Utilization of a portable glucometer for the measurement of tissue glucose as a stress indicator in ornamental fish

Abstract

The stress response in vertebrates is determined by measuring cortisol production following acute or chronic exposure to various environmental stimuli. Cortisol assays as responses to stressful events are done on blood samples using ELISA or radio-immunoassays. However, these procedures require expensive reagents and special equipment that are not available to most fish growers or hobbyists. A portable glucometer, which is a point-of-care (POC) device to monitor blood glucose levels, was assessed in terms of its usefulness in assessing the stress response in vertebrates by quantitating whole body (tissue) glucose. Using ornamental fish as our model species, glucose levels from tissue homogenates were measured in swordtail (Xiphophorus hellerii) following handling stress by exposure to air for 3 min. Tissue glucose was measured before air exposure (control), immediately after air exposure for 3 min, and at 30 min post-air exposure (recovery). There was an increase in tissue glucose immediately after exposure of the fish to air for 3 min. At 30 min post-exposure, the levels of tissue glucose were still elevated, but may be moving towards returning to the pre-air exposure levels (control), which were measured prior to the application of the stressor. Our results have shown that a portable glucometer has good potential in monitoring stress response in vertebrates using ornamental fish as a model by quantifying tissue glucose in lieu of a more expensive cortisol assay.

Full text

Poeciliid Research, 2021, Volume 11, Issue 1.
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Utilization of a portable glucometer for the
measurement of tissue glucose as a stress
indicator in ornamental fish
1Christopher M. A. Caipang, 1Joel E. Deocampo Jr., 1Jehannie T. Fenol,
1Francis B. Onayan, 1Edda Brenda S. Yerro, 2Christian Le Marjo A.
Caipang, 3Rolando V. Pakingking Jr.
1 Department of Biology, College of Liberal Arts, Sciences, and Education, University of
San Agustin, Iloilo City, Philippines; 2 Electronics Technology Department, College of
Industrial Technology, Iloilo Science and Technology University, LaPaz, Iloilo City,
Philippines; 3 Aquaculture Department, Southeast Asian Fisheries Development Center
(SEAFDEC AQD), Tigbauan, Iloilo, Philippines. Corresponding author: C. M. A. Caipang,
cmacaipang@yahoo.com
Abstract. The stress response in vertebrates is determined by measuring cortisol production following
acute or chronic exposure to various environmental stimuli. Cortisol assays as responses to stressful
events are done on blood samples using ELISA or radio-immunoassays. However, these procedures
require expensive reagents and special equipment that are not available to most fish growers or
hobbyists. A portable glucometer, which is a point-of-care (POC) device to monitor blood glucose levels,
was assessed in terms of its usefulness in assessing the stress response in vertebrates by quantitating
whole body (tissue) glucose. Using ornamental fish as our model species, glucose levels from tissue
homogenates were measured in swordtail (Xiphophorus hellerii) following handling stress by exposure to
air for 3 min. Tissue glucose was measured before air exposure (control), immediately after air exposure
for 3 min, and at 30 min post-air exposure (recovery). There was an increase in tissue glucose
immediately after exposure of the fish to air for 3 min. At 30 min post-exposure, the levels of tissue
glucose were still elevated, but may be moving towards returning to the pre-air exposure levels
(control), which were measured prior to the application of the stressor. Our results have shown that a
portable glucometer has good potential in monitoring stress response in vertebrates using ornamental
fish as a model by quantifying tissue glucose in lieu of a more expensive cortisol assay.
Key Words: aquaculture, aquatic, cortisol, handling stress, ornamental fish, physiology, point of care.
Introduction. Stress in fish results from husbandry activities that the animals are
subjected to, as well as any imbalances in the rearing environment. Responses to these
stressors, in extreme cases, may have significant negative effects on the physiological
status of the fish, including growth, reproduction, flesh quality, and susceptibility to
disease (Wedemeyer 1996; Barton 1997; Pankhurst & van der Kraak 1997). Because of
these consequences, fish biologists have used a variety of methods for evaluating the
effects of stress on fish (Adams 1990; Wedemeyer et al 1990). These methods for
monitoring metabolic indicators of stress in fish have the obvious potential for improving
husbandry protocols and product quality of the fish upon harvest (Wells & Pankhurst
1999). However, most of these techniques have been designed and optimized for
laboratory research work and require sophisticated and expensive procedures and
equipment. Fish culturists and fisheries managers need reliable field methods that can
accurately detect stress in fish and must be easy to use and low cost.
Iwama et al (1995) and Morgan & Iwama (1997) tested the possibility of
detecting and monitoring stress conditions in fish under field conditions. They concluded
that by analyzing blood glucose with a portable instrument, the data can provide a
reliable measure of stress in fish, and thus, have practical uses in aquaculture and field

