Item No. 1 of 1

INVESTIGATOR: Lemley, C.

PERFORMING INSTITUTION:
MISSISSIPPI STATE UNIV
MISSISSIPPI STATE, MISSISSIPPI 39762

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

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 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.

OBJECTIVES: 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. 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. Currently, we have completed the majority of the animal work for this USDA transfer project and we are in the process of analyzing samples via bioassays and histological procedures. We observed an increase in umbilical artery blood flow, fetal aorta blood flow, and fetal growth parameters following melatonin infusion during pregnancy (objective 2). 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). The melatonin receptor mediated alterations in umbilical artery blood flow and fetal descending aorta blood flow may be dependent on increasing nitric oxide bioavailability; therefore, we are in the process of analyzing total nitrites (an indicator of nitric oxide production) in the placental vascular bed. For the remainder of this USDA transfer grant we will be analyzing melatonin receptor 1 and melatonin receptor 2 protein expression as well as immunolocalization of melatonin receptors in the placentome, caruncular (maternal placentome) artery, cotyledonary (fetal placentome) artery, and umbilical artery. We anticipate a greater expression of melatonin receptor 2 in the fetal portion of placenta compared to the maternal portion of the placenta. In addition, melatonin receptors will be localized to both endothelial cells and vascular smooth muscle cells of the caruncular, cotyledonary, and umbilical artery. 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.

APPROACH: The majority of animal work for this USDA transfer grant has been completed. The scientific approach describes both completed and ongoing research for both objectives. For objective 1, Melatonin supplementation and nutritional plane treatments began on day 50 of gestation. Ewes were supplemented with 5 mg of melatonin (MEL) or no melatonin (CON) and provided 100% (ADQ; adequate nutrition) or 60% (RES; restricted diet) of nutrient recommendations for the remainder of the experiment (2 x 2 factorial design). The primary output for this experiment was fetal size, which was not different at day 90 of gestation (mid-gestation) based on ultrasonography examination. When treatments (melatonin or nutrient restriction) were allowed to progress to day 130 of gestation (late gestation) we observed alterations in fetal size amongst treatment groups. At day 130 of gestation fetal weight was decreased in nutrient restricted dams compared to adequate fed dams. Moreover, adequate fed dams (100% of nutrient recommendations) supplemented with melatonin showed an increased fetal weight, fetal ponderal index, and fetal abdominal girth compared to the adequate fed dams with no melatonin supplementation. Therefore, melatonin supplementation did not rescue fetal growth in nutrient restricted dams, but caused an overgrowth of the fetuses in adequate fed dams. This overgrowth was partially attributed to an increase in umbilical artery blood flow in dams supplemented with melatonin. The mechanisms behind this pathway are currently being examined using the experimental design for objective 2 (see below). Objective 2 Approach: Beginning on day 62 of gestation, the uterus of adequate fed ewes was fitted with Alzet mini osmotic pumps attached to a catheter and infused with vehicle (control), melatonin, or luzindole (melatonin receptor 1 and 2 antagonist). Osmotic pumps delivered treatments chronically for a duration of 4 weeks. The following experiment allowed us to determine uteroplacental blood flow and placental and fetal development during chronic melatonin and/or melatonin receptor antagonist infusion. Therefore, direct effects of placental melatonin receptors on gestational performance will be examined. So far we have 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 luzindole, decreased umbilical artery blood flow and fetal descending aorta blood flow (or fetal cardiac output). Currently we are examining placental melatonin receptor 1 and 2 protein expression at day 90 of gestation following the 4 week chronic infusions. In addition, we are optimizing procedures to localize melatonin receptor 1 and 2 in the placental vascular bed, including the caruncular, cotyledonary, and umbilical arteries. This will allow us to determine both expression patterns and localization following the chronic 4-week infusions of melatonin or luzindole.

