Ảnh hưởng của nhiệt độ và hàm lượng oxy thấp lên cấu trúc cơ quan hô hấp của cá tra (pangasianodon hypophthalmus)

MINISTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY LE MY PHUONG THE EFFECTS OF ELEVATED TEMPERATURE AND HYPOXIA ON THE RESPIRATORY ORGANS OF Pangasianodon hypophthalmus PHD DISSE

MINISTRY OF EDUCATION AND TRAINING

CAN THO UNIVERSITY

LE MY PHUONG

THE EFFECTS OF ELEVATED TEMPERATURE AND

HYPOXIA ON THE RESPIRATORY ORGANS OF

Pangasianodon hypophthalmus

PHD DISSERTATION MAJOR: AQUACULTURE MAJOR CODE: 62 62 03 01

MINISTRY OF EDUCATION AND TRAINING

CAN THO UNIVERSITY

LE MY PHUONG

THE EFFECTS OF ELEVATED TEMPERATURE AND

HYPOXIA ON THE RESPIRATORY ORGANS OF

Pangasianodon hypophthalmus

PHD DISSERTATION MAJOR: AQUACULTURE MAJOR CODE: 62 62 03 01

SUPERVISORS Supervisor: Assoc Prof Dr DO THI THANH HUONG

Co-supervisor: Assoc Prof Dr MARK BAYLEY

2017

Data sheet

Title: The effects of elevated temperature and hypoxia on the respiratory

organs of Pangasianodon hypophthalmus

Subtitle: PhD Dissertation

Affiliation: College of Aquaculture & Fisheries, Can Tho University,

Vietnam; Zoophysiology, Department of Bioscience, Aarhus University, Denmark

Publication year: 2017

Cite as: Phuong L.M (2017) The effects of elevated temperature and

hypoxia on the respiratory organs of Pangasianodon

hypophthalmus PhD Dissertation College of Aquaculture &

Fisheries, Can Tho University, Vietnam and Zoophysiology, Department of Bioscience, Aarhus University, Denmark

Keywords: climate change, hypoxia, temperature, morphometric, swim

bladder, gill remodelling, air-breathing fish, Pangasianodon

hypophthalmus

Supervisor: Associate Professor Do Thi Thanh Huong, Deparment of Aquatic

Nutrition and Products Processing, College of Aquaculture and Fisheries, Can Tho University, Vietnam

Co-supervisor: Associate Professor Mark Bayley, Zoophysiology, Deparment of

Bioscience, Aarhus University, Denmark

Co-supervisor: Professor Jens Randel Nyengaard, Core Center for Molecular

Morphology, Section for Stereology and Microscopy, Centre for Stochastic Geometry and Advanced Bioimaging, Aarhus University, Denmark

Table of Contents

Data sheet 1

Table of Contents 2

ACKNOWLEDGEMENTS 3

SUMMARY 4

Chapter 1: INTRODUCTION 6

1.1 Oxygen requirement and adaptive mechanisms of fish to hypoxia 6

1.2 Striped catfish (Pangasianodon hypophthalmus) 7

1.3 General gill structure in teleosts 8

1.4 Osmo-respiratory compromise in fish gills 11

1.5 Gill plasticity and environmental factors causing gill plasticity 12

1.6 Methods applied in morphometric studies 14

1.7 Objectives of dissertation…… ………… ……….17

REFERENCES 17

Chapter 2 (PAPER 1) 26

Recovery of blood gases and haematological parameters upon anaesthesia with Benzocaine, MS-222 or Aqui-S in the air-breathing catfish (Pangasianodon hypophthalmus) 26

Chapter 3 (PAPER 2) 48

Gill remodelling and growth rate of striped catfish Pangasianodon hypophthalmus under impacts of hypoxia and temperatures 48

Chapter 4 (PAPER 3) 76

Ontogeny and morphometric of the gill and swim bladder of air-breathing striped catfish Pangasianodon hypophthalmus 76

Chapter 5 (PAPER 4) 104

Gill remodelling does not affect the rate of acid-base regulation in the striped catfish Pangasianodon hypophthalmus 104

Chapter 6: Conclusions and Perspectives… 121

APPENDICE……… ………… 125

ACKNOWLEDGEMENTS

Foremost, I would also like to give my thanks to Assoc Prof Do Thi Thanh Huong and Prof Nguyen Thanh Phuong in Can Tho University for always supporting, encouraging, and watching my steps during my study I would also like to express my gratitude to Assoc Prof Mark Bayley who has found and built my passions and directions for science as who I am and what I have today His guidance and enthusiasm have inspired and supported me moving forward and working by my best during my research and writing this thesis I am also grateful

to Prof Jens Randel Nyengaard for teaching me stereological methods and supporting me during my working in his laboratory

My sincere thanks also go to Prof Tobias Wang, Prof Atsushi Ishimatsu, and Prof Hans Malte for giving me special advices and precious and initiative suggestions which contribute importantly in my thesis Also, I acknowledge Christian Damsgaard who greatly contributed in interpreting and discussing data of the last manuscript in this thesis

I would like to thank the staffs at the College of Aquaculture and Fisheries, Can Tho University (Vietnam), at the Zoophysiology Section, Department of Biological Sciences, and

at Core Center for Molecular Morphology, Section for Stereology and Microscopy, Centre for Stochastic Geometry and Advanced Bioimaging, Aarhus University, Denmark where my projects were carried out Especially, I would like to thank Maj-Britt Lundorf for teaching me with helpful skills in histology and Per Guldhammer Henriksen for helping me during my experiments in Aarhus University

I would also like to thank my fellow friends in iAQUA project: Nguyen Thi Kim Ha,

Le Thi Hong Gam, Dang Diem Tuong, and Phan Vinh Thinh for their friendship and all the funs we have during the time we worked together in the project

Finally, I would like to thank my family and my friends for their love and spiritually supporting me throughout my research, my writing, and my whole life

My project was funded by The Danish International Development Agency (DANIDA), Ministry Affairs of Foreign Denmark, iAQUA project

