The modernization of the US swine industry has been critical to the sustainability and global competitiveness of US swine production. At the same time, modernization has also required dramatic changes in animal management. By housing pigs indoors, producers have caused the unintended problem of iron deficiency anemia in neonatal pigs. This problem has been further exacerbated by advances in genetics and nutrition, which resulted in faster growth rates and efficiency of growth. In order to prevent anemia in neonatal pigs, therefore, all U.S. producers use injectable iron dextran. While the use of any injectable raises concerns for meat animal production, recent shortfalls in iron dextran supplies have added tremendously to the burden on U.S. swine producers. As a result, there is a significant unmet need for a safe and effective substitute for iron dextran. An orally administered iron product that was safe and effective in preventing anemia in neonatal pigs would be ideal. The proposed SBIR Phase I research consists of two pivotal studies that will assess key attributes of the low molecular weight iron chelate, sodium ferricitropyrophosphate (FCP), as an oral iron nutrient and the importance of lactoferrin/transferrin iron delivery systems in the intestine to support hemoglobin production and rapid growth in neonatal piglets. Proposed Study 1 is an in vitro study of the efficiency of transfer of iron from FCP to lactoferrin. Preliminary data indicate the transfer will occur rapidly and effectively, saturating the iron-binding capacity of the protein. In Proposed Study 2, we will assess the ability of FCP, supplied in sow's milk formula, to provide sufficient iron to maintain hemoglobin levels in neonatal pigs. The data will identify, for the first time, the bioavailability of FCP to neonatal pigs and will enable estimate of the overall iron absorptive capacity of the neonates. In addition, the data will enable comparison of the iron delivery capabilities of FCP with the capabilities of iron dextran and other iron fortificants. Preliminary data suggest that robust hemoglobin production and rapid, sustained growth will be observed after administration of FCP to neonates in this way. The proposed SBIR Phase 1 research is expected to verify that providing iron as FCP in sow's milk formulas will more effectively support both the hemoglobin concentration and rapid growth of the neonatal pig, while substantially reducing the need for iron dextran injections, injections which contribute to the expenses of both veterinary care and labor. In addition, use of FCP is expected to result in a decreased risk of disease transmission and enhanced biosecurity, since administration of FCP is not expected to require exposure of piglets to either used needles or pen-to-pen transmission of pathogens. Phase 2 research will extend applicability to large swine herds by employing special FCP-containing feeds provided in neonatal feeders to eliminate manual labor. In summary, if the expected results are obtained, the United States will not only maintain its competitive leadership but will also lead other hog-producing nations to more effective resource utilization. OBJECTIVES: A growing body of evidence supports the hypothesis that (a) Both the lactoferrin- and transferrin-iron-trafficking systems in enterocytes of neonatal piglets have the potential to provide a supply of iron to the systemic circulation sufficient for robust hemoglobin production and optimal growth. (b) These transport systems are stimulated by iron deficiency and are not shut down by hepcidin. Moreover, iron will be supplied by both systems to the body as transferrin-bound iron, the delivery system which is used physiologically for hemoglobin and red cell production. The proposed SBIR Phase I research consists of year-long pivotal studies that will assess key attributes of FCP as an oral iron nutrient and the importance of the lactoferrin/transferrin iron delivery systems to support hemoglobin production in neonatal piglets. Specific Aim 1. Determine the rate and efficiency of iron transfer to lactoferrin. The sow's colostrum and milk do not provide quantities of iron adequate to maintain hemoglobin levels in piglet blood. Sows in confinement express colostrum having an iron concentration of about 2 ppm and milk having an iron concentration of about 1 ppm. A quart of sow's milk is estimated to contain 1 mg iron. However, if the 25 micromoles of lactoferrin that is present in a liter of sow's milk were fully iron saturated, it could provide almost 3 mg of iron to the piglet. This difference in iron concentration suggests that lactoferrin in sow's milk is not fully iron-loaded. Preliminary studies have shown that FCP saturates the iron-binding capacity of a related