PROGRESS: 2012/10 TO 2014/04
Target Audience: The major target audiences include academic and industry personnel involved with advancements in animal sciences, physiology, reproductive biology, and endocrinology. In addition, both undergraduate and graduate students in the agriculture and life sciencesdisciplineserveas a target audience. Research data have been presented at local workshops targeting animal agriculture producers in Mississippi as well as nationalscientific meetings, such as the Joint Annual Meeting with the American Society of Animal Science held in Indianapolis, IN (2013 meeting) and Kansas City, MO (2014 meeting). Research has been disseminated through formal classroom instruction at Mississippi State University, including ADS/PHY 8243 “Advanced Physiology of Reproduction”. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Several collaborators and their graduate/undergraduate studentshave contributed to this project: Drs. Kimberly Vonnahme, Kendall Swanson, Joel Caton, Dale Redmer, Larry Reynolds, Steve O'Rourke, Allison Meyer, Mathew Wilson, and Dennis Hallford. During the project period the following graduate/undergraduate students have presented data directly related to the project experimental design and/or treatments: L.E. Camacho, F.E. Doscher, L.D. Prezotto, K.E. Brockus, A.W. Eifert,L.J. Grossner,M.A. Lein, and E.A. Nere. How have the results been disseminated to communities of interest? Results have been disseminated as peer-reviewed publications. In addition, the results have been presented at several local producer events, universities, regional meetings (Midwest Section of ASAS), and national (JAM ASAS/ADSA) scientific conferences. The PI did attend a NIFA Fellows Project Directors (PD) meeting in August of 2012. The PD meeting was cancelled for 2013; however, the PD has presented several of the results at National meetings in both 2013 and 2014. The PD has decided to present the results at a national scientific conference (JAM ASAS/ADSA meeting) instead of attenting the 2015 NIFA Fellows Project Directors meeting in Washington, DC. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

IMPACT: 2012/10 TO 2014/04
What was accomplished under these goals? Objective 1: Determine uteroplacental blood flow and fetal growth during mid-gestation in undernourished and control ewes supplemented with or without melatonin. 1.) We have completed an experiment examining umbilical blood flow and fetal growthduring melatonin supplementation as a 2 x 2 factorial design. At day 50 of gestation, 32 ewes were supplemented with 5 mg of melatonin or no melatonin and were allocated to receive 100% or 60% of nutrient requirements until day 130 of gestation. 2.) Umbilical blood flow and fetal growth were determined every 10 days using Doppler ultrasonography. On day 130 of gestation all ewes underwent a terminal surgery to examine amino acid uteroplacental uptake. 3.) Dietary melatonin supplementation increased umbilical artery blood flow by 20% compared to no melatonin groups. Dietary nutrient restriction decreased umbilical artery blood flow by 20% compared to adequate fed ewes. As anticipated nutrient restriction decreased fetal weight; however, melatonin supplementation did not rescue fetal weight compared to adequately fed ewes. Interestingly, fetal uptake of branched chain amino acids during late gestation was decreased by maternal nutrient restriction and rescued to the level of control ewes by supplementingdietary melatonin. In general melatonin supplementation did not impact maternaluterine nutrient uptake; however, fetal uptake of nutrients was increased, which is primarily a result of the increase in umbilical artery blood flow. 4.) A key outcome from the current objectives was the consistent increase in umbilical artery blood flow following dietary melatonin supplementation. Moreover, compromised pregnancies experiencing a decrease in nutrient exchange to the fetus or a decrease in placental blood perfusion could be rescued by supplementing the dam with dietary melatonin. In addition, melatonin is an inexpensive supplement that could easily be supplied during specific time periods of gestation. 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. 1.) We have completed an experiment examining ovine placental hemodynamics during mid-gestation following a 4 week uterine infusion of melatonin (n = 5), luzindole (a melatonin receptor antagonist; n = 5), or vehicle control (n =4).Multiparous singleton pregnant ewes were surgically implanted with mini osmotic pumps on day 62 of gestation. Pumps and catheters were sutured under perimetrium and catheterswere advanced to the gravid uterine vascular network of the mesometrial region. 2.) On day 90 of gestation fetal blood flow was determined and animals were slaughtered for tissue collection. 3.) After 4 weeks of infusion umbilical artery blood flow and umbilical artery blood flow relative to placentome weight were increased (P < 0.05) in MEL versus CON and LUZ infused dams. Fetal descending aorta blood flow was increased (P < 0.05) in MEL versus CON and LUZ infused dams, while fetal descending aorta blood flow relative to fetal weight was increased in MEL versus CON and decreased in LUZ versus CON infused dams. Following the 4 week infusion we observed an increase in placental efficiency (fetal weight: placentome weight ratio) in MEL versus LUZ infused dams. 4.) This is the first study examining endocrine mediators of placental function in vivo by surgically implanting mini-osmotic pumps directly to the gravid uterine vasculature. Therefore, a major outcome to this proposal was the development of a feasible technique to examine in vivo placental functional capacity. Moreover, this experiment determined the increase in umbilical artery blood flow due to chronic uterine melatonin infusion is potentiated by an increased fetal cardiac output through the descending aorta. Melatonin receptor antagonism decreased fetal descending aorta blood flow relative to fetal weight. Therefore, melatonin receptor activation partially mediates the observed increase in fetal blood flow following dietary melatonin supplementation.