SUMMARY

The striped catfish Pangasianodon hypophthalmus is one of the most important species

in terms of both economy and physiology The overall objective of this study is to provide input to the assessment of the effects of elevated temperature and/or hypoxia on the respiratory

organs of air-breathing catfish Pangasionodon hypophthalmus by implementing stereological

methods to reveal plasticity This research will play a key role in a better understanding the capacity for adaptation of air-breathing fish to temperature increases

Four main projects were investigated in this study In the first project, the disturbances

in blood gasses and haematological parameters, caused by the three different anaesthetics, commonly used in aquaculture during transport as well as for surgical procedures, were

investigated during recovery in Pangasianodon hypophthalmus We found that these parameters were normalized within 24h and this was the first indication that P hypophthalmus

is unusual among air-breathing fish with its strong capacity for acid-base regulation In addition, this study demonstrated that this species lacks the β-adrenergic swelling responses in red blood cells

In the second project, by applying vertical sections in stereology, gill morphometrics of

P hypophthalmus exposed to the average Mekong river present temperature (27°C) as well as

to a constant 6°C elevation were investigated These temperature treatments were combined with normoxic and hypoxic oxygen levels We found strong plasticity in gills lamellar surface areas (SA), with highest SA under elevated temperature and hypoxia whereas almost eliminated This plasticity was due to proliferation of cell mass between the secondary lamellae (ILCM), which was thus most developed in the normoxic low temperature group Further, the diffusion distance from water to blood (HM) was thinnest (approximately 1.0µm) in the fish exposed to hypoxia and high temperature At their largest, the gills of this species are on a weight specific basis similar to active water-breathers, such as trout, but with significantly shorter HM This is the first documentation of ILCM in catfish and is similar to that found in cyprinids and seems to present support for the osmo-respiratory compromise phenomenon In this study it was also demonstrated for the first time that this species grows much faster at higher than present temperatures, with an 8-fold higher growth rate at 33°C than 27°C

The third project, we examined the development of the gill and air-breathing organ (ABO) SA and HM with body size By calculating the anatomical diffusion factor, it was possible to evaluate the importance of gills and ABO for oxygen uptake and to make

interspecies comparisons Here I found that the ADF of the gills is high (comparable to an active water-breather such as rainbow trout), even with fully developed ILCM Further, when required, the gills can change rapidly (<20h) elevating ADF by a factor 3 In addition, the dimensions (respiratory SA, volume) of respiratory organs scaled with body mass, with the scaling slope for gills being in the range found for active water-breathing fish and with the swim bladder scaling as a mammalian lung

It has been argued that there is a compromise between gas exchange and ion regulation functions in fish gills In the fourth project, we exposed the fish in two different oxygen levels (hypoxia and hyperoxia) to induce gill remodelling and to test whether such branchial

remodelling affects the rate of acid/base regulation in response to aquatic hypercapnia in P

hypophthalmus We found that there was no difference in the rate of acid-base regulation in

these two groups, and suggest that despite its well-developed gills that there is a functional separation in this species occurring where the respiratory gill surfaces do not function in ion exchange

Chapter 1: INTRODUCTION

1.1 Oxygen requirement and adaptive mechanisms of fish to hypoxia

Climate change, hypoxia, and its effects

It has been predicted that the environmental temperature is increasing as a result of global climate change (IPPC, 2014) It is clear that the projected increase in environmental temperature may lead to negative effects on fish populations as a result of disturbances on fish physiology, metabolism, immune functionality, reproductive capacity, behavior, growth

performance, or mortality (Watts et al., 2001; Cnaani, 2006; Dalvi el al., 2009; khoshnevis Yazdi and Shakouri, 2010; Singh et al., 2013; Reid et al., 2015) Temperature elevation causes

a reduction in oxygen concentration in water (hypoxia), and at the same time an increase in the metabolic demand for oxygen by living organisms, which has been argued to lead to aerobic scope reductions These are the features underlying the oxygen- and capacity-limited thermal tolerance (OCLTT) hypothesis, which proposes that oxygen delivery is the key mechanism underlying the negative effects of increased temperatures in aquatic organisms (Pörtner, 2001; Pörtner and Farrell, 2008; Pörtner, 2010) It has also been noted that elevated temperature

interacting with hypoxia could result in larger effects on organism performance (McBryan et

al., 2013) Tropical regions are predicted to be seriously affected by the climate change Indeed,

there are more than 7000 freshwater fish species living in tropical regions, and it has been argued that since these animals are likely stenothermal and since they already live closer to their upper thermal limit, they may actually be the most vulnerable (Nelson, 1994; Tewksbury

et al., 2008) Besides, tropical areas are known for significant fluctuations in oxygen content

in the water, where water can be richly oxygenated in wet seasons and severely hypoxic or even anoxic in dry season (Welcomme, 1979; Lucas and Baras, 2008; Nguyễn Lâm Anh, 2016) In addition, as a result of primary production in organically rich water with poor mixing, oxygen can fluctuate on a dual basis from deep hypoxia at night to hyperoxia during the day

Fish adaptive mechanisms to hypoxia

Fish have developed a variety of adaptive mechanisms in their physiology,

morphology, or behaviour to cope with hypoxia in the environment (Wu, 2002; McBryan et

al., 2013) Firstly, fish have some biochemical and physiological responses to maintain oxygen

delivery, such as increasing gill ventilation to increase water flow over the gills, increase gill perfusion (Randall, 1970, 1982; Wu, 2002), increase the amount of RBC (Randall, 1982;

Soldatov, 1996; Paper 1), or increase affinity for O2 in haemoglobin (Randall, 1982; Val et al.,

1995; Damsgaard et al., 2015b) The fish may reduce their swimming activities to reduce oxygen demand and conserve energy expenditure under hypoxia (Schurmann and Steffensen,

1994) Besides, numerous fish have been found with some mechanisms to increase capacity for oxygen uptake under hypoxic conditions by changing their respiratory structures (discussed

in the following section)

