Performing Department
(N/A)
Non Technical Summary
Eggs are an affordable, high-quality protein source produced by the commercial laying hen industry. Like all sectors of agriculture, this industry faces challenges surrounding ways to increase food production to meet the needs of a growing global population in an environmentally sustainable and economically viable manner. One way the laying hen industry has tried to meet this challenge is by extending flock production cycles to increase the total number of eggs laid per hen from about 320 at 72 weeks of age to 500 at 100 weeks of age. These "long-life" layers allow for decreased carbon footprint and increased profitability on a per egg basis, since the 18-week period when the birds are raised prior to when egg production starts is a smaller fraction of the flock's life. Laying hens produce about one egg every 24 hours once they reach sexual maturity, a process involving daily mobilization of calcium and phosphorus stores from bone for use in eggshell formation. As hens get older, they become less efficient at using these minerals for eggshell formation and bone remodeling, leading to osteoporosis, increased incidence of bone fractures, keel deformities, and reduced egg quality from thinning shells. As a result, managing hens through an extended production cycle leads to challenges involving the aforementioned welfare and economic concerns. Our overall hypothesis is that the dynamic changes that occur during the 24h egg formation cycle will change from the onset of egg production, through the peak egg laying period, and into extended production. The hens are expected to become less efficient at absorbing and utilizing calcium and phosphorus for bone and eggshell formation as they age. We further hypothesize that dietary supplementation with a more active form of vitamin D3, Alpha D3, will improve mineral utilization for bone and eggshell formation, ultimately enhancing animal welfare and egg quality during extended production. This project will identify key biological systems and processes associated with laying hen utilization of calcium and phosphorus for eggshell and bone mineralization. It will further evaluate how these systems and processes change with age and are influenced by dietary supplementation with a more biologically active form of vitamin D3. The mechanisms to be studied are associated with actions of hormones regulating mineral utilization by important tissues that include shell gland, kidney, intestine, and liver. Parameters associated with eggshell quality and bone health will also be evaluated. In addition, the impact of feeding AlphaD3 on biological processes, eggshell parameters, and bone characteristics will be determined. It is expected that important processes and tissues associated with mineral utilization will be identified, and these processes will involve metabolism of dietary vitamin D3, hormonal signaling, and mineral uptake and transport. The impact of age and source of dietary vitamin D3 will also be determined. Together, results from this project will elucidate fundamental biological processes regulating laying hen calcium and phosphorus homeostasis at the cellular, tissue, and whole-bird level and identify novel biomarkers for improved eggshell and skeletal strength. Further, they will reveal which of the systems change most substantially with age and could contribute to the reduction in efficiency of calcium and phosphorus utilization for bone and eggshell formation in older hens, making them key targets for strategies to improve both eggshell quality and hen skeletal welfare during extended lay. Successful development and implementation of such strategies would substantially improve consumer perception of the laying hen industry as well as increase the number of salable eggs resulting from each production cycle. Both of these outcomes will allow for improved efficiency and sustainability of egg production that would be passed on to the consumer in the form of a reduced cost for poultry products.
Animal Health Component
30%
Research Effort Categories
Basic
70%
Applied
30%
Developmental
(N/A)
Goals / Objectives
Our long-term goal is to elucidate fundamental physiological mechanisms regulating laying hen Ca and P homeostasis at the cellular, tissue, and whole-bird level to facilitate development of these strategies. To this end, Specific Objectives of the proposed research are to:(1) Determine how dietary supplementation with 1α(OH)D3 influences physiological regulation of Ca and P metabolism in laying hens from onset of lay through extended production.(2) Determine how dietary supplementation with 1α(OH)D3 influences eggshell quality and skeletal integrity from onset of lay through extended production.(3) Investigate regulation of vitamin D3 metabolism in primary kidney and liver cells in vitro.
Project Methods
Two different experiments will be conducted for this project. In the first, how dietary supplementation with a commercially available 1α-hydroxycholecalciferol product (AlphaD3) influences physiological regulation of calcium (Ca) and phosphorus (P) metabolism, eggshell quality, and skeletal integrity from the onset of lay through extended production will be determined. A total of 336 commercial laying hens of uniform body weight (BW) will be transferred to individual cages in the laying facility at 16 weeks of age and housed through 100 weeks of age. From 17 weeks through the end of the trial at 100 weeks, hens will be fed either a basal or AlphaD3 supplemented (3.5 μg/kg feed) diet formulated according to nutritional recommendations to support early (onset), peak (layer 1), and extended (layer 2) production. Feed mixed for each diet that is fed for the week immediately prior to each sampling period will contain an indigestible marker (0.3% titanium dioxide). Standard flock-level production parameters will be determined for hens fed each diet. Individual egg production for all hens will be monitored daily to determine hen-day egg production [HDEP, %; (# eggs produced/# hens present)*100]. At 18 weeks of age, 4 hens from each of the 4 