Combined effects of temperature and salinity and induced stress on some hematological parameters of tra catfish (pangasianodon hypophthalmus) fingerlings

Three separate experiments were carried out to assess combined effects of temperature and salinity and induced stress on blood variables of Tra catfish (Pangasianodon hypophthalmus) fingerlings. Some hematological parameters included Haemoglobin concentration, glucose and pH levels which were tested in different conditions. The experiment 1 aimed to test how blood variables change in different temperature (24, 28-30, 32 oC) and salinity (0, 6 and 12 ppt) while the experiment 2 induced catfish fingerlings to different stress time (5, 15 and 30 min.).

COMBINED EFFECTS OF TEMPERATURE AND SALINITY AND INDUCED STRESS ON

SOME HEMATOLOGICAL PARAMETERS OF TRA CATFISH (Pangasianodon

hypophthalmus) FINGERLINGS

Dang The Luc1

1 An Giang University, VNU - HCM

Information:

Received: 17/10/2018

Accepted: 08/07/2019

Published: 11/2019

Keywords:

Pangasianodon hypophthalmus,

temperature, salinity,

Haemoglobin, glucose, stress

ABSTRACT

Three separate experiments were carried out to assess combined effects of temperature and salinity and induced stress on blood variables of Tra catfish (Pangasianodon hypophthalmus) fingerlings Some hematological parameters included Haemoglobin concentration, glucose and pH levels which were tested

in different conditions The experiment 1 aimed to test how blood variables change in different temperature (24, 28-30, 32 oC) and salinity (0, 6 and 12 ppt) while the experiment 2 induced catfish fingerlings to different stress time (5, 15 and 30 min.) Additionally, combined effects of all factors (temperature, salinity and induced stress) on fish blood parameters were also determined in the experiment 3 Results showed that in normal condition, the mean Hb concentration and glucose levels of blood varied from 4.36 – 4.90 g/dL, and from 3.83 – 5.23 mmol/L, respectively and mean pH ranged from 7.73 – 8.20

The concentration of Hb, glucose and pH levels changed when temperature increased from 24 to 32oC After 30 stress minutes, there was only glucose levels influenced Moreover, there were significant interaction effects among temperature, salinity and stress level on blood variables The findings revealed that the physiology of Tra catfish fingerling could be affected after 5 stress minutes at temperature of 24oC and 32oC with salinity of 6 and 12 ppt All data

of experiments showed that a positive correlation between Hb concentrations and glucose levels and negative correlation between Hb concentrations and pH levels were found Results also showed that changes in blood variables could cause stress for aquatic animal, especially Tra catfish fingerlings

1 INTRODUCTION

Catfish is one of the most important species

cultured in the MeKong Delta, which brings the

main income to An Giang farmers However,

despite of climate change, there are some negative

impacts on catfish farming, especially when

temperature and salinity have changed

Many previous studies have been carried out to

assess effects of rising temperatures, salinity

levels and the interaction between temperature and salinity on fishes (Kemp, 2009; Wright and Tobin, 2011; Nguyen et al., 2015) These studies have all concluded that fish health including growth rates, survival rates and physiological responses could be negatively affected by significant changes

The increase of temperature could affect the metabolism of aquatic animals as well as the

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growth rate of fish (Kemp, 2009) Modified

salinity is a factor causing stress and affecting fish

physiology (Fashina-Bombata & Busari, 2003;

Konstantinov & Martynova, 1993; Partridge &

Jenkins, 2002; Sink, 2010) Stress is a popular

physiological status in fish or other species in

order to deal with changes of the environment to

survive and maintain internal balance of animals

(Fuzzen et al., 2011)

Previously, stress indicators included changes in

plasma glucose and pH levels in fish, especially

Tra catfish, were examined (Kirchhoff et al.,

2014; Nguyen et al., 2014) In addition,

Haemoglobin concentration is also considered as

an indicator to evaluate overall physiology and

general health in fish (Houston, 1997) However,

it is necessary to fully collect more data on blood

variables in Tra catfish's fingerlings in the

condition of climate change in Mekong Delta,

Vietnam

Therefore, the study “Combined effects of

temperature and salinity and induced stress on

some hematological parameters of Tra catfish

(Pangasianodon hypophthalmus) fingerlings”

was conducted to fully understand about Tra

catfish fingerling’s health This also contributes to

establish some blood variables’ baseline for Tra

catfish, which helps to determine and manage fish

health status more effectively As a result, the

production cycle of Tra catfish could be enhanced

and sustainably developed in the condition of

climate change in Mekong Delta

2 MATERIALS AND METHODS

2.1 Experimental system and source of catfish

Catfish fingerlings (25.96 ± 0.67 g/fish) were

obtained from a hatchery farm located in Dong

Thap province Fish were in good conditions and

fed commercial feed (40% crude protein) during

the experiment Individuals were acclimated for a

month before transfered to 80L tanks in

freshwater at 28-30°C (maintained by heaters)

with a continuous supply of well-aerated water

After the acclimatation period, fish were arranged

into different experiments

The study was carried out from December 2016 to October 2017 at the wetlab of An Giang University, Long Xuyen city, An Giang province