PUBLICATIONS (not previously reported): 2012/10 TO 2014/04
1. Type: Journal Articles Status: Accepted Year Published: 2014 Citation: Shukla, P., C. O. Lemley, N. Dubey, A. M. Meyer, S. T. O?Rourke, and K. A. Vonnahme. 2014. Effect of maternal nutrient restriction and melatonin supplementation from mid to late gestation on vascular reactivity of maternal and fetal placental arteries. Placenta. 35:461-466.
2. Type: Journal Articles Status: Accepted Year Published: 2014 Citation: Shukla, P., S. Ghatta, N. Dubey, C. O. Lemley, M. L. Johnson, A. Modgil, K. A. Vonnahme, J. S. Caton, L. P. Reynolds, C. Sun, and S. T. O?Rourke. 2014. Maternal nutrient restriction during pregnancy impairs an endothelium-derived hyperpolarizing factor-like pathway in sheep fetal coronary arteries. American Journal of Physiology ? Heart and Circulatory Physiology. (DOI: 10.1152/ajpheart.00595.2013).
3. Type: Journal Articles Status: Accepted Year Published: 2014 Citation: Prezotto, L. D., C. O. Lemley, L. E. Camacho, F. E. Doscher, A. M. Meyer, J. S. Caton, K. A. Vonnahme, K. C. Swanson. 2014. Effects of nutrient restriction and melatonin supplementation on maternal and fetal hepatic and small intestinal energy utilization. Journal of Animal Physiology and Animal Nutrition. (DOI: 10.1111/jpn.12142).
4. Type: Journal Articles Status: Under Review Year Published: 2014 Citation: Eifert, A. W., M. E. Wilson, K. A. Vonnahme, P. P. Borowicz, D. A. Redmer, S. Romero, S. Dorsam, J. Haring, and C. O. Lemley. 2014. Effect of melatonin or maternal nutrient restriction on vascularity and cell proliferation in the ovine placenta. Journal of Reproduction and Development. (Submitted).
5. Type: Conference Papers and Presentations Status: Accepted Year Published: 2014 Citation: Vonnahme, K. A., M. E. Wilson, S. T. Dorsam, J. Harring, P. P. Borowicz, D. A. Redmer, and C. O. Lemley. 2014. Effect of melatonin (MEL) or maternal nutrient restriction on vascularity of the ovine placenta. Journal of Animal Science. (Accepted).

PROGRESS: 2012/10/15 TO 2013/10/14
Target Audience: Academic and industry personnel involved with advancements in animal sciences, physiology, reproductive biology, and endocrinology. In addition, both undergraduate and graduate students in the agriculture and life sciencesdisciplineserveas a target audience. Research data has been presented at local workshops targeting animal agriculture producers in Mississippi as well as nationalprofessional societies such as the American Society of Animal Science. Research has been disseminated through formal classroom instruction at Mississippi State University, including ADS/PHY 8243 “Advanced Physiology of Reproduction”. Moreover, research results were disseminated through the Mississippi State University“Farm and Family Radio Program”. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Several collaborators and their graduate/undergraduate studentshave contributed to this project: Drs. Kimberly Vonnahme, Kendall Swanson, Joel Caton, Dale Redmer, Larry Reynolds, Steve O'Rourke, Allison Meyer, Mathew Wilson, and Dennis Hallford. During the project period the following graduate/undergraduate students have presented data directly related to the project experimental design and/or treatments: L.E. Camacho, F.E. Doscher, L.D. Prezotto, K.E. Brockus, A.W. Eifert,L.J. Grossner,M.A. Lein, and E.A. Nere. How have the results been disseminated to communities of interest? Results have been disseminated as peer-reviewed publications. In addition, the results have been presented at several local events, universities, regional meetings, and national scientific conferences. What do you plan to do during the next reporting period to accomplish the goals? We are currently examining the steroid hormone flux across the uteroplacenta from melatonin treated and nutrient restricted dams. This project is nearing completion and will be submitted for publication in 2014. In addition, we are examining in vitro melatonin receptor dependent modulation of nutrient uptake using a choriocarcinoma cell-line as well as primary placental cell cultures. These experiments will help solidify melatonin's role in modulating placental functional capacity.