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monitoring activities. Comparative studies done by Wells & Pankhurst (1999) on the
efficiency of portable devices and laboratory-based assays in monitoring blood glucose
and lactate in fish showed strong correlation between the two methods. Moreover, they
pointed out that these portable devices offer advantages in terms of portability, simplicity
of use, and the ability to use fresh, unprocessed blood in micro-volumes.
From the standpoint of aquaculture, the industry will greatly benefit from
standardizing the methods in assessing stress conditions through the use of these
devices. However, the aquaculture industry is not only composed of rearing food fish.
This industry also encompasses the ornamental fish industry, which is a multibillion-dollar
business with a demand equivalent of at least 10 billion USD (Dey 2016). Given the
importance of the ornamental fish industry, the role of stressors in the husbandry of
ornamental fish must not be overlooked because these can affect productivity. There is
limited information on stress physiology in ornamental fish, more so on the use and
impacts of these portable devices in assessing stress in ornamental fish. To answer these
research gaps, this study aimed to determine the feasibility of using a portable
glucometer in measuring glucose as an index of the stress response in ornamental fish
using swordtail (Xiphophorus hellerii) as a model organism. In addition, this study aimed
to determine the usefulness of the whole fish as the biological material for measuring
glucose levels from small-sized fish.
Material and Method. A portable battery-operated blood glucose meter designed for
personal use was evaluated for measuring whole body tissue glucose in fish. The portable
glucometer (OneTouch Select Plus Simple®, LifeScan Europe GmbH, Switzerland) records
glucose levels in the range 20–600 mg dL-1 (approximately 1–30 mmol L-1). The
disposable test strip works when glucose in the blood or homogenized tissue sample
mixes with the enzyme glucose oxidase in the test strip and a small electric current is
produced. The strength of this current is correlated with the amount of glucose in the
sample. The glucometer measures the current, calculates the glucose level and displays
the result. Approximately 1 l of the sample may be analyzed in the temperature
operating range of 10 to 40oC.
Mixed sex juvenile swordtail (X. hellerii, average weight: 0.34 g) were subjected
to severe handling stressor by holding them out of water in a dipnet for 3 min (Caipang
et al 2014) and then returned back to the rearing container for recovery and monitoring.
7 fish were sampled before (non-stressed fish, control), immediately after air exposure
and 30 min after air exposure for whole tissue glucose. All procedures employed in the
study followed the institutional and national guidelines on responsible handling and
welfare of fish in research. Figure 1 shows the schematic diagram of the procedure that
was employed in the study.
Data are presented as means ±1 SD where appropriate. Student’s t-test for
independent samples was used to compare the tissue glucose levels immediately after
the application of the stressor and recovery with the pre-stress (control) levels. All
statistical computations were performed at the 0.05 level of significance using the
statistical package of Microsoft Excel 2010.
Results and Discussion. A portable glucometer was used to measure glucose levels
from tissue homogenates of ornamental fish as a means of assessing responses of the
host following exposure to air as a handling stress. Figure 1 shows the protocol that was
developed using this approach.

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Figure 1. Schematic diagram of measuring tissue glucose in ornamental fish using a
portable glucometer.
Briefly, individual fish from the control (pre-stress), immediately after air exposure and
recovery (30 min post-air exposure), were extracted immediately from the container,
killed and weighed. The fish were placed in individual 1.5 mL microfuge tubes and an
equal volume of normal saline solution (0.9% NaCl w/v) was added. The fish were
homogenized on ice using a plastic pestle, centrifuged at 4000 rpm for 30 sec and the
supernatant was transferred to a new microfuge tube. A disposable glucose strip was
attached to the glucometer, then the tip of the strip was dipped into the tissue
homogenate. Glucose level was immediately read and recorded. After recording, the
glucose strip was carefully removed and properly disposed. The surface of the glucometer
was disinfected with 75% ethanol after every reading to prevent contamination. All tissue
homogenates were placed on ice during the assays.
Tissue glucose was monitored in swordtail before air exposure, immediately after
air exposure for 3 min and during recovery (30 min post-air exposure). Figure 2 shows
the levels of glucose in the fish. Swordtail exhibited a significant rise in tissue glucose
immediately after exposure to air for 3 min. During recovery, the tissue glucose
decreased and the levels were returning to the pre-air exposure (control) levels.
In the present study, tissue glucose in swordtail increased following exposure to a
handling stress. In this case, handling stress was in the form of air exposure for 3 min.
The results obtained in this study were consistent with the observed increase in blood
glucose following a stress episode (Iwama et al 1995; Morgan & Iwama 1997; Wells &
Pankhurst 1999; Gomes 2007; Caipang et al 2014). The increased level of tissue glucose
that were observed in fish immediately after exposure to a stressor is an indicator that
the organism is likely undergoing a stress hyperglycemia, which is an evolutionarily
preserved response that enables the host to survive during periods of severe stress
(Barreto & Volpato 2006; Soeters & Soeters 2012; Marik & Bellomo 2013). However,
tissue glucose did not remain at elevated levels during recovery, which were in contrast
to earlier studies on fish (Gomes 2007; Caipang et al 2014). Gomes (2007) observed an
elevated blood glucose in pirarucu (Arapaima gigas) even during recovery at 24 hours
post-air exposure, while Caipang et al (2014) observed two peaks in juvenile Atlantic cod
(Gadus morhua) during recovery following acute handling stress. The differences on the
regulation of glucose levels in fish as a response to a stressor could be due to the species
of fish, the type and duration of the stress episode and the sampling times that were
employed during the conduct of the studies (Swift 1983; Barton & Schreck 1987).

