Hematology and Plasma Biochemistry in Whale Sharks (Rhincodon typus): Baseline Reference Intervals Based on Captivity Status, Blood Sampling Sites, and Handling Methods

Abstract

This study aimed to establish reference intervals for red and white blood cell counts, hematocrit levels, mean corpuscular volume, and 25 key plasma biochemistry parameters in captive and wild whale sharks (Rhincodon typus). Blood samples were collected from the caudal vein (CV) and dorsal cutaneous vein (DCV) of 30 wild sharks caught in fixed nets off the Kochi Prefecture coast, Japan, and from 24 captive sharks between 2007-2023. Samples were obtained from restrained captive and wild sharks as well as unrestrained captive sharks trained for husbandry. Comparative analyses considered three factors: captivity status (wild vs. captive sharks under restraint), blood sampling sites (CV vs. DCV under restraint), and handling methods (DCV sampling under restrained vs. unrestrained conditions). Analysis of captivity status revealed significant differences in 12 of 29 parameters, with triglyceride levels significantly lower in wild sharks, possibly indicating nutritional deficiencies due to their prolonged migrations. Comparisons of blood sampling sites revealed significant differences in 11 parameters, including red and white blood cell counts and hematocrit levels, with most CV-derived parameters being higher than those from the DCV. A strong correlation (r > 0.7) was found between the CV and DCV for 19 parameters, indicating predictive values between these vessels. Additionally, the relationship between RBC, Ht, and MCV indicates that the RBC and MCV results may not be entirely reliable and should therefore be interpreted with caution. In the handling method comparison, eight parameters exhibited significant differences; specifically, aspartate aminotransferase, ammonia, and creatine phosphokinase levels were likely influenced by stress effects, including restraint-induced muscle damage. These findings emphasize the importance of unrestrained blood collection, facilitated through husbandry training, for accurate blood parameter evaluations. Integrating statistical results across the three studied factors allowed for the establishment of reference intervals, means, and medians for whale sharks, contributing to health management in captive sharks and conservation in wild populations.

Keywords: Captive vs. wild comparison; Comparative blood analysis; Husbandry training; Shark health assessment.

Fig. 1.

Blood sampling from the caudal and dorsal cutaneous veins of a restrained whale shark (Rhincodon typus). An underwater blood collection kit was used for sampling.

Fig. 2.

Blood sampling from the dorsal cutaneous vein of an unrestrained whale shark (Rhincodon typus). Husbandry training was applied to facilitate sampling.

Fig. 3.

Box-and-whisker plots of 29 blood parameters for the groups Wild (CV, Restrained), Wild (DCV, Restrained), Captive (CV, Restrained), Captive (DCV, Restrained), and Captive (DCV, Unrestrained), categorized by captivity status, blood sampling site, and handling method. “Wild” and “Captive” represent data from wild and captive sharks, respectively. “CV” and “DCV” indicate data from the caudal vein and dorsal cutaneous vein, respectively. “Restrained” and “Unrestrained” denote data from restrained and unrestrained sharks, respectively. The bottom and top of each box represent the first quartile and third quartile, respectively, with the line inside the box indicating the median. Whiskers extend to the smallest and largest values within 1.5 times the interquartile range from the first and third quartiles. Data points outside this range are considered outliers and are represented as individual dots. The × symbol indicates the mean value for each group. Groups with significant differences (p < 0.05) in captivity status and blood sampling sites, based on two-way ANOVA, and in handling methods, based on Student’s t-test, are marked as follows: * for captivity status, † for blood sampling sites, and § for handling methods.

Fig. 3.

