TECHNOLOGY AREA(S): Biomedical

ACQUISITION PROGRAM: Explosive Ordnance Disposal Underwater Programs (SEA00 EOD/CREW-2)

OBJECTIVE: Develop a safe and stable oral dietary supplement to support bottlenose dolphin health including normalization of such things as white blood cell count, neutrophil count, erythrocyte sedimentation rate, hematocrit, and levels of insulin, triglycerides, ferritin, iron, globulins, alanine aminotransferase, gamma-glutamyl transferase, and/or aspartate aminotransferase toward reference levels.

DESCRIPTION: The U.S. Navy works with bottlenose dolphins (Tursiops truncatus) in the fleet�s operational Marine Mammal Systems to protect harbors and Navy assets, and to detect and/or mark underwater mines. Mission success depends on the sustained fitness and health of the U.S. Navy marine mammals. Recently, studies have described metabolic conditions in U.S. Navy dolphins, including metabolic syndrome, iron overload, and nephrolithiasis [Refs 1-4]. While most of these metabolic conditions are also observed in wild, free-ranging dolphins, studies suggest that there may be a higher prevalence of metabolic conditions in U.S. Navy dolphins, likely due to their advanced age, and differences in fish diets and feeding strategies [Refs 5, 6]. Recently, it has been identified that a change in the primary fish types fed to U.S. Navy dolphins correlated with changes in blood-based indicators of chronic, low-level inflammation, anemia, liver conditions, and iron overload (i.e., white blood cell count, neutrophil count, erythrocyte sedimentation rate, hematocrit, and concentration of globulins, alanine aminotransferase, gamma-glutamyl transferase, aspartate aminotransferase and iron) suggesting the possibility that diet impacted the animals� health.

The U.S. Navy Marine Mammal Program (MMP; Biosciences Division, Space and Naval Warfare Systems Center Pacific) with support from the Office of Naval Research is pioneering the investigation of how diet impacts a number of disease processes in bottlenose dolphins. They have obtained evidence that providing a more native fish diet may help to protect dolphin health [Ref 7]. However, changing ocean conditions and fishery volatility have led to both unreliable availability and nutrient quality of given fish stocks from year to year. Brokers providing fish to the MMP have recently expressed concern regarding availability and sustainability regarding many current fish sources. It is becoming increasingly difficult for the MMP to control the quantity of specific nutrients in available fish diets, and an oral supplement is desired to support bottlenose dolphin health including normalization of such things as white blood cell (WBC) count, neutrophil count, erythrocyte sedimentation rate, hematocrit, and levels of� insulin, triglycerides, ferritin, iron, globulins, alanine aminotransferase (ALT), gamma-glutamyl transferase (GGT), or aspartate aminotransferase (AST) toward reference levels (i.e., 4,275 - 11,405 WBC cells/ul, 2,737- 8,224 neutrophils/ul, 0 - 20 mm/h erythrocyte sedimentation rate, 38 - 46% hematocrit, < 12 ulU/ml insulin, <175 mg/dl triglycerides, < 600 ng/ml ferritin, 100 - 300 ug/dl iron, 1.8 - 3.1 g/dl globulin, 13 - 55 U/L ALT, 17 - 44 U/L GGT, and 118 - 395 U/L AST). Currently, U.S. Navy dolphins receive a daily, commercially available supplement providing water and fat-soluble vitamins.

Proposed concepts should generate a safe and stable product that can be used as an oral supplement by veterinarians to help maintain Navy dolphin health. The product should be based on dolphin-specific data and have a known dose and a means to monitor the effect, including measurable blood levels and health indices. The end result of this effort could lead to extended service life and enhanced welfare of Navy dolphins. Ultimately, a commercially-viable oral dietary supplement that can be used routinely as part of the MMP�s preventive medicine program that corrects dietary deficiencies in managed animal diets is sought.

PHASE I: Phase I Base
Determine the feasibility for developing a safe and stable, oral dietary supplement that can support bottlenose dolphin health including normalization of such things as white blood cell count, neutrophil count, erythrocyte sedimentation rate, hematocrit, and levels of insulin, triglycerides, ferritin, iron, globulins, alanine aminotransferase, gamma-glutamyl transferase, or aspartate aminotransferase toward levels detailed in the Description. Archived Navy dolphin serum and liver samples are available for analysis along with any available wild dolphin serum and liver samples for identifying candidate target molecules or compounds that could augment managed diets and provide quantifiable health benefits to dolphins. Understand the differences in wild versus managed bottlenose dolphin diets, and define the composition and dosing scheme of the proposed oral supplement. Conduct a literature search on the known safety and efficacy of the supplement in normalizing a factor(s) toward reference levels. Generate a plan for developing the oral dietary supplement in Phase II with performance goals, key technology milestones, and a strategy for testing the safety and health benefits of the product.