IMPACT: 2012/10/15 TO 2013/10/14
What was accomplished under these goals? The incidence of morbidity and mortality is increased during the neonatal period in offspring of low birth weight, while surviving low birth weight offspring are associated with poor growth performance and lower daily rates of gross energy accretion. Lamb mortality rate can increase as much as 20 to 50% when birth weights are below average. Several factors contribute to lamb survivability and low birth weights could account for approximately 5% of total lamb loss rate. In the U.S., which has nearly 5.5 million sheep, we could expect an annual loss of nearly 275,000 lambs due to low birth weights. Assuming 30 to 40 lbs of meat per lamb harvested, we could extrapolate a total annual loss of nearly 10 million lbs of meat due to low birth weights alone. The long-term goal is to determine the possible mechanisms that dietary supplements, particularly melatonin, may modify placental function and fetal development. Moreover, we are examining the potential mechanisms behind these melatonin mediated responses using an ovine compromised pregnancy model. Our major findings includeblood flow dataat both mid- and late gestation following melatonin supplementation. For example, dietary melatonin supplementation consistently increases fetal sheep umbilical blood flow by approximately 20% compared to non-supplemented controls. This increase in umbilical blood flow alters amino acid uptake by theplacenta resulting in disproportionatefetal growth during lategestation.Moreover, these responses have been determined to be partially melatonin receptor mediated. Objective 1: Determine uteroplacental blood flow and fetal growth during mid-gestation in undernourished and control ewes supplemented with or without melatonin. 1.) We have completed an experiment examining umbilical blood flow and fetal growthduring melatonin supplementation as a 2 x 2 factorial design. At day 50 of gestation, 32 ewes were supplemented with 5 mg of melatonin or no melatonin and were allocated to receive 100% or 60% of nutrient requirements until day 130 of gestation. 2.) Umbilical blood flow and fetal growth were determined every 10 days using Doppler ultrasonography. On day 130 of gestation all ewes underwent a terminal surgery to examine amino acid uteroplacental uptake. 3.) Dietary melatonin supplementation increased umbilical artery blood flow by 20% compared to no melatonin groups. Dietary nutrient restriction decreased umbilical artery blood flow by 20% compared to adequate fed ewes. As anticipated nutrient restriction decreased fetal weight; however, melatonin supplementation did not rescue fetal weight compared to adequately fed ewes. Interestingly, fetal uptake of branched chain amino acids during late gestation was decreased by maternal nutrient restriction and rescued to the level of control ewes by supplementingdietary melatonin. In general melatonin supplementation did not impact maternaluterine nutrient uptake; however, fetal uptake of nutrients was increased, which is primarily a result of the increase in umbilical artery blood flow. 4.) A key outcome from the current objectives was the consistent increase in umbilical artery blood flow following dietary melatonin supplementation. Moreover, compromised pregnancies experiencing a decrease in nutrient exchange to the fetus or a decrease in placental blood perfusion could be rescued by supplementing the dam with dietary melatonin. In addition, melatonin is an inexpensive supplement that could easily be supplied during specific time periods of gestation. 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. 1.) We have completed an experiment examining ovine placental hemodynamics during mid-gestation following a 4 week uterine infusion of melatonin (n = 5), luzindole (a melatonin receptor antagonist; n = 5), or vehicle control (n =4).Multiparous singleton pregnant ewes were surgically implanted with mini osmotic pumps on day 62 of gestation. Pumps and catheters were sutured under perimetrium and catheterswere advanced to the gravid uterine vascular network of the mesometrial region. 2.) On day 90 of gestation fetal blood flow was determined and animals were slaughtered for tissue collection. 3.) After 4 weeks of infusion umbilical artery blood flow and umbilical artery blood flow relative to placentome weight were increased (P < 0.05) in MEL versus CON and LUZ infused dams. Fetal descending aorta blood flow was increased (P < 0.05) in MEL versus CON and LUZ infused dams, while fetal descending aorta blood flow relative to fetal weight was increased in MEL versus CON and decreased in LUZ versus CON infused dams. Following the 4 week infusion we observed an increase in placental efficiency (fetal weight: placentome weight ratio) in MEL versus LUZ infused dams. 4.) This is the first study examining endocrine mediators of placental function in vivo by surgically implanting mini-osmotic pumps directly to the gravid uterine vasculature. Therefore, a major outcome to this proposal was the development of a feasible technique to examine in vivo placental functional capacity. Moreover, this experiment determined the increase in umbilical artery blood flow due to chronic uterine melatonin infusion is potentiated by an increased fetal cardiac output through the descending aorta. Melatonin receptor antagonism decreased fetal descending aorta blood flow relative to fetal weight. Therefore, melatonin receptor activation partially mediates the observed increase in fetal blood flow following dietary melatonin supplementation.

