Abstract

Nutritional status of captive dolphins (Tursiops truncatus) was examined using biochemical analysis. Voluntary blood samples (obtained from four healthy, captive bottlenose dolphins at Brookfield Zoo) were analyzed for vitamin D metabolites [25(OH)D and 1,25(OH)₂D], lipids (total cholesterol, triacylglycerides, HDL-cholesterol, and LDL-cholesterol), and fatty acids. Fish fed to these dolphins were analyzed for dry matter, crude protein, fat, energy, vitamin D, cholesterol, and fatty acids. Plasma values for 25(OH)D were higher than those published for a variety of cetacean species, but similar to published bottlenose dolphin data. Total cholesterol was lower than that reported for captive beluga, but similar to wild beluga. Palmitic, oleic, eicosapentaenoic, and docosahexaenoic acids were high in both dolphin plasma and the fish sampled. Vitamin D in herring was similar to that found in other species of fish and was reflected in the high circulating levels of 25(OH)D. Capelin was found to be considerably lower in vitamin D than other species of fish. Total cholesterol in fish samples was higher than that of fillets of other fish species.

Introduction

Determinations of circulating levels of vitamins, lipids, and fatty acids can provide a base for examining nutritional status of captive cetaceans.⁹ Limited information exists regarding circulating concentrations of these nutrients in cetaceans. This study examines vitamin D metabolites, lipids, and fatty acids in captive bottlenose dolphins and relates those values to the fish they consumed.

Methods

Blood

Voluntary blood samples were collected from two male (17 and 19 years) and two female (18 and seven years) bottlenose dolphins at Brookfield Zoo. Both females were gestating at the time blood samples were obtained. Animals were housed in their usual exhibit and fed their usual fish diet with supplemental thiamin, vitamin E, and a multivitamin. Venous blood was drawn by animal management staff into tubes containing either heparin or EDTA and chemically analyzed. Plasma was separated by centrifugation and frozen for less than two months at -80°C until thawed for analysis. Samples from all four animals were analyzed for fatty acids. Samples for vitamin D metabolites and lipids were not obtained from the seven-year-old female.

Samples were analyzed for the vitamin D metabolites 25(OH)D and 1,25(OH)₂D at Boston University School of Medicine (Boston, MA). The 25(OH)D was extracted from 0.05 ml plasma with 100% ethanol, followed by a protein-binding assay using rat serum vitamin D-binding protein which has a high affinity for 25(OH)D and using a high specific activity [³H]25(OH)D₃ as a tracer.⁶ To determine 1,25(OH)₂D, 1.0 ml of plasma was extracted with acetonitrile. The extracts were then applied to C-18-OH reversed-phase cartridge to separate 1,25(OH)₂D from 25(OH)D and tri-hydroxylated vitamin D metabolites. The quantitation of 1,25(OH)₂D was accomplished by a radio-receptor binding assay using calf thymus receptor and high specific activity [³H]1,25(OH)₂D.^3,7

Total cholesterol^1,4 triacylglycerides, high-density lipoprotein cholesterol (HDL-cholesterol), and low-density lipoprotein cholesterol (LDL-cholesterol)⁴ concentrations were measured at Loyola University Medical Center (Maywood, IL) using the Synchron Delta CX7 Analyzer (Beckman-Coulter, Inc., Brea, CA).

Fatty acid analyses were conducted at the Kennedy Krieger Institute (Baltimore, MD). Fatty acids were determined by capillary gas chromatography of fatty acid methyl esters as described by Moser et al.¹⁴

Diet

Proximate analysis of whole capelin (Mallotus villosus) and herring (Clupea sp.) was conducted at the Brookfield Zoo Nutrition Laboratory. Fish were pureed with liquid nitrogen until they formed a fine powder, then dried at 60°C using a forced-draft oven. Crude protein was determined using the Kjeldahl method ×6.25.² Crude fat was determined by Soxhlet ether extraction and energy by bomb calorimetry (Parr Instruments, Moline, IL). Vitamin D, cholesterol, and fatty acid analyses of fish were performed by Covance Laboratories (Madison, WI). Vitamin D was determined by HPLC using AOAC method 45.1.22, modified 2. Total cholesterol was analyzed according to AOAC method 994.102. Fatty acids were determined by gas chromatography according to the American Oil Chemists Society.³