Phase I Option
Complete and submit U.S. Navy required documentation for conducting research involving vertebrate animals, and gain approval during the Phase I option period.

PHASE II: Develop the oral dietary supplement and establish its dosing, toxicity (using in vitro and laboratory animal studies), pharmacokinetics, and pharmacodynamics. Determine guidelines for storage of the supplement as well as the dolphin dosing amount, dosing frequency, and therapeutic blood levels. Demonstrate in at least one laboratory animal study and one dolphin field study that the oral dietary supplement successfully and safely raises blood levels of the targeted nutrients and improves white blood cell count, neutrophil count, erythrocyte sedimentation rate, hematocrit, and levels of insulin, triglycerides, ferritin, iron, globulins, alanine aminotransferase, gamma-glutamyl transferase, and/or aspartate aminotransferase. Refine the oral dietary supplement into an initial supplement formulation based on the results of the Phase II proof-of-concept study. Begin the process to ensure that the product meets all U.S. Food and Drug Administration (FDA) and/or United States Pharmacopeia (USP) certification for quality and safety [Ref 8].

PHASE III DUAL USE APPLICATIONS: Support the U.S. Navy in transitioning the oral supplement for its intended use in Navy dolphins. Commercialize the oral dietary supplement. In addition to the military market, the technology could have applicability in non-military marine mammal populations, with the potential to benefit other domestic and exotic animal populations.

REFERENCES:

1. Venn-Watson, S., et al. �Blood-based Indicators of Insulin Resistance and Metabolic Syndrome in Bottlenose Dolphins (Tursiops truncatus).� Frontiers in Endocrinology, Vol. 4, Article 136 (2013). doi: 10.3389/fendo.2013.00136

2. Mazzaro, L., et al. �Iron Indices Among Bottlenose Dolphins (Tursiops truncatus): Identifying Populations at Risk for Iron Overload.� Comparative Medicine, Vol. 62, pp. 508-515 (2012).

3. Johnson, S., et al. �Use of Phlebotomy Treatment in Atlantic Bottlenose Dolphins with Iron Overload.� Journal of the American Veterinary Medical Association, Vol. 235, pp. 194-200 (2009).� doi: 10.2460/javma.235.2.194

4. Smith, C., et al. �Pathophysiological and Physicochemical Basis of Ammonium Urate Stone Formation in Dolphins.� The Journal of Urology, Vol. 192, pp. 260-266 (2014). doi: 10.1016/j.juro.2014.01.008

5. Venn-Watson, S., et al. �Adrenal Gland and Lung Lesions in Gulf of Mexico Common Bottlenose Dolphins (Tursiops truncatus) Found Dead Following the Deepwater Horizon Oil Spill.� PLOS ONE Vol. 10, pp. e0126538 (2015). doi: 10.1371/journal.pone.0126538

6. Wells, R., et al. �Evaluation of Potential Protective Factors Against Metabolic Syndrome in Bottlenose Dolphins: Feeding and Activity Patterns of Dolphins in Sarasota Bay, Florida.� Frontiers in Endocrinology, Vol. 4, Article 139 (2014).� doi: 10.3389/fendo.2013.00139

7. Venn-Watson, S., et al. �Increased Dietary Intake of Saturated Fatty Acid Heptadecanoic Acid (C17:0) Associated with Decreasing Ferritin and Alleviated Metabolic Syndrome in Dolphins.� PLOS ONE Vol. 10, pp. e0132117 (2015).� doi: 10.1371/journal.pone.0132117

8. �Guidance for Industry: Current Good Manufacturing Practice in Manufacturing, Packaging, Labeling, or Holding Operations for Dietary Supplements; Small Entity Compliance Guide�. U.S. Food and Drug Administration, December 2010. https://www.fda.gov/Food/GuidanceRegulation/GuidanceDocumentsRegulatoryInformation/ucm238182.htm

KEYWORDS: Marine Mammal Health; Bottlenose Dolphin; Metabolic Syndrome; Iron Overload; Nephrolithiasis; Diet; Dietary Supplement