PUBLICATIONS: 2012/10/15 TO 2013/10/14
1. Type: Journal Articles Status: Accepted Year Published: 2013 Citation: Vonnahme, K. A., C. O. Lemley, P. Shukla, and S. T. O?Rourke. 2013. 2011 AND 2012 EARLY CAREERS ACHIEVEMENT AWARDS: Placental programming: How the maternal environment can impact placental function. Journal of Animal Science. 91:2467-2480.
2. Type: Conference Papers and Presentations Status: Accepted Year Published: 2013 Citation: Reynolds, L. P., J. S. Caton, P. P. Borowicz, K. A. Vonnahme, C. O. Lemley, and A. J. Conley. 2013. Maternal melatonin treatment alters expression of fetal hypothalamic 5alpha-reductase type 1. Journal of Pediatric Research. (accepted).
3. Type: Conference Papers and Presentations Status: Accepted Year Published: 2013 Citation: Brockus, K. E., L. E. Camacho, K. A. Vonnahme, and C. O. Lemley. 2013. Chronic uterine infusion of melatonin or melatonin receptor antagonist during mid-gestation alters ovine placental nitrites and superoxide dismutase activity. Journal of Animal Science. 91 (Supplement 2):T344.
4. Type: Conference Papers and Presentations Status: Accepted Year Published: 2013 Citation: Grossner, L. J., L. E. Camacho, D. M. Hallford, K. A. Vonnahme, and C. O. Lemley. 2013. Uterine flux of estrone sulfate in an ovine maternal nutrient restriction model during melatonin supplementation. Journal of Animal Science. 91 (Supplement 2):W174.
5. Type: Journal Articles Status: Accepted Year Published: 2013 Citation: Lemley, C. O., L. E. Camacho, and K. A. Vonnahme. 2013. Uterine infusion of melatonin or melatonin receptor antagonist alters ovine feto-placental hemodynamics during mid-gestation. Biology of Reproduction. 89:40, 1-9.
6. Type: Journal Articles Status: Accepted Year Published: 2013 Citation: Lemley, C. O., L. E. Camacho, A. M. Meyer, M. Kapphahn, J. S. Caton, and K. A. Vonnahme. 2013. Dietary melatonin supplementation alters uteroplacental amino acid flux during intrauterine growth restriction in ewes. Animal. 7:1500-1507.
7. Type: Journal Articles Status: Accepted Year Published: 2013 Citation: Reynolds, L. P., K. A. Vonnahme, C. O. Lemley, D. A. Redmer, A. T. Grazul-Bilska, P. P. Borowicz, and J. S. Caton. 2013. INVITED REVIEW. Maternal stress and placental vascular function and remodeling. Current Vascular Pharmacology. Hot topic issue ?Uteroplacental Circulation and Fetal Vascular Function and Development?. L. Zhang (ed.). 11:564-593.
8. Type: Conference Papers and Presentations Status: Accepted Year Published: 2013 Citation: Eifert, A. W., M. E. Wilson, K. A. Vonnahme, P. P. Borowicz, D. A. Redmer, S. T. Dorsam, J. Harring, C. O. Lemley. 2013. Effect of melatonin (MEL) or maternal nutrient restriction on cell proliferation in the ovine placenta. Journal of Animal Science. 91 (Supplement 2):P106.
9. Type: Conference Papers and Presentations Status: Accepted Year Published: 2013 Citation: Doscher, F. E., C. O. Lemley, L. E. Camacho, P. Borowicz, J. S. Caton, A. M. Meyer, K. A. Vonnahme, and K. C. Swanson. 2013. Influence of nutrient restriction and melatonin supplementation of pregnant ewes on maternal and fetal pancreatic beta-cell morphology. Journal of Animal Science. 91 (Supplement 2):P139.