Results and Discussion

Blood

Vitamin D metabolite, lipid, and fatty acid concentrations in bottlenose dolphin samples are listed in Table 1. There was little variation among dolphins in the concentrations of vitamin D metabolites. Plasma concentrations of 25(OH)D in Brookfield Zoo dolphins were similar to those published for wild bottlenose dolphins.¹¹ Values were higher than published values for wild beluga whales (Delphinapterus leucas), wild pilot whales (Globicephala malaena), and wild white-sided dolphins (Lagenorhynchus acutus).¹¹ Concentrations of 1,25(OH)₂D have not been reported in cetaceans. Total cholesterol and HDL cholesterol showed less individual variation than did triacylglycerides and LDL cholesterol. Total cholesterol was lower than previously noted for captive beluga⁸ but within the ranges obtained for wild beluga.⁸ No data published for other cetaceans were found.

Table 1. Circulating concentrations of vitamin D metabolites, lipids, and selected fatty acids^a in the plasma total lipids of captive bottlenose dolphins

^aFatty acids that exceeded 1% were listed.

Monounsaturated and polyunsaturated fatty acid percentages in dolphin plasma were similar at 36% and 38%, respectively, with saturated fatty acids accounting for roughly 20% of the total. Fatty acid composition of dolphin plasma was similar to that previously published for captive bottlenose dolphins¹⁵ with oleic, eicosapentaenoic, and palmitic as the three highest fatty acids for both groups. Omega 3 and omega 9 fatty acids made up more than 50% of the fatty acids measured.

Diet

Composition of fish is known to vary with species, gender, age, season, and the location where they are caught.⁵ Nutrient composition of the capelin and herring fed to Brookfield Zoo dolphins on a dry matter basis (DMB) is presented in Table 2. Protein content was similar between the fish types; however, fat content was higher in herring. Vitamin D content of the herring was higher than that for capelin. Fish liver oils are known to be a good source of vitamin D, yet few studies have examined vitamin D content of whole fish. Published values for Baltic herring fillets with skin had higher levels of vitamin D than the herring sampled in this report (3828 IU/100 g and 1257 IU/100 g, respectively DMB). Fresh water perch fillets without skin had lower levels of vitamin D (326–1527 IU/100 g DMB) than the Baltic herring, with an upper range similar to the sampled herring.¹³

Table 2. Selected^a nutrient composition of fish fed to Brookfield Zoo dolphins on a dry matter basis

^aFatty acids that exceeded 0.05 g/100 g were listed.

Total cholesterol in capelin was higher than the herring. Lie et al.¹² examined the fatty acid and cholesterol content of a variety of fish species but analyzed fillets, not whole fish. Cholesterol of the sampled fish was twice that of the fish fillets examined by Lie, which was to be expected.

Myristic, palmitic, palmitoleic, oleic, eicosenoic, eicosapentaenoic, and docosahexaenoic acids were the highest in both herring and capelin. Eicosenoic was highest in capelin, while palmitic was highest in herring. Overall, herring had higher levels of both saturated and polyunsaturated fatty acids. Palmitic and oleic acids were high in many fish species examined by Lie et al.,¹² as it was in the sampled fish. However, herring and capelin were considerably higher in eicosenoic acid compared to most species reported.

Plasma concentrations of vitamin D metabolites, lipids, and fatty acids in captive bottlenose dolphins appear to be reflective of the fish diet consumed. The supplemental multivitamins contributed to the vitamin D concentrations in plasma but were consumed at such low levels that the impact should not have been substantial. Diet was noted as the primary factor for difference found in the fatty acid composition of the liver of freshwater and marine ringed seals.¹⁰

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