During long-term exposure to hypoxia, many fish species have formed an adaptive behaviour that they can migrate to more oxygenated waters or move to the surface of the water for aquatic surface respiration or aerial respiration when the water becomes hypoxic (Lewis, 1970; Petersen and Petersen, 1990; Wannamaker and Rice, 2000; Kramer and Mehegan, 1981;

Chapman et al., 1995) Air-breathing is one of the adaptive responses of fish to aquatic hypoxia (Johansen et al., 1970; Randall et al., 1981; Graham, 1997) Many species especially in tropical

areas have evolved a transition from aquatic to aerial respiration (Graham, 1997) When the gills of fish fail to meet oxygen requirement for their metabolic demands in hypoxic water, there has been a strong evolutionary stimulous to develop air-breathing using a diversity of accessory respiratory organs to enable them uptake oxygen from the air (Graham, 1997), which has occurred on more than 60 separate events (Shartau and Brauner, 2014) In this thesis, I focus on quantifying the morphological plasticity of respiratory structures of air-breathing fish under conditions that impact oxygen uptake (temperature and water oxygen content)

1.2 Striped catfish (Pangasianodon hypophthalmus)

The facultative air-breathing striped catfish Pangasianodon hypophthalmus belongs to Pangasidae, a recently evolved group of siluriform catfishes (Pouyard et al., 2000) In contrast

to other air-breathing fish, P hypophthalmus is a highly active species known for its extreme

seasonal migration over at least 2000 km along the Mekong River between their spawning and

feeding grounds (Poulsen and Valbo-Jørgensen, 2000; Zalinge et al., 2002; So et al., 2006)

During the long migration, there are a variety of environmental constraints such as temperature fluctuations, high organic matter, severe hypoxic and hypercapnic water, and a high risk of

predation (Lucas and Baras, 2008; Li et al., 2013) Furthermore, P hypophthalmus is also

important species in aquaculture throughout South-East Asia where it is cultured extensively

in ponds characterizing with severe hypoxia or even anoxia and hypercapnia water (Lefevre et

al., 2011b; Phạm Quốc Nguyên và ctv., 2014; Damsgaard et al., 2015a)

Recent studies have found P hypophthalmus unusual compared to other air-breathing

fish, because it possesses both well-developed gills and a highly trabeculated swim bladder,

indicating a high capacity for oxygen uptake by both water- and air-breathing (Lefevre et al

2011a, 2013; Paper 2; Paper 3) In addition, it has and high capacity for pH regulation evident both during recovery from anesthesia (Paper 1) and during exposure to environmental

hypercapnia (Damsgaard et al., 2015a) However, quantitative analysis of the respiratory

structures has not been performed, making it interesting to investigate the respiratory organs in

P hypophthalmus, and further, to look for evidence of plasticity in these metrics under

changing temperature or oxygen availability

1.3 General gill structure in teleosts

The teleost gill structures have been well described in previous studies (Laurent, 1984;

Olson, 1991; Olson, 2002; Evans et al., 2005) Briefly, there are four gill arches on each side

of the head Each gill arch includes anterior and posterior hemibranches connected with a gill raker The number and the length of gill filaments vary during the development between species Each gill filament is supported by a cartilaginous rod and striated abductor and adductor muscles adjusting the filament position during ventilation, and it is covered by thick layers of non-respiratory epithelium including mucous cells, chloride cells, and pavement cells Secondary lamellae are perpendicularly distributed along the length of the primary filaments from their both sides, and consist of two epithelial layers separated by a network of pillar cells forming a sheet through which the blood flows Secondary lamellae are covered with a two-layered epithelium consisting mostly of pavement cells, and less common mucous cells, and mitochondria rich ion exchange cells Pillar cells contain collagen fibers and are contractile; therefore, it has been suggested that the width of lamellar blood spaces could be adjusted by these cells (Morgan and Tovell, 1973)

Blood circulation and gas exchange in fish gills

The deoxygenated blood from the ventral aorta is supplied to branchial arteries into each gill arch and to the gill filaments At each gill filament, the blood crosses secondary lamellae from afferent filamental artery (AFA) extending along the filament into efferent

filamental artery (EFA) which is opposite to AFA (Olson, 2002; Evans et al., 2005) When the

blood crosses the secondary lamellae in the opposite direction of the water flow, the gas

exchange takes place Oxygenated blood is collected and reaches the EFA towards the branchial efferent artery, which join together in the dorsal aorta The gill vasculature is controlled by both neural and hormonal factors (Olson, 2002) In the secondary lamellae, the

blood flow through lamellar sinusoids constructed by pillar cells It has been shown that these

cells can regulate the amount of blood flows through the lamellae by their contraction (Evans

et al., 2005) In addition to the lamellar sinusoids, blood may flow across the lamellae via the

inner marginal channel (IMC) or outer marginal channel (OMC) The IMC is embedded within

the body of the filament body and is suggested as a non-respiratory shunt which does not contribute to gas exchange; whereas, blood flows continuously throughout the OMC which has

a larger diameter compared to the lamellar sinusoids and has a greater surface area in contact

with the ambient water (Olson, 1991)

Morphometric and anatomic diffusion factor in fish respiratory structures

The efficiency of respiratory gas exchange at the fish gill depends on the gill’s diffusion capacity (Hughes and Morgan, 1973; Hughes, 1980) Oxygen diffuses from water to blood through the epithelial layers of the secondary lamellae along a gradient in oxygen tension existing between ambient water and the blood in the capilliaries of the respiratory surfaces (Hughes and Morgan, 1973) The morphometric diffusion capacity (Dmorphology) of the gills can

be calculated by the equation:

𝛕 𝒉

where k is tissue specific Krogh’s diffusion coefficient for respiratory gases and ADF is the anatomical diffusion factor calculated as respiratory surface areas (SA) divided by harmonic mean diffusion distance (τh) (Hughes and Morgan, 1973; Perry, 1978; da Costa et al., 2007; Fernandes et al., 2012) The respiratory surface area and the diffusion distance are therefore

the most important dimensions in evaluationg the capacity of the respiratory structure for gas exchange