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Figure 2. Tissue glucose in swordtail as monitored by a portable glucometer following
exposure to air; the column bar with an asterisk indicates significant difference from the
initial group at p<0.05 (N=7).
Recent improvements in the design of portable glucometers allow the measurements of
glucose levels below 2 mmol L-1 (Wells & Pankhurst 1999). In our study, the minimum
level of glucose that could be detected by the device was 1 mmol L-1, indicating that this
can detect even the relatively low glucose values typical of unstressed fish (Wood et al
1983; Madsen 1990). Because of this, the use of glucose as a stress indicator may be
monitored far better than the range of tests that were conducted by Morgan & Iwama
(1997). Portable glucometers have been utilized to measure glucose levels in fish during
field samplings and in areas where there are limitations to access with laboratory-based
assays in assessing the stress response (Iwama et al 1995; Wells & Pankhurst 1999). In
these studies, the feasibility of using these handheld devices was tested in foodfish,
namely, coho salmon (Oncorhynchus kisutch) and rainbow trout (Oncorhynchus mykiss)
respectively. These were in contrast with the present study, where whole body (tissue)
glucose was measured from ornamental fish. Because of its size, whole body was used to
obtain samples in monitoring glucose levels from swordtail. Our results were in
accordance with the expected pattern of increase, namely glucose levels significantly
elevated following handling stress. In addition, this also indicates that for small fishes,
the use of whole body as the biological material to measure various indices of stress is
feasible both for laboratory assays and using portable devices.
Conclusions. Taken together, our results demonstrated that a portable glucometer has
the potential to measure glucose levels in small fish using whole body (tissue) as the
biological material. Monitoring tissue glucose in small ornamental fish using a glucometer
can be used as an index of the secondary stress response in fish. This handheld device is
easy to use and is readily accessible in resource-limited areas where regular field
samplings for monitoring health and welfare in fish are conducted. Future studies should
focus on establishing the correlation between cortisol levels and tissue glucose in
response to stress. Moreover, there is also the need to validate the robustness of
measuring tissue glucose using portable devices in comparison with the values obtained

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using more sensitive laboratory-based assays. It is noteworthy to mention that these
portable devices will only be suitable for use in the ornamental fish industry if reference
values are well-established for particular species or strains of fish.
Acknowledgements. This study was supported by the University of San Agustin
Professorial Chair Research Grant titled, “Gut Microbiome: A Potential Source of Probiotic
Candidates for Ornamental Fish” awarded to CMA Caipang.
Conflict of Interest. The authors declare that there is no conflict of interest.
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Received: 12 November 2021. Accepted: 10 December 2021. Published online: 30 December 2021.
Authors:
Christopher Marlowe Arandela Caipang, Department of Biology, College of Liberal Arts, Sciences, and Education,
University of San Agustin, 5000 Iloilo City, Philippines, e-mail: cmacaipang@yahoo.com
Joel Escalada Deocampo Junior, Department of Biology, College of Liberal Arts, Sciences, and Education,
University of San Agustin, 5000 Iloilo City, Philippines, e-mail: jjdeocampo@usa.edu.ph
Jehannie Traigo Fenol, Department of Biology, College of Liberal Arts, Sciences, and Education, University of
San Agustin, 5000 Iloilo City, Philippines, e-mail: jfenol@usa.edu.ph
Francis Bernasol Onayan, Department of Biology, College of Liberal Arts, Sciences, and Education, University of
San Agustin, 5000 Iloilo City, Philippines, e-mail: fonayan@usa.edu.ph
Edda Brenda Somes Yerro, Department of Biology, College of Liberal Arts, Sciences, and Education, University
of San Agustin, 5000 Iloilo City, Philippines, e-mail: eyerro@usa.edu.ph
Christian Le Marjo Arandela Caipang, Electronics Technology Department, College of Industrial Technology,
Iloilo Science and Technology University, LaPaz, Iloilo City 5000, Philippines, e-mail: clmacaipang@gmail.com
Rolando Villarente Pakingking Jr., Aquaculture Department, Southeast Asian Fisheries Development Center
(SEAFDEC AQD), 5021 Tigbauan, Iloilo, Philippines, e-mail: rpakingking@seafdec.org.ph
This is an open-access article distributed under the terms of the Creative Commons Attribution License, which
permits unrestricted use, distribution and reproduction in any medium, provided the original author and source
are credited.
How to cite this article:
Caipang C. M. A., Deocampo J. E. Jr., Fenol J. T., Onayan F. B., Yerro E. B. S., Caipang C. L. M., Pakingking R.
V. Jr., 2021 Utilization of a portable glucometer for the measurement of tissue glucose as a stress indicator in
ornamental fish. Poec Res 11(1):30-35.