blood flow during mid-gestation. Journal of Animal Science. 90 (supplement 3): abstract 522.
• Lemley, C.O., L.E. Camacho, C.A. Zimprich, L.A. Lekatz, J.S. Caton, P. Shukla, S.T. O'Rourke, and K.A. Vonnahme. 2012. Maternal nutrition and dietary supplements that impact uteroplacental blood flow and fetal development. Journal of Animal Science. 90 (supplement 2): abstract 177.
• Lemley, C.O., L.E. Camacho, A.M. Meyer, M. Kapphahn, J.S. Caton, and K.A. Vonnahme. 2012. Uteroplacental flux of amino acids (AA) in adequately fed or nutrient restricted ewes supplemented with melatonin. Journal of Animal Science. 90 (supplement 2): abstract 90.
• Lein M.A., C.O. Lemley, L.E. Camacho, A.M. Meyer, and K.A. Vonnahme. 2012. Uteroplacental uptake of glucose and fetal liver glycogen in adequately fed or nutrient restricted ewes supplemented with melatonin. Journal of Animal Science. 90 (supplement 2): abstract 47.
• Meyer, A.M., K.C. Swanson, B.J. Awda, M. Kapphahn, R.D. Yunusova, L.E. Camacho, J.S. Caton, K.A. Vonnahme, and C.O. Lemley. 2012. Effects of nutrient restriction and melatonin supplementation during mid- to late gestation on ewe and fetal lamb small intestinal and hepatic in vitro oxygen (O2) consumption. Journal of Animal Science. 90 (supplement 2): abstract 176
• Swanson, K.C., A.M. Meyer, C.O. Lemley, J.S. Caton, and K.A. Vonnahme. 2012. Influence of nutrient restriction and melatonin supplementation of pregnant ewes on maternal and fetal pancreatic α-amylase and trypsin activity. Journal of Animal Science. 90 (supplement 2): abstract 315P.