Fig 1.1 presents branchial respiratory SA and water-blood barrier thickness diffusion distance morphometric partitioning of respiratory structures of several water-breathing, obligate and facultative air-breathing fish (Paper 2) Generally, the respiratory surface areas of gills of water-breathing fish are much higher than in air-breathing species (both obligate and facultative species) It has been suggested that low gill surface area in air-breathing fish is an adaptation to minimize branchial oxygen loss to hypoxic water when the oxygenated blood

from the swim bladder passes through the gills (Randall et al., 1981) In contrast to other breathing species, P hypophthalmus is shown in this thesis to have a potential for large

air-branchial respiratory SA, especially in hypoxic water and/or at high temperature as well as having a thin water-blood diffusion distance (Fig 1.1; Paper 2), which combined give a high ADF (Table 1.1; Paper 3)

Fig 1.1 Respiratory branchial surface area and water-blood diffusion distance of different fish

species Figure is adapted from Paper 2

Branchial surface area (mm 2 g -1 )

Obligate air-breathing fish Facultative air-breathing fish Striped catfish

Human

Dogfish

Brown Bullhead Climbing perch

Snakehead Walking catfish

Striped catfish Mud Skipper

Horse Mackerel

Striped catfish Striped catfish

Striped catfish Striped catfish

Tuna Human

Table 1.1 Anatomic diffusion factors (ADF, cm 2 µm -1 kg -1 ) of gills and air-breathing organs (ABO) of different fish species

Heteropneustes fossilis 156.42 Sk 20.27 Hughes and Munshi (1973)

1.4 Osmo-respiratory compromise in fish gills

The gills of fish present a multifunctional organ playing important roles in gas

exchange, ion regulation, acid-base regulation, and nitrogenous excretion (Evans et al., 2005)

Among these functions, it has been argued that there is a compromise between the osmoregulation and respiratory functions because to optimise gas diffusion there is a strong evolutionary pressure to minimise diffusion resistance (thin layer), while at the same time minimising unwanted water and ion movement (thick layer) It has been shown that during exercises in rainbow trout, an increase in fish oxygen consumption through gill ventilation and perfusion was associated with a significant increase in the rate of ion loss and water uptake

across the gills (Randall et al., 1972; Wood and Randall, 1973a, 1973b) This phenomenon was

then termed the “osmo-respiratory compromise” (Nilsson, 1986) Gonzalez and McDonald (1992) compared the increasing ratios between oxygen consumption (MO2) and ion loss after exhaustive exercise of the rainbow trout acclimated in freshwater and found that the ratio of ion loss across the gills was disproportionally greater than that of the MO2 Concerning fish with different lifestyles, however, Gonzalez and McDonald (1994) found that this pattern was

not the same for all the fish The authors found that active fish were able to achieve higher rates

of MO2 while effectively minimizing ion losses compared to less active species

Various mechanisms have been investigated in minimizing the compromise For example, during severe hypoxia there was no iono-regulatory imbalance found in the water-

breathing Amazonian oscars Astronotus ocellatus, with suggested mechanisms inluding

channel arrest, reduction of functional gill SA, or alterations in leakiness of gill membranes to

prevent ion imbalance in severe hypoxia (Wood et al., 2007) Further several freshwater fishes

have been shown to reduce their functional gill SA by developing an inter-lamellar cell mass (ILCM) between the secondary lamellae, thus minimising water uptake and preumably ion loss

across the respiratory epithelium (Sollid et al., 2003, 2005; Nilsson et al, 2012), as discussed

below In air-breathing fish, the gills are normally reduced to avoid the branchial oxygen loss

into hypoxic water but they are thought to retain their role in ion exchange, pH regulation, and

CO2 and nitrogenous excretion; whereas ABO assumes the oxygen uptake function (Graham,

1997; Randall et al., 1981; Brauner et al., 2004; Fernandes et al., 2012) Therefore, it has been

argued that air-breathing fish in general have a reduced compromise between gas exchange and

ion-regulation in their gills In addition, some air-breathing fish such as lungfish (Protopterus

aethiopicus) and snakeheads (Channa maculata and C argus), as well as amphibians such as

tadpoles (e.g Rana temporaria and Bufo bufo) have vascular shunts within their gills, which

offers them a non-respiratory blood flow path to the dorsal aorta to avoid branchial oxygen loss

into hypoxic water (Johansen et al., 1970; Laurent et al., 1978; Ishimatsu et al., 1979; Saint‐ Aubain and Louise, 1981) In Pangasianodon hypophthalmus, we found reduced gill SA (as a

result of ILCM growth) under low temperature and in normoxia whereas large gill SA at elevated temperature and hypoxia (Paper 2) However, when compare between the groups with ILCM and without ILCM gills during the exposure to hypercapnia, we found no differences in the rate of acid-base regulation between these two groups (Paper 4)

1.5 Gill plasticity and environmental factors causing gill plasticity

Gill morphological changes in response to changes in a variety of environmental

parameters have been found in numerous fish species Tuurala et al (1998) found that

temperature acclimation (5 and 25ᵒC) induced morphometric changes in gills of freshwater

adapted eel (Anguilla anguilla) Gill tissue volume increased by as much as 50%, associated

with an increase in epithelial volume density and in the relative size of the basal channels when exposed the eels to 5ᵒC It was also found that exposure to 5ᵒC led to a 2.5-fold increase in

to 60% of that found in fish at 25ᵒC Such increases in epithelial water-blood barrier thickness were considered an adaptive response for overwintering torpor (Tuurala et al., 1998) Soivio

and Tuurala (1981) found a significant decrease in lamella water-blood barrier thickness in rainbow trout during acute or short-term hypoxia associated with an increase in the lamellar

surface area Laurent and Perry (1991) supposed that an increase in respiratory lamellae would

be the most direct adaptive evidence for long-term exposure to hypoxia; and suggested that morphometric scaling may be influenced by oxygen requirements rather than oxygen availability

