5.1 Effect of heat stress on the hematological parameters
The study of hematological characteristics in cultured fish species is an important tool in the development of aquaculture system (Mauri et al. 2011). It is necessary to know how the basic environmental factors influence fish health.
In this study, there was a significant increase in counts of RBC and WBC, concentration of hemoglobin (HGB) and the percentage of hematocrit (HCT) in the heat stress group. The concentration of RBC and HGB increased because the high water temperature increased the body metabolic rate in the experimental fish. Therefore, the fish responded by producing more red blood cells and hence hemoglobin so as to transport more oxygen and meet the increased demand for oxygen in the fish body to supply sufficient oxygen for the increased metabolic rate (Dagoudo et al. 2021). Also as the temperature increases, oxygen absorption by RBC decreases. Thus, the body compensates this reduction by increasing the amount of red blood cells in circulation. This was also reported by De et al. (2019) on the study on effects of water temperature and diet on blood parameters and stress levels in juvenile hybrid grouper. Hemoglobin (HGB) is the main oxygen-carrying protein in RBC, and affects the transport capacity of blood oxygen and the numbers and function of RBCs (Bao et al. 2018). Yellow catfish is a freshwater ectothermic fish and its metabolism is highly temperature-dependent (Avvakumov al. 2010). Furthermore, this study showed that the observed increases in RBCs are combined with increased levels of HGB to satisfy a higher oxygen demand needed for higher metabolic requirements under heat stress (Dagoudo et al. 2021). It also was noted that the RBC counts and HGB levels were decreased after 96 hours of heat stress, probably because the fish anti-stress mechanism gradually weakened with time hence the slight decrease at 96 h. Bao et al., (2018), demonstrated similar changes in the GIFT tilapia. Blood is an important part of the immune system, and changes in blood parameters can be used to assess the physiological health of fish (Li et al. 2010). WBCs are another important component of blood is which are involved in cellular immunity.
In the current study, increases in the numbers of WBCs showed that heat stress affected the immune function in the juvenile Yellow catfish. The increase in numbers of WBCs within this study hint at possible stimulation of the nonspecific immune system of the experimental fish. Similar results have been reported by Huang et al. (2018) who found a decrease in non-specific immunity after heat stress in Prenant's Schizothoracin (Schizothorax prenati). Therefore, the significant increase of WBC in the fish under heat stress (350C) may be due to cope up the stress.
In this study the concentration of hematocrit (HCT) increased due to increase in the number of erythrocytes (Red blood cells) and level of hemoglobin or a decrease in plasma volume. The increased HCT enhances the function of RBCs within the normal range of viscosity, and the greater number of RBCs and higher HGB concentration promote the transport of oxygen (Bao et al. 2018). This explains why the changes in HCT in the heat stress group showed the same trend as the changes in concentration of Red Blood Cells (RBC) and hemoglobin. In other comparative studies, the seawater flathead grey mullet (Mugil cephalus) and the freshwater goldfish (C. auratus) presented significant hematological variations. Higher values of RBC and HCT, were reported in the grey mullet in respect to goldfish (Parrino et al. 2018).
4.2 Effect of heat stress on Serum biochemical parameters
Serum biochemical parameters, such as serum glucose concentration, ALT, AST activity and TP & TC levels, are related to temperature stress in fish (Liu et al. 2016). Alanine aminotransferase (ALT) and Aspartate aminotransferase (AST) in the mitochondria can be released to the plasma following the tissue damage and organ dysfunction. Several studies have shown that changes in the activities of ALT and AST in serum usually directly indicate cell damage in specific organs, because the two aminotransferases are present in mitochondria and can be released into the serum as a result of tissue damage and organ malfunction (Liu et al. 2016).
The increased activities of ALT and AST observed under heat stress group treatment in this study indicate that the enhanced ALT and AST activities could be due high water temperature stress. Several findings have showed that environmental stressors can lead to an increase of the plasma ALT and AST activities in the fish, implying that both ALT and AST can serve as an indispensable indicator capable of monitoring the changed fish physiology following the exposure to environmental stressors such as high water temperature (Li et al. 2019). In this study, ALT and AST levels significantly increased in the heat stress group from 6h up to 96 h. Thus, the increased levels of AST and ALT activities in blood plasma at higher temperature indicated that organ dysfunction occurred in the experimental fish under heat stress. Similarly, previous studies indicate that a sharp increase of ALT and AST is observed in bream after thermal stress (Ming et al. 2012).
ALT is an important liver enzyme that reflects liver damage (Senior 2012). In this study, ALT activity increased significantly from 6 hours to 96 hours. When liver and myocardial cells are impaired or when their permeability rises, AST and ALT are released into the blood, leading to the increase of blood transaminase activity. The ALT and AST are both very important transaminases that are transformed between catalytic amino acid and ketonic acid. In our study, when the activity of ALT and AST both increase under heat stress for 6 to 96 hours, the damage by high temperature to the fish's liver cells may have caused the emission of ALT and AST, which is similar to the previous studies on catfish (Horabagrus brachysoma) and rainbow trout (Oncorhynchus mykiss). Therefore, the activities of serum AST and ALT can be used to monitor the health status of fish (Javed and Usmani 2019).
Serum TC is the sum of all cholesterol in the blood in the form of different lipoproteins, and the liver is the main organ for synthesis and storage of cholesterol (Komprda et al. 2014). In this current study, there was a significant increase in serum TC levels under heat stress in the juvenile yellow catfish. The changes in serum TC show that high temperature stress triggered some liver damage.