rows of cages fed each diet will be randomly selected to serve as "barometer" birds to evaluate production parameters on a longitudinal basis (n = 16 birds per diet). These birds will be used throughout the trial to assess feed intake (FI), egg weight, feed conversion ratio (FCR)/dozen, and FCR/kg egg every 2 weeks through 30 weeks of age and every 4 weeks thereafter, as well as for eggshell quality measurements described below. Flock-level HDEP and barometer bird production parameters and eggshell quality measurements will be carried out through 100 weeks of age. Eight randomly selected hens will be sampled at 17 weeks of age, prior to when treatment diets and photostimulation begin (n = 8 replicate hens). At 30, 60, and 90 weeks of age when flock-level HDEP is >95%, 85-90%, and 70-75%, respectively, 8 hens from each diet will be sampled at 1.5, 6, 15, and 21 hours post-oviposition (HPOP; n=8 replicate hens/diet/HPOP). For each age, approximate individual oviposition times of all hens will be recorded for 2 days prior to sampling and used to group them into possible sample collection groups at 1.5, 6, 15, and 21 HPOP. The day immediately before and on sample collection, exact individual oviposition times will be noted for each hen, and they will be assigned to collection times at each age. Body weights will be recorded and ~4-mL of blood will be collected from the brachial vein of each hen immediately prior to euthanasia by administration of pentobarbital into the medial metatarsal vein. Electrolytes (iCa, Na+, K+), blood gases (pCO2, pO2), acid-base status (pH, HCO3-, base excess), glucose, hematocrit, total P, albumin, total protein, uric acid, creatine kinase, aspartate aminotransferase, and bile acids will be analyzed in whole blood immediately after collection using portable devices. In plasma, levels of vitamin D3 metabolites [25(OH)D3, 1,25(OH)2D3, 24,25(OH)2D3] will be measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) at a commercial lab (Heartland Assays, Ames, IA) and hormones will be assessed using commercially available ELISAs [parathyroid hormone (PTH; Cusabio), calcitonin (CALC; Cusabio), fibroblast growth factor 23 (FGF23; abbexa)] or RIAs [estradiol (E2); MP Biomedicals). Kidney, shell gland, liver, and mucosal scrapings from proximal ileum will be collected from the same area of all hens at each of the four HPOP at each age. Genes associated with vitamin D metabolism (CYP2R1, CYP24A1), hormonal signaling (VDR, RXRA, RXRG, PTH1R, CALCR, ERα, FGFR1-4, KL), Ca and P uptake and utilization (NCX1, PMCA1, TRPV6, CALB1, Pit-1, Pit-2), and bicarbonate formation and transport (CA2, SLC26A9) will be measured in the four tissues by reverse-transcription quantitative PCR. At 1.5 and 15 HPOP, intestinal contents from the distal 2/3 of the ileum will be collected for analysis of dry matter, Ca, and phosphorus digestibility using titanium dioxide as the indigestible marker.Eggshell characteristics will be measured in the barometer hens at 30, 60, & 90 weeks of age and from all sampled hens used to assess physiological parameters. These include egg weight, egg volume, egg specific volume, shell length, shell width, egg surface area, shell breaking strength, shell deformation, shell thickness, shell weight, and % shell. Morphometric measurements (volume, surface area, length, width) and mechanical properties (breaking strength, deformation) will be determined using surface scanning and a texture analyzer, respectively. Dry and demembranated shell weight, shell %, shell Ca and P, and shell Ca and P % will also be determined.Keel bone and right and left humerus and tibia will be collected from birds at 17 weeks of age (n=8) and from the 6 and 21 HPOP at 30, 60, and 90 weeks of age (n=8/diet/HPOP at each age; 8 birds x 2 diets x 2 HPOP x 3 ages = 96 birds). These HPOP were chosen to represent times of full bone mineral repletion (6 HPOP; transition from bone to eggshell mineral deposition) and depletion (21 HPOP; transition from eggshell to bone mineral deposition). Weight of each bone type relative to individual body weight be determined. Length and width at the midpoint of the shaft for each tibia and humerus will be evaluated using digital calipers. All three bones will be analyzed for bone mineral content (BMC) and density (BMD) using dual X-ray absorptiometry. Left tibia samples will also be analyzed using a high-resolution peripheral quantitative computed tomography scanner to allow 3-dimensional reconstruction of skeletal microarchitecture, including detection of BMC and BMD for cortical and medullary regions and total cross-sectional area, medullary area, cortical area, and cortical thickness. For keel bone scoring, presence of fractures, deviations, and deviation distance will be assessed on a 3-point scale (0-no fracture or deviation on the ventral spine of the keel, 1-single fracture with or without a deviation of <0.5 cm, 2-multiple fractures or single fracture with a deviation of >0.5 cm). Bone-breaking strength will be analyzed for right tibia and humerus using a texture analyzer. Using digital calipers, cortical thickness will be measured in the anterior, posterior, medial, and lateral positions at the fracture site.The second study will be an in vitro experiment to investigate the site and hormonal regulation dietary vitamin D3 metabolism. Primary liver and kidney cells will be isolated from 17-week-old pullets just prior to the onset of egg laying. For each replicate, liver and kidney cells will be isolated and pooled from 3 hens, and a total of 6 experimental replicates will be performed (n=6). Cells will be left untreated or treated with vitamin D3 (50 ng/mL), 25(OH)D3, (50 ng/mL), PTH (200 ng/mL), FGF23 (200 ng/mL), or E2 (1 nM) alone or in combinations of vitamin D3 plus each hormone (PTH, FGF23, or E2) and 25(OH)D3 plus each hormone in serum-free media for 12h. Media will be analyzed by LC-MS/MS (Heartland Assays) for 25(OH)D3 (untreated and vitamin D3-treated wells only), 1,25(OH)2D3 (all wells), and 24,25(OH)2D3 (all wells). Expression of CYP2R1, CYP24A1, and 18S by RT-qPCR will also be determined.