Box-and-whisker plots of 29 blood parameters for the groups Wild (CV, Restrained), Wild (DCV, Restrained), Captive (CV, Restrained), Captive (DCV, Restrained), and Captive (DCV, Unrestrained), categorized by captivity status, blood sampling site, and handling method. “Wild” and “Captive” represent data from wild and captive sharks, respectively. “CV” and “DCV” indicate data from the caudal vein and dorsal cutaneous vein, respectively. “Restrained” and “Unrestrained” denote data from restrained and unrestrained sharks, respectively. The bottom and top of each box represent the first quartile and third quartile, respectively, with the line inside the box indicating the median. Whiskers extend to the smallest and largest values within 1.5 times the interquartile range from the first and third quartiles. Data points outside this range are considered outliers and are represented as individual dots. The × symbol indicates the mean value for each group. Groups with significant differences (p < 0.05) in captivity status and blood sampling sites, based on two-way ANOVA, and in handling methods, based on Student’s t-test, are marked as follows: * for captivity status, † for blood sampling sites, and § for handling methods.

Fig. 3.

Box-and-whisker plots of 29 blood parameters for the groups Wild (CV, Restrained), Wild (DCV, Restrained), Captive (CV, Restrained), Captive (DCV, Restrained), and Captive (DCV, Unrestrained), categorized by captivity status, blood sampling site, and handling method. “Wild” and “Captive” represent data from wild and captive sharks, respectively. “CV” and “DCV” indicate data from the caudal vein and dorsal cutaneous vein, respectively. “Restrained” and “Unrestrained” denote data from restrained and unrestrained sharks, respectively. The bottom and top of each box represent the first quartile and third quartile, respectively, with the line inside the box indicating the median. Whiskers extend to the smallest and largest values within 1.5 times the interquartile range from the first and third quartiles. Data points outside this range are considered outliers and are represented as individual dots. The × symbol indicates the mean value for each group. Groups with significant differences (p < 0.05) in captivity status and blood sampling sites, based on two-way ANOVA, and in handling methods, based on Student’s t-test, are marked as follows: * for captivity status, † for blood sampling sites, and § for handling methods.

Fig. 3.

Box-and-whisker plots of 29 blood parameters for the groups Wild (CV, Restrained), Wild (DCV, Restrained), Captive (CV, Restrained), Captive (DCV, Restrained), and Captive (DCV, Unrestrained), categorized by captivity status, blood sampling site, and handling method. “Wild” and “Captive” represent data from wild and captive sharks, respectively. “CV” and “DCV” indicate data from the caudal vein and dorsal cutaneous vein, respectively. “Restrained” and “Unrestrained” denote data from restrained and unrestrained sharks, respectively. The bottom and top of each box represent the first quartile and third quartile, respectively, with the line inside the box indicating the median. Whiskers extend to the smallest and largest values within 1.5 times the interquartile range from the first and third quartiles. Data points outside this range are considered outliers and are represented as individual dots. The × symbol indicates the mean value for each group. Groups with significant differences (p < 0.05) in captivity status and blood sampling sites, based on two-way ANOVA, and in handling methods, based on Student’s t-test, are marked as follows: * for captivity status, † for blood sampling sites, and § for handling methods.

Fig. 3.

Box-and-whisker plots of 29 blood parameters for the groups Wild (CV, Restrained), Wild (DCV, Restrained), Captive (CV, Restrained), Captive (DCV, Restrained), and Captive (DCV, Unrestrained), categorized by captivity status, blood sampling site, and handling method. “Wild” and “Captive” represent data from wild and captive sharks, respectively. “CV” and “DCV” indicate data from the caudal vein and dorsal cutaneous vein, respectively. “Restrained” and “Unrestrained” denote data from restrained and unrestrained sharks, respectively. The bottom and top of each box represent the first quartile and third quartile, respectively, with the line inside the box indicating the median. Whiskers extend to the smallest and largest values within 1.5 times the interquartile range from the first and third quartiles. Data points outside this range are considered outliers and are represented as individual dots. The × symbol indicates the mean value for each group. Groups with significant differences (p < 0.05) in captivity status and blood sampling sites, based on two-way ANOVA, and in handling methods, based on Student’s t-test, are marked as follows: * for captivity status, † for blood sampling sites, and § for handling methods.