iron-binding protein, transferrin, within minutes. No data are available regarding the kinetics of iron saturation of lactoferrin. Therefore, this study will provide data concerning the kinetics and efficiency of iron transfer from FCP to lactoferrin, the iron-binding protein with the highest concentration in sow's milk. Specific Aim 2. Determination of the safety and bioavailability of FCP supplied to neonates via the diet. A growing body of evidence suggests that lactoferrin may be involved in iron absorption from the gut during early infancy. Likewise, a transferrin shuttling system, which also is present at birth and maintained during maturation, may be responsible for as much as 50% of the iron absorbed from the diet. Both systems are upregulated in iron deficiency and bind ferric iron. No prior study has verified that a sow's milk formula, appropriately supplemented with ferric iron, will adequately support hemoglobin production in the neonatal pig. Therefore, to understand the importance of this means for iron delivery to neonates and to evaluate its potential for supplying sufficient iron to the rapidly growing animal, we will study FCP supplements in neonatal swine. This study will be completed under a subcontract to North Carolina State University, College of Veterinary Medicine. If the results of these studies demonstrate effective support for hemoglobin production and rapid neonatal growth, SBIR Phase 2 studies will evaluate commercial feeders and pellets as a cost-effective means for mass implementation of oral iron delivery as FCP to piglets and sows. APPROACH: This year-long SBIR Phase 1 study will provide specific data about the effectiveness of ferricitropyrophosphate (FCP) as an oral iron source for neonatal pigs. Study 1: Kinetics and effectiveness of iron transfer from FCP to lactoferrin. Bates' method will be used to determine the rate and extent of iron uptake from FCP to the iron binding sites of apolactoferrin. A solution containing nitrilotriacetic acid (NTA) and ferric sulfate will be used as a control solution, since this chelate has been reported to react immediately with apotransferrin, another member of the transferrin family and similar reactivity with apolactoferrin is expected. A blank reaction using buffer instead of a test or control sample will also performed to correct for interfering iron from the diluent. After admixture, the binding of iron to apolactoferrin will be monitored every 10 seconds at 25 C and 460 nm, the absorption maximum for ferric lactoferrin, until there is no significant change. The molar absorptivity will be used to calculate the kinetics and efficiency of iron transfer from FCP to lactoferrin. Study 2: Safety and bioavailability of FCP to neonates. This study will be completed at North Carolina State University, College of Veterinary Medicine. Thirty day-old piglets (N=5/group) will be fed one of six dietary treatments as liquid milk replacer for 21 d. These pigs will not receive an injection of 100 mg iron as iron dextran. The dietary treatments will contain three different concentrations of iron from either ferrous sulfate (control) or FCP. The base milk replacer will meet all of the nutritional needs of newborn pigs, with the exception of iron. The iron treatments will be added to the milk replacer at reconstitution to provide dietary iron concentrations that are Adequate (100 mg iron/kg diet), Deficient (50 mg iron/kg diet), or Excessive (150 mg iron/kg diet). Thus, all piglets will receive the same nutrition (except for iron) and dietary lipid peroxidation by ferrous sulfate will be minimized. Piglets will be fed 4 times/day via a liquid feed delivery system to insure equal consumption between groups and to generate growth rates similar to sow-reared pigs. Feed intake and body weight will be recorded daily. Blood will be collected on day 7, 14, and 21 of the feeding trial for determination of red cell volume, hemoglobin concentration, serum ferritin, transferrin saturation, catalase, SOD, and other metabolic markers. At the completion of the trial, samples of liver, spleen, small intestinal and colonic epithelium will be collected for analyses of gene expression and markers of oxidative stress, ferritin, and iron concentration. Data will be analyzed using a general linear model with dietary treatment considered as a fixed effect. The study design provides the statistical power needed for a 1 g/dL whole blood difference in circulating levels of hemoglobin to be statistically different (P < 0.05). The proposed research is expected to verify that providing iron as FCP in sow's milk formulas (SBIR Phase 1) or pellets (SBIR Phase 2) will effectively support the rapid growth of the neonatal pig and make the farmer more competitive in the global market.