The phenomenon of gill remodeling has been discovered in several fish species, where the spaces between the secondary lamellae are partly or completely filled with a cell mass called interlamellar cell mass (ILCM), making radical changes in respiratory gill surface area

It has been suggested that with gill remodeling, fish would benefit from reduced regulatory costs, limiting uptake of toxic substances, and reducing the risks of pathogen

osmo-infections (Nilsson et al., 2012) This phenomenon was firstly discovered on crucian carp

Carassius carassius exposed to hypoxia by Sollid et al (2003), and subsequently explored in

three other species goldfish Carassius auratus, mangrove killifish Kryptolebias marmoratus, scaleless carp Gymnocypris przewalski when the fish were exposed to different temperatures, air exposure, hypoxia, or exercise (Sollid et al., 2005; Ong et al., 2007; Matey et al., 2008) To this group can now be added the striped catfish Pangasianodon hypophthalmus, where the

ILCM similarly responds to temperature and oxygen level (Paper 2, 4) but also to swimming

(Paper 3) In crucian carp (Carassius carassius) exposed to changes in the oxygen availability, Sollid et al (2003) found that the gills lacked protruding lamellae when the fish were exposed

to normoxic water; however, after exposure to hypoxic water for one day, lamellae protruded and reached the greatest extent after 7 days, increasing the respiratory surface area i by 7.5-fold in the process After one more week of recovery in normoxic water, the gills reverted to their original state and the whole gill filaments were found without protruding lamellae (Sollid

et al., 2003) It is supposed that such gill remodeling in carp is an adaptive mechanism to

promote higher capacity for oxygen uptake under hypoxic conditions and to reduce problems

related to osmoregulation cost under normoxic conditions (Sollid et al., 2003) The effect of hypoxia on gill morphology was also observed on scaleless carp (Gymnocypris przewalskii) from Lake Qinghai, China by Matey et al (2008) It was found that the gill structure changed

significantly within 8h of acute hypoxia exposure (0.3 mgO2 l-1) and changed almost completely within 24h, which was characterized with more than 50% reduction of filament

epithelial thickness, more than 60% expansion of lamellar respiratory surface area, and less than 50% reduction in epithelial water–blood diffusion distance Such changes probably also increase the capacity for gas exchange during hypoxia Here again, the changes were reversible following a 12 hours recovery of the fish in normoxic water Gill remodeling also occurs in the

crucian carp, goldfish, and striped catfish as a result of exercise (Brauner et al., 2011; Fu et al.,

2011, Perry et al., 2012) Perry et al (2012) found that at 27ᵒC goldfish gills were filled with

ILCM, but that these cells were rapidly lost during excersise and following 3h of swimming

the SA had increased by 44.8% Similarly, P hypophthalmus at 27ᵒC & normoxia have their

gills partly embedded by ILCM, but swimming for ca 20h at 30ᵒC caused a dramatic almost three-fold increase in respiratory gill SA, from 109±15 to 301±29 mm2g-1 (Paper 4)

In addition to these oxygen-related changes, environmental pollutants including heavy metals, detergents, phenol as well as unfavourable water pH also cause morphological changes However, the nature of these changes are slightly different mostly consisting of inflammation

of epithelial layers or damage to gill structures (Mueller et al., 1991; Lappivaara et al., 1995; Schjolden et al., 2007; Huang and Lin, 2011)

1.6 Methods applied in morphometric studies

It is essential to have unbiased quantitative methods when evaluating the respiratory surface area or epithelial diffusion distance to compare or evaluate the effectiveness of the respiratory organs for gas exchange among different species, and among the species in different environments Orientation differences in tissue can potentially have dramatic effects on quantitative estimates and introduce systematic errors A large number of studies have involved

in measuring gill dimensions such as gill surface area and diffusion distance of water-blood barrier thickness (Price, 1931; Gray, 1954; Hughes, 1966) There are three main measurements from these investigations: measuring the total length of gill filaments (L), estimating the number of secondary lamellae on the filaments (n), separating several specific secondary lamellae and measuring area value of these lamellae (bl) The total surface area of lamellae in fish is calculated by multiplying these values (Total area = L.n.bl) This method has been

widely used in gill morphometric studies (Hughes, 1972; Hakim et al., 1978; Mazon et al.,

1998) However, this method has potential errors It is very difficult to apply, very consuming, cannot be shown to be mathematically unbiased and is not necessarilly applicable

time-to the gills of air-breathers, which can be dramatically modified and much more irregular

structures than the classic teleost gill (da Costa et al., 2007)

Stereological methods, which have been applied for the quantitative evaluation of lungs for more than 40 years (Weibel, 1963), more recently, have been applied in gill morphometric studies Stereology is a collection of methods using geometrical and statistical principles to provide unbiased and quantitative estimates of metrics (such as volume, area, length, or number) of three-dimensional structures from measurements of its two-dimensional sections

(Boyce et al., 2010) When combined with random and systematic sampling, design-based

stereology allows for dramatic reductions in the amount of tissue required to give reliable

estimates (Boyce et al., 2010) Stereology has been applied to evaluate the respiratory surface

of highly heterogeneous lungs (Perry, 1978) Recently a stereology–base method which combined the principles of Cavalieri and surface area estimation in vertical uniform random

sections - VUR (Baddeley et al., 1986) has been applied to investigate different dimensions of the multiple gas exchange organs of the South American lungfish Lepidosiren paradoxa,

Arapaima gigas (de Moraes et al., 2005; da Costa et al., 2007; Fernandez et al., 2012) I also

applied this method (as described in details below) in measuring dimensions of respiratory

structures of P hypophthalmus

Stereological method applied in P hypophthalmus gills and swim bladder

Gills and swim bladder were carefully removed from the experimental fish and immersed in 4% phosphate-buffered formalin for at least 24h These organs were subsampled

using a smooth fractionator and systematic uniform random sampling (SURS) (Gundersen et

al., 1988) before processing for stereological analysis After histological processing, the chosen

tissues were embedded in methyl methacrylate Technovit®7100 solutions (Heraeus Kulzer,

