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Inefficient calcium utilization in laying hens results in thinner eggshells and increased incidences of skeletal disorders. Thin eggshells are a major cause of egg breakage, resulting in economic loss for the producer and increased susceptibility to food-borne illness to the consumer. Furthermore, skeletal disorders in laying hens are a major animal welfare concern. <P>
We hypothesize that dyssynchrony of biological clocks controlling calcium uptake and utilization promotes inefficient calcium utilization and therefore we seek to improve egg shell quality and bone health in laying hens by optimizing calcium utilization through the manipulation of the endogenous circadian clocks controlling calcium uptake, calcium sequestering and calcium deposition. <P>
We will demonstrate how circadian clocks mediate the effects of estrogen, progesterone and melatonin on calcium metabolism, calcium utilization and calcium deposition in the bones of laying hens, on proper shell gland function, and on the ability of the shell gland to form a proper eggshell matrix. <P>
We will demonstrate the mechanisms by which the circadian clock controls the efficiency of calcium uptake in the intestine. Finally, we will determine the concentration of melatonin in feed required for increasing eggshell thickness and for improving bone health in layers. <P>
Our work will increase our collective knowledge of how calcium is used in laying hens and how biological clocks regulate calcium homeostasis. <P>
The knowledge gained from this work will be used to develop protocols for designed for improving eggshell quality and bone health in laying hens. Specifically, this knowledge will help in the devising of improved protocols for hen house lighting, improved feeding regimens for layers and potential nutraceuticals for improving calcium utilization efficiency.
NON-TECHNICAL SUMMARY: The inability of some laying hens to form a fully protective eggshell is a large economic cost for the egg industry. Cracked eggs are also one of the major causes of food poisoning caused by eggs. Furthermore, mobilization of calcium for the purposes of forming an egg can cause weakened bones in hens over time. Implementing a program to increase egg shell strength would therefore have a tremendous monetary impact as well as significance for public health and animal welfare. We seek to improve the regulation of calcium in laying hens in order to increase the thickness and strength of their eggshells while simultaneously reducing the incidences of weakened bones and skeletal defects during and after the lay period. Our efforts will focus on determining how biological clocks influence the ability of laying hens to use calcium most efficiently by examining how biological clocks and the hormones they regulate influence calcium uptake within the digestive system, control calcium mobilization at different times of the day and influence the deposition of calcium into bone and eggshell. Our findings will provide increased knowledge about how eggshell formation is regulated in laying hens and increased knowledge about calcium is used. We will use this knowledge to develop novel lighting regimens for hen houses, improved feeding protocols for layers and potential feed additives designed for improving calcium utilization efficiency.
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APPROACH: This project seeks to identify the neural and hormonal mechanisms by which the circadian clock controls calcium homeostasis in order to improve egg shell strength and increase bone integrity. Because many physical properties within bone and shell gland are influenced by ovarian steroids, dysynchrony of the ovary with the rest of the body likely affects the normal calcium utilization in bone and the shell gland. We will directly examine how synergism between time of day effects and the hormonal signals from the pineal gland (melatonin) and the ovary (estrogen and progesterone) influence calcium transport and utilization by documenting their abilities to affect calcium uptake and calcium deposition at different times of the day in medullary bone and shell gland. Calcium uptake and deposition will be measured using a radioactive calcium tracer. We will also examine the ability of these hormones to directly activate transcription of clock genes by employing a firefly luciferase reporter assay. Because most calcium in laying hens is absorbed in the duodenum, we will elucidate the mechanisms by which the circadian clock controls calcium uptake in the intestine. This will be achieved by directly manipulating the molecular circadian clock in duodenal tissue by transfecting with expression vectors containing clock gene constructs or by "knocking down" clock gene expression with small interfering RNA's and then measuring the amount of radioactive calcium absorbed by duodenal tissue. We will also examine the effects of melatonin on calcium uptake in the duodenum using similar in vitro methods. Finally, we will determine the effects of melatonin on egg shell thickness and bone health in laying hens by adding melatonin to their feed. Eggshell thickness and calcium content will be measured and bone integrity determined by measuring mineral content after ashing. Knowledge gained from these experiments will be used to develop new feeding and lighting protocols for layers to maximize calcium uptake from feed, maximize calcium deposition in eggshell matrix and most efficiently influences calcium mobilization from internal stores.