2.2 Experimental design

The experiment 1: A two-factor designs, where

three temperature (T) levels (24°C, 28°C – 30°C and 34°C) and three salinity (S) levels (0 ppt, 6 ppt and 12 ppt) were tested in triplicate combinations (TxS)

In this experiment, with a density of 30 individuals/tank, fish fingerlings were accustomed gradually to required salinity rates by replacing freshwater in each tank with saline water in a stepwise fashion, which was 2‰ per day to prevent shock to fish, until all tanks had reached their target salinities Individuals were then kept within 24 hours to be stable

Finally, the temperature levels were modified by ice and heaters in a stepwise fashion, which was not higher than 2°C per 3 hours until all tanks had reached their target temperature levels Fish blood was immediately collected from the caudal veins within 5 min of sampling with 1 mL heparin-coated syringes after being anaesthetised at the following time intervals: 0h, 1h and 24h

The experiment 2: According to Lopez-Patino et

al (2014), fish was stressed by chasing (using hand-nets to catch up fish) within 5, 15 and 30 minutes After stressed time, fish blood was immediately collected from the caudal veins as in the experiment 1

The experiment 3: Fish was challenged by a

three-factor design which combined different temperature (T), salinity rates (S) and stress levels (St) in triplication This experiment included 27 treatments Temperature and salinity rates were adjusted as the experiment 1 before fish was chased as in the experiment 2 After that, fish blood was immediately collected from the caudal veins within 5 min of sampling with 1 mL heparin-coated syringes after being anaesthetised

2.3 Sampling collection and analysis

Fish fingerlings were anaesthetised by 0.3 ppm of MS-222 Fish blood was collected from caudal veins Each sample contained 2 mL in heparinised

Vacutainer® tube (BD, USA) All samples were

stored with ice before being analysed

Haemoglobin (Hb) concentration was determined

by using a haemoglobin meter, ACON Mission®

Hb Plus Meter (ACON Laboratories Inc., USA)

Glucose concentration was determined by using a

glucose meter test, ACON On Call Plus Meter

(ACON Laboratories Inc., USA) Blood pH was

determined by using a pH meter, Horiba 11

(LAQUAtwin, Japan)

2.4 Statistical analysis

All data were analysed by SPSS 20.0 for

Windows (SPSS Inc., Chicago, IL, USA) Means

and standard errors were calculated for blood

variables including Hb concentration, levels of

glucose and pH The differences among

treatments within experiment were determined by

two-way and three-way analysis of variance

(ANOVA) at a significance α=0.05 for significant

main effects (temperature, salinity or stressed

time) and their interactions on blood variables All variances were homogenous by Levene's test Relationships between blood variables were determined using all data and Spearman's rank correlation coefficients Homogeneity of variance was tested with Levene’s test and the means of blood variables were compared using Duncan test

3 RESULTS

3.1 The experiment 1

3.1.1 Haemoglobin

There was an interaction between temperature and salinity on Hb concentrations Hb concentrations were unstable during this study, especially there was an increase in Hb concentration at 24 0C, 0 ppt after 24 hrs (shown in Table 1) when Hb concentration reached the highest concentration (6.03 ± 0.45 g/dL) The lowest one (2.90 ± 0.10 g/dL) was at 240C, 6 ppt, 0 hr The Hb concentration in a normal condition was in range

of 4.36 – 4.90 g/dL

Table 1 Mean Hb concentrations (g/dL) between treatments in the experiment 1

Mean values having different letters within columns are significant difference (p<0,05)

3.1.2 Glucose

An significant interaction effect between

temperature and salinity (TxS) was found on

glucose level Glusoce level was unstable during

this study (shown in Table 2) The highest level

(6.60 ± 0.20 mmol/L) was found at 340C, 0 ppt, 0

hr while the lowest (2.16 ± 0.35 mmol/L) was at

340C, 6 ppt after 1 hr The glucose level in a normal condition was in range of 3.83 – 5.23 mmol/L

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Table 2 Mean glucose levels (mmol/L) between treatments in the experiment 1

Mean values having different letters within columns are significant difference (p<0,05)

3.1.3 Blood pH

There was a change in pH level in fish blood The

pH level in a normal condition was in range of

7.73 – 8.20 In addtion, an interaction between

temperature and salinity (TxS) was found on pH

level As Hb concentration and Glucose level, pH

level was also unstable during this study (shown

in Table 3) At the control temperature treatment,

pH level increased and reached the the highest one (9.16 ± 0.25) when fish were in 12 ppt, 0 hr However, after after 1 hr of acclimation, that level decreased significantly to the lowest (3.33 ± 0.05)

Table 3 Mean pH levels between treatments in the experiment 1

Mean values having different letters within columns are significant difference (p<0,05)

3.2 The experiment 2

It is showed that in the experiment 2, after 5, 15

and 30 stressed minutes, significant differences in

Haemoglobin and pH levels between treatments

were not found However, there was a significant

difference in glucose levels after 30 stressed minutes (shown in Table 4) Glucose levels increased gradually after a period of time (from 5

to 30 min.) chased by hand-nets

Table 4 Mean glucose levels (mmol/L) between treatments in the experiment 2

Mean values having different letters within columns are significant difference (p<0,05)

3.3 The experiment 3

3.3.1 Haemoglobin

There was an interaction between temperature,

salinity and stress levels (TxSxSt) on Hb

concentration in Tra catfish fingerlings The

highest Hb concentration (6.45  0.35 g/dL) was

found at 24 0C, 12 ppt after 15 min while the lowest (2.93  0.12) was found at control temperature, 12 ppt also after 15 min It is indicated that Hb concentration in treatments was different significantly and differed from each other after 30 stressed minutes (shown in Table 5)

Table 5 Mean Hb concentrations (g/dL) between treatments in the experiment 3

Mean values having different letters within columns are significant difference (p<0,05)

3.3.2 Glucose

There was an interaction between temperature, salinity and stress levels (TxSxSt) on glucose levels in Tra catfish fingerlings The highest glucose level (10.93  3.91 mmol/L) was found at 24 0C, 0 ppt after 30 min while the lowest (2.97  0.06) was found at 34 0C, 6 ppt after 5 min It is statistically confirmed that glucose levels in treatments was different significantly and fluctuated after 30 stressed minutes (shown in Table 6)

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Table 6 Mean glucose levels (mmol/L) between treatments in the experiment 3

Mean values having different letters within columns are significant difference (p<0,05)

3.3.3 Blood pH

It is indicated that an interaction between

temperature, salinity and stress levels (TxSxSt)

was found on pH level in Tra catfish fingerlings

The highest pH level (8.85  0.67) was found at

34 0C, 12 ppt after 5 minutes In contrast, the

lowest (4.90  1.29) was found at control temperature, 6 ppt after 30 minutes In addition,

pH levels in treatments was different significantly and differed from each other after 30 stressed minutes (Table 7)

Table 7 Mean pH levels between treatments in the experiment 3

Mean values having different letters within columns are significant difference (p<0,05)

3.4 The relationship between blood variables

In this study, there was a significant correlation

between Hb concentrations and glucose levels

examined at 0h and 1h in the experiment 1 In the

experiment 3, there was also a significant

correlation between Hb concentrations and pH levels when combined all figures However, at different stressed times, no siginificant correlation was found

Figure 1, 5 The correlation between Hb concentrations and glucose levels in the experiment 1

Figure 6 The correlation between Hb concentrations and pH levels in the experiment 3

4 DISCUSSION AND RECOMMENDATION

Haemoglobin is a complex protein including a

globin (96%) combined 4 Heme (4%) Red blood

cells contain 90% Hb which makes these cells

become red (Nguyen Van Tu, 2005) Hb

concentration in blood is considered as a factor to

evaluate carrying oxygen ability, also to satisfy

the essential oxygen demand and determine fish

health and physiology, especially anaemia in fish

(Houston, 1997)

In this research, when the temperature and salinity

changed, there was a variation in Hb

concentration At 240C after 24 hours being kept

in the experiment 1, fish was likely to be in oxygen deficiency In this condition, fish boosted the respiratory system to concentrate a large amount of Hb to carry oxygen to tissues Therefore, Hb concentration increased and was higher than fish in normal condition However, the low Hb concentration could explain that fish might be in anemia or parasite infection

There was a significant difference in Hb concentration (p<0.05) in the experiment 1 and 3 This would be warned that in the global changes,

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some disturbances happened in short terms could

affect fish health When fish in unhealthy

condition, transportation fingerlings from nursery

to grow-out ponds could lead to the low survival

rate and diseases

According to Kirchhoff et al., (2014), as

compared to tuna, Hb concentration changed from

22.29 – 28.68 g/dL within 2 years from 2010 to

2012, which showed the unstableness of blood

variables in aquatic animals This is similar to

results when Hb concentration varied from 2.90 –

6.45 g/dL Hb concentration of Tra catfish is

lower than tuna because tuna is an active species

in ocean

There was an interaction between temperature and

salinity on glucose level in Tra catfish Glucose

level increased when fish gets stressed and needs

a large amount of glucose to use Nguyen Van Tu

(2005) pointed that glucose is one of main

ingredients in plasma and its level changes

depended on the environment and fish health

condition In freshwater species, the relationship

of glucose level and fish behaviour is not clear but

there is difference between species When fish

becomes active, glucose level would increase but

at the some points, glucose level would decrease

This result is also supported by Nguyen et al

(2015) when the interaction between temperature

and salinity was described to affect blood

parameters on Tra catfish Hb concentration

increased to higher than 8 g/dL when temperature

was higher than 30 0C Besides that, their study

also confirmed cortisol levels increased with

temperature and salinity

This is also similar to findings of Nguyen Loan

Thao et al., (2013) when they concluded that the

higher salinity is, the more plasma glucose level

increases in Tra catfish (Pangasianodon

hypophthalmus) After 141 days, plasma glucose

level changed from 1.38 – 1.83 mmol/L

However, in this research, glucose level was

higher than previous study from 0.77 – 4.76

mmol/L Results showed that glucose level was

affected significantly by the interaction of temperature and salinity

According to Nguyen Thi Kim Ha and Do Thi Thanh Huong (2014), the increase of plasma glucose level of Tra catfish fingerlings

(Pangasianodon hypophthalmus) could be caused

by acute or chronic stress which is due to the conversion from glycogen to glucose This is related to catecholamine and cortisol concentrations The difference of treatments in the experiment 2 could be explained by disturbance of stress activity Stress can cause the increase of CO2 concentration The lack of oxygen in blood leads to the increase of respiration rate in fish to satisfy oxygen demand Plasma glucose level will increase to deal with the environmental changes (i.e temperature and salinity changes) Previous study pointed that when fish got stressed after 48 hours, it also remained a high glucose level in blood, accounted for 3.3 mmol/L (Moraes & Bidinotto, 2000)

The glucose level of fish in the experiment 2 is similar to findings of Nguyen Thi Kim Ha & Do Thi Thanh Huong (2014) They found that glucose levels of Tra catfish was 3.33 – 6.11 mmol/L after 2h, 4h and 6h transportation This research showed that only 30 minutes of induced stress by chasing, Tra catfish fingerlings got stressed through visual observation as fingerlings started to swim and react slowly after 15 stressed minutes After that, on the surface of tanks, there was a number of air bubbles As compared to eels, glucose levels were higher In eels, this level was from 1.95 – 4.24 mmol/L (Nguyen Huong Thuy

& Do Thi Thanh Huong, 2010)

Plasma pH is one of important factors to reflect physiological status of animals and changes of the environment pH levels depend on the ratio of blood H+ and OH- Mean pH levels varied from 7.52 – 7.71, not stable as mammals (Nguyen Van

Tu, 2015) During this study, plasma pH levels changed unstably and were influenced when fish got stressed because of changes of acid lactic concentration According to Dang (2015), there

was a negative correlation of pH levels and lactate

concentration in blood When lactate

concentration was high, there was also an increase

of metabolic rate to deal with disturbance This

leads to a decrease of pH level

Other finding to support this research is that if

CO2 concentration increases, this could lead to a

low pH level After a physiological process, CO2

concentration increases followed by a decrease of

carbonic acid, which causes higher pH level This

could explain the reason why pH level in this

study varied However, there is not enough

information to describe in detail how pH level

changes in fish in bad condition The variation in

pH level of this study was in accordance with the

research of Dang (2015)

Relationships between Hb concentration, glucose

and pH levels in this study were found to support

that when fish gets stressed, blood variables could

be influenced This result is similar to findings of

Dang (2015), Pearson & Stevens (1991), Wells et

al (1986) and Graham & Dickson (2001) All

previous studies concluded that Hb concentration

increased when Rainbow Trout, Yellowtail Tuna,

Snapper got stressed There was over 20% of Hb

concentration stored at normal condition When

fish got stressed, there was over 90% of stored Hb

to be released In Snapper, Hb concentration in

stressed fish was 40% higher than healthy fish

(Wells et al., 1986)

In summary, blood variables could be used to

evaluate changes of health and physiological

status in fish, especially Tra catfish However, it is

essential to repeat this research at different

temperature and salinity rates in different sizes of

Tra catfish Also, it is recommended to compare

how different blood variables would be when

using kit test and lab analysis

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