Germany) followed the designs of de Moraes et al (2005) and da Costa et al (2007), applying vertical uniform random sections - VUR (Baddeley et al., 1986) (Fig 1.2A, B) Point and line

testing systems from the newCast stereological software VIS® (Olympus, Denmark) were applied for analysing dimensions (volume, surface area, water- or air- blood diffusion distance)

of these respiratory organs (Fig 1.2C), where volume and surface area measurements were

determined following (Gundersen et al., 1988; Howard and Reed, 1998; Michel and

Cruz-Orive, 1988), and harmonic mean of epithelial barrier thickness was determined based on

Jensen et al (1979)

Fig 1.2 Schematic drawing and light micrographs show the sampling method for stereological

observation applying VUR design for gills (A) and swim bladder (B) of Pangasianodon

hypophthalmus (C) Portion of the test array that was superimposed on the microscopic image for the

evaluation of the Cavalieri volume and surface areas The arrows indicate the vertical axis of

measurements

1.7 Objectives of dissertation

First, the disturbance caused by these three anaesthetics on blood gases and

haematological parameters of P hypophthalmus and the time course for normalisation when

allowed to recover in clean normoxic and normocapnic water were investigated

Second, we aimed to investigate the branchial surface area and how its metrics changed during growth at present and elevated temperatures in normoxic and hypoxic conditions by implementing stereological methods In addition, fish growth was assessed under these cultured conditions

Third, we aimed to investigate the ontogeny of the respiratory SA of the gills and swim

bladder of P hypophthalmus at different sizes, in addition to the harmonic mean diffusion

distance (τh) of these organs using stereology methods Then, we tested the hypothesis that

Final, we tested whether branchial remodelling affects the rate of acid/base regulation in

response to aquatic hypercapnia by acclimating P hypophthalmus to hypoxia or hyperoxia to

induce gill remodelling

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Chapter 2 (PAPER 1)

Recovery of blood gases and haematological parameters upon anaesthesia with Benzocaine, MS-222 or Aqui-S in the air-breathing catfish

(Pangasianodon hypophthalmus)

Le My Phuong, Christian Damsgaard, Do Thi Thanh Huong, Atsushi Ishimatsu,

Tobias Wang, Mark Bayley

This chapter has been published as: Phuong, L.M., Damsgaard, C., Ishimatsu, A., Wang, T and Bayley, M., 2017 Recovery of blood gases and haematological parameters upon

anaesthesia with benzocaine, MS-222 or Aqui-S in the air-breathing catfish Pangasianodon

hypophthalmus Ichthyological Research 64, pp.84-92

Abstract

Fish anaesthesia is used to minimize handling stress and damage during harvesting, transportation, and surgical procedures Through depression of cardiovascular and respiratory functions it causes significant changes in blood gases and pH Here, we present the effects of Benzocaine (100 mg l-1), MS-222 (100 mg l-1), and Aqui-S (30 mg l-1) on blood gases and

haematological parameters of commercial sized (≈1 kg) striped catfish (Pangasianodon

hypophthalmus) and the time course of recovery Blood was taken through a dorsal aorta

catheter immediately after catheterization, and regularly during the following 72 h recovery in

aerated water All anaesthetics caused increases in PCO2 and lactate resulting in a decrease in

pHe closely mirrored by RBC pHi, as well as a marked rise in Hct, associated with elevated [cortisol] and [glucose] and increased RBC counts but no change in RBC volume, as confirmed

by the lack of an adrenergic response of RBC in vitro All anaesthetics showed similar efficacy

and blood parameters were normalized within 24 to 48 h

Keywords Pangasianodon hypophthalmus · Anaesthesia induction · Haematology ·

Catheterization

List of Abbreviations

[Cl-]e plasma Cl- concentration

[Cl-]i red blood cell intracellular Cl- concentration

[cortisol] plasma cortisol concentration

[glucose] plasma glucose concentration

[lactate] plasma lactate concentration

[HCO3-] bicarbonate concentration

[HbO2] concentration of haemoglobin bound oxygen

PCO2 partial pressure of carbon dioxide

2.1 Introduction

Anaesthetics are widely used in aquaculture to diminish stress and physical injuries during harvesting or transport, and proper anaesthesia is absolutely essential to alleviate pain

during surgeries and health examinations (Coyle et al., 2004; Kiessling et al., 2009; Abdolazizi

et al., 2011) For aquatic organisms, soluble anaesthetics can be added directly to the water and

are readily absorbed across the gills, and transported by the blood to the nervous system; loss

of equilibrium and mobility follows rapidly (Coyle et al., 2004; Popovic et al., 2012) However, anaesthesia also disturbs respiratory and cardiac function causing hypoxemia (Soivio et al., 1977; Fredricks et al., 1993; Andersen and Wang, 2002) Thus disturbances of blood gases and

haematological parameters occur within seconds of administration, due to splenic contraction and adrenergic activation of the RBC sodium-proton exchanger and hence extracellular acidosis and red cell swelling (Jensen, 2004) These respiratory, haematological and metabolic disturbances during anaesthesia have been studied extensively in water-breathing fish, such as

Atlantic salmon, rainbow trout, kelp grouper, red drum, perch (Soivio et al., 1977; Iwama et

al., 1989; Thomas and Robertson, 1991; Iversen et al., 2003; Park et al., 2008; Velíšek et al.,

2009), but little is known about such responses in air-breathing fish, which often thrive at

higher temperatures than water breathers (Lefevre et al., 2014) Furthermore, it has been

suggested that air-breathers, with their reduced branchial ion and respiratory gas exchange, are less able to regulate pHe than water breathers (Shartau and Brauner, 2014) and therefore, the restoration of acid-base status and blood gases may be different from those of water-breathers