Similarly, in this study, serum TP increased significantly from 6 hours to 48 hours in the heat stress group. Serum TP maintains the balance of plasma colloid osmotic pressure and pH, and functions in transportation, coagulation, immunity, and energy supply. Thus, increases in serum TP are due to decreased water content in serum (Li et al. 2013), which reflects the reduction in water content of blood and hence the concentration of blood during stress. This result also confirms the increase in blood density reflected in the HCT changes. Serum total protein levels can be used as a diagnostic tool and a valuable test for evaluating the general physiological state in fish.
Glucose is a significant nutrient in serum, which is controlled by the external and internal environment. In this, study, the rapid increase in the concentration serum glucose at 6h in the heat stress group could have been due to mobilization of glucose from muscle and liver sources. When fish are exposed to heat stress, muscle and hepatic sources of glucose are rapidly mobilized which results in the sharp increase in serum glucose to supply the required energy in counteracting stress. It was reported that serum glucose levels in silver catfish (Rhamdia quelen) and Coilia ectenes Jordan (Coilia nasus) increase at high water temperature (Qiang et al. 2016). Stressful stimuli stimulate rapid secretion of both glucocorticoids and catecholamine from the adrenal tissue offish both hormones produce a rapid hyperglycemia. Frequent occurrence of accelerated metabolism during exposure to environmental stressors has been demonstrated in several studies about hyperglycemia during stress, because plasma glucose levels are positively correlated with metabolic rate.
In this current study, the level of serum TG increased significantly with increasing time duration (P < 0.05) in the heat stress group. The concentrations of TG increased with probably because the high temperatures accelerated metabolic rate of the fish thus causing a rise in the demand for more energy of the juvenile yellow catfish. Therefore, the observed significant increase in the serum TG, was probably due to the mobilization of lipid reserves to cope with an increased energy demand in the juvenile yellow catfish under heat stress (Stress et al. 2005). Acute and chronic stress is typically associated with increased metabolic rate. The slight decrease in TG levels at 12h was probably to inhibition of lipid metabolism by high temperature. Caijuan (Li et al. 2019), similarly demonstrated that the concentration of TG decreased with increasing temperatures, suggesting that high temperatures may inhibit the lipid metabolism of pikeperch. It was noticed that the concentration of serum glucose, TP, ALT activity, and TC slightly decreased at the end of the experiment. This could be that the fish could no longer adapt their physiology in response to high temperature stress by the end of the experiment.
4.3 Effect of Heat Stress on the Liver Biochemical Parameters
In vertebrates, stress negatively affects body homeostasis and triggers a series of physiological and immune responses, with liver playing a key vital role. The Liver responds with changed metabolism, enabling the stressed animal to cope with the stress situation, which involves carbohydrate and lipid mobilization. The liver of fish is a sensitive indicator of metabolic adaptation to varying environmental influences such as high water temperature.
The total protein is a measure of the total amount of two classes of proteins found in the fluid portion of blood, albumin and globulin. Albumin protein which accounts for half of the total protein found in blood plasma helps to prevent fluid from leaking out of blood vessels. It also regulates the osmotic pressure in the plasma to prevent water from leaking out of the blood vessels. While globulins are an important part of the immune system. Total Proteins are essential for overall health of fish. During stress the immune activity decreases in the fish body.
In this study, a significant decrease in the levels of Liver TP was observed in the Heat stress group after 12 hours up to 96 hours during the experiment. The significant decrease (P < 0.05) in liver protein level might be due to impaired protein synthesis caused by liver disorder during heat stress. The TP concentrations decreased with time at high temperature (350 C). The decline in the concentrations of TP could have been caused by changes in the structure of the fish liver, thereby reducing aminotransferase activity, decreasing deamination capacity, and impairing the control of fluid balance (Coz-Rakovac et al. 2005). Total serum protein is also used as an indicator of liver impairment (Firat and Kargin 2010). The TP content of fish reflects the animals’ nutritional status and indirectly reflects their nonspecific immune status (Huang et al. 2018).
This study also showed that heat stress induced a significant increase in the liver TG after 24, 48, and 96 hours, which was probably due to the mobilization of lipid reserves to cope with an increased energy demand in fish (Stress et al. 2005). Similar results were demonstrated on the effect of thermal stress on the biochemical parameters in Pufferfish (Takifugu obscurus) (Cheng et al. 2018).
One of the first responses to environmental stressors, such as high water temperature, is the release of stress hormones like Adrenaline, Noradrenaline and cortisol. The release of these hormones especially Cortisol, triggers a range of biochemical changes in the fish body known collectively as secondary stress responses. Their metabolic effects may include hyperglycemia and depletion of glycogen tissue reserves. The catabolic effects catecholamines and corticosteroids on the energy reserves stored in the body tissues may result in reduced growth in stressed fish. The changes seen in the muscle and liver tissue agree well with the general picture of secondary responses, whereas the catecholamine are thought to cause the initial elevation in plasma glucose levels by mobilizing the glycogen reserves (glycogenolysis) corticosteroids may contribute to the maintenance of hyperglycemia via the stimulation of gluconeogenesis. The primary stress reaction in fish are characterized by raised levels of stress hormones, cortisol and catecholamine in the blood (Dagoudo et al. 2021). Cortisol is the most commonly used indicator of stress in fish (Saravanan et al. 2011). In the present study, the levels of cortisol hormone increased and rose higher in the heat stress group than the control group. The releases of these catecholamines and cortisol trigger a broad collection of biochemical changes known collectively as secondary stress responses. The metabolic effects may include hyperglycemia and depletion of glycogen tissue reserves. The catabolic effects catecholamine and corticosteroids on the