Striped catfish Pangasianodon hypophthalmus is a tropical facultative air-breathing fish, which is effective at both air- and water-breathing (Lefevre et al., 2011) Pangasianodon

hypophthalmus is intensively cultured in South East Asia, and while various anaesthetics are

widely used for transport, there is no information on the rate of recovery or physiological effects In aquaculture, MS-222 (tricaine methanesulfonate), benzocaine (ethyl para-

aminobenzoate), and Aqui-S (50 % isoeugenol) are the most commonly utilised anaesthetics (Coyle et al., 2004; Kiessling et al., 2009; Weber et al., 2009; Abdolazizi et al., 2011) Both

benzocaine and MS-222 are local anaesthetics that provide general anaesthesia in fish by blocking voltage-gated sodium channels and hence inhibit neural transmission within the central and peripheral nervous systems (Attili and Hughes, 2014) Eugenol is a widely used local analgesic agent to alleviate tooth pain that shares several pharmacological actions with local anaesthetics, including inhibition of voltage-gated sodium channel as well as activation

of transient receptor potential vanilloid subtype 1 (TRPV1) (Park et al., 2009) Here we

evaluate the disturbance caused by these three anaesthetics on blood gases and haematological

parameters of P hypophthalmus and the time course for normalisation when allowed to recover

in clean normoxic and normocapnic water In addition, since adrenergic swelling responses have been found in erythrocytes of numerous water-breathing fish (Nikinmaa and Huestis, 1984), leading to an increase in cell volume and changes in hematocrit, and since we expected

pH and hematocrit changes, we also investigate the existence of this β-adrenergic response in the present study

2.2 Materials and Methods

Fish

Striped catfish Pangasianodon hypophthalmus weighing 700-1000 g were transferred

from local farms to tanks with aerated water at the Department of Aquaculture, Can Tho University (Vietnam) several weeks before the study commenced During this period, they were fed to satiation with commercial pellets on a daily basis, but fasted for 24 h pre-instrumentation

Anesthetics preparation

Benzocaine was pre-dissolved in 3ml ethanol 70 % and mixed within water (100 mg l

-1, Florindo et al., 2006) Aqui-S was dissolved directly into the water at 30 mg l-1 (Iversen et

al., 2003) MS-222 was dissolved at 100 mg l-1 tank water with 100 mg l-1 NaHCO3 used as a

buffer (Iwama et al., 1989)

Experimental procedures

Each fish was kept in the induction chamber until total loss of equilibrium and reaction

to touch (Iwama et al., 1989; Coyle et al., 2004) When anaesthetized, the fish was catheterized

into the dorsal aorta using polyethylene tubing (I.D 0.58 mm, O.D 0.96 mm) containing heparinized saline (50 IE ml-1) (Soivio et al., 1975), whilst the fish gills were constantly

irrigated with aerated water containing one third of the initial dose of anaesthesia Times required for induction time and catheterization time were recorded for each fish An arterial blood sample was collected from the catheter immediately after catheterization (0 h) and subsequently at 3, 6, 24, 48, and 72 h of recovery, whilst the fish were maintained in a 200 l tank containing aerated water Great care was taken not to disturb the fish during recovery Water temperatures during recoveries were 21.6±0.2, 24.2±0.1, and 25.1±0.2 ºC in Benzocaine, Aqui-S, and MS-222, respectively

Blood collection and treatment

Each blood sample (0.3 ml) was collected using a 1 ml syringe, carefully avoiding air

bubbles, for measurements of PCO2, pHe, and [lactate] using an iSTAT blood gas analyser (Abbott Laboratories, Abbott Park, Illinois, USA); 0.7 ml of blood was transferred to a 1.5 ml Eppendorf tube and kept on ice for immediate determination of other haematological parameters, such as Hct, [Hb], and RBC counts The remaining blood was centrifuged at 6000 rpm for 6 min, to separate plasma and RBC and stored at -80ºC for subsequent analysis of [glucose], [cortisol], [Cl-]e, and [Cl-]i

Measurement of the haematological and biochemical parameters

Hct was determined by centrifugation in a standard microhaematocrit centrifuge Hb concentration was determined by Drabkin’s method; spectrophotometrically at 540 nm using

an extinction coefficient of 10.99 mmol-1 cm-1 (Zilstra et al., 1983) Plasma glucose

concentration was determined according to Huggett and Nixon (1957), and [cortisol] was determined using a DRG Salivary Cortisol ELISA commercial Kit (USA) RBC counts were determined by counting the number of RBC in a Neubauer chamber under a microscope after diluting 200 times of 0.5 µl blood sample with Natt & Herrick’s stain solution [Cl-]e was measured using a chloride titrator (Sherwood model 926S MK II chloride analyser) For [Cl-]i,

a known mass of RBC pellet was transferred to a known volume of distilled water to induce cell lysis, and Cl- measured in the haemolysate RBC water content was measured gravimetrically in another RBC aliquot from the same centrifuged cell pellet before and after drying at 60ºC for 16 h

In vitro assessment of red cell adrenergic response

Four fish were catheterized under anaesthesia with benzocaine and allowed to recover for at least 48 h before a 3 ml blood sample was taken Blood was placed in an Eschweiler tonometer (Kiel, Germany) and equilibrated with humidified gas mixtures supplied from two serially linked Wösthoff gas mixing pumps (Bochum, Germany) Initially, blood was equilibrated with 30 % O2 (PO2 = 216 mmHg) and 3 % CO2 (PCO2 = 21.6 mmHg) to determine blood O2 carrying capacity, and then reduced to a PO2 of 15.1 mmHg (approximately 10 % air)

at 3 % CO2, resulting in HbO2 saturations of 15-30 % At this PCO2, pHe is expected to be 7.35

(Damsgaard et al., 2015) The beta-adrenergic agonist isoprenaline was added to the blood to

a final concentration of 10-5 mol l-1 (Brauner et al., 2002; Koldkjær et al., 2002) At both steps

the concentration of Hb bound O2 ([Hb-O2]), Hct and [Hb] were determined

PCO2 (true) [mmHg] = -2.17 + 1.20 PaCO2 (iStat)

Plasma [HCO3-] was calculated from the Henderson Hasselbach equation

[HCO3−] = αCO2 ∙ 𝑃CO2∙ 10pHe −pK

where αCO2 is the temperature compensated CO2 solubility in trout plasma (Boutilier et

al., 1985) and pK is the pHe corrected dissociation exponent for CO2 in P hypophthalmus plasma (Damsgaard et al., 2015)

[Hb-O2] was determined by measuring [O2]total and subtracting physically dissolved O2

[Hb − O2] = [O2]total− 𝛼O2∙ 𝑃O2where [O2]total was determined according to Tucker (1967), αO2 is the temperature compensated solubility of O2 (Dejours, 1981) and PO2 the partial pressure of O2 in the gas mixture

O2 saturation of Hb (HbO2 sat) during equilibration with 10% air was calculated as

Saturation = [Hb − O2]10% air

[Hb − O2]30% O2MCHC was calculated as

isoprenaline exerted significant effects on the red cells in vitro A probability (P) value at the

0.05 level was considered as significant

2.3 Results

Time required for induction of anaesthesia, surgery and recovery stage

A surgical plane of anaesthesia was 3.1±0.2 min for benzocaine, whereas it took 9.3±0.9 and 8.1±0.7 min for MS-222 and Aqui-S, respectively (Fig 2.1) Regardless of anaesthetic, the catheter was inserted and secured within the dorsal aorta in less than 15 min and the fish regained equilibrium within 2-4 min (Fig 2.1)

Fig 2.1 Time recorded for duration of anesthesia to reach the surgical plane, cannulation, and

post-operative recovery of Pangasianodon hypophthalmus with the three anesthetics Time values are presented as means ± S.E.M (n = 7) Significant differences between anesthesia treatments,

cannulation, and post-operative recover are indicated with *, #, and +, respectively

Haematological and biochemical parameters

Following full anesthesia, Hct, RBC counts and [Hb] changed in a similar manner, with maximal disruption immediately after catheterization, followed by a rapid decrease within 6h

of recovery (Fig 2.2a‒c) MCHC was stable around 25 mmol l-1 throughout the entire recovery period except for with MS-222 (Fig 2.2d)

All three anaesthetics caused high [glucose] and [cortisol] immediately after surgery Plasma glucose was high initially within 0-3 h, then decreased gradually during recovery and normalized at approximately 3 mmol l-1 at 72 h (Fig 2.2e) Cortisol concentrations fell by more than a factor of 5 during recovery, and stabilized at approximately 30 mmol l-1 within 24 h (Benzocaine and MS-222) to 48 h (Aqui-S) (Fig 2.2f)

(d) (c)

(f) (e)

m

a

b a

b a

m

b b

a

a

m

a a b

b

b a

a a

m m

m

b b a

a a

Fig 2.2 Hematological and biochemical parameters of arterial blood following anesthesia in

Pangasianodon hypophthalmus with Aqui-S (green), MS-222 (red) and Benzocaine (blue) with (a)

Hct, (b) RBC counts, (c) [Hb], (d) MCHC, (e) [Glucose], (f) [Cortisol] Letters a,m,b indicate

significant difference (P < 0.05) from 72 h, for Aqui-S, MS-222, and Benzocaine respectively (one

way RM ANOVA) Values are presented as means ± S.E.M (n = 7)

Acid-base status and chloride ions

Immediately after surgery, there was a significant plasma acidosis, which rapidly returned to normal values over the subsequent 6-24h (Fig 2.3a); pHi followed a similar pattern,

with a difference between pHe and pHi of around 0.2-0.3 units throughout the experiment for

all three anaesthetics (Fig 2.3b) Arterial PCO2 was elevated during this time and this elevation was most pronounced in Benzocaine (up to 6 mmHg) During recovery from anaesthesia with

all three compounds, PCO2 decreased to approximately 2.5 mmHg (Fig 2.3c) Similarly, all three anaesthetics caused elevated [lactate] which remained high at 6-8 mmol l-1 immediate

after catheterization In line with PCO2, lactate also recovered almost completely to approximately 0.3 mmol l-1 at 24h post operation (Fig 2.3d) Immediately after surgery with Aqui-S and MS-222, [HCO3-] was depressed, but stabilized within 6 h of recovery at approximately 9 mmol l-1 (Fig 2.3e) No significant changes were observed in [Cl-]e and [Cl-]i

after anaesthesia, with the concentrations being approximately 100 and 60 mmol l-1, respectively (Fig 2.3g, h) Davenport diagrams (Fig 2.4a, b, c) show the respiratory status of

Pangasianodon hypophthalmus during and after anesthesia with the three anesthetics and show

that the low pHe immediately after surgery can be largely ascribed to a metabolic acidosis with

a minor respiratory component

7.0 7.2 7.4 7.6 7.8 8.0 8.2 8.4

Aqui-S MS-222 Benzocaine

(a)

(d)

(e)

(h) (f)

a

b b

Fig 2.3 Acid-Base status and chloride ions of arterial blood following anaesthesia in Pangasianodon

hypophthalmus with Aqui-S (green), MS-222 (red), and Benzocaine (blue) with (a) pHe , (b) pH i , (c)

PCO2 , (d) [Lactate], (e) [HCO 3-], (f) gH 2 O/g RBC dried weight, (g) [Cl - ] e , and (h) [Cl - ] i Letters a,m,b

indicate significantly different (P < 0.05) from 72 h for Aqui-S, MS-222, and Benzocaine, respectively (one way RM ANOVA) Values are presented as means± S.E.M (n = 7)

14 Benzocaine

0h

6h

3h 72h

3h

6h

72h

Fig 2.4 Davenport diagrams of (a) Aqui-S, (b) MS222, and (c) Benzocaine with the curved dotted

lines indicating PCO2-isopleths and dashed lines indicating in vitro buffer lines taken from

Damsgaard et al (2015) Values are presented as means ± S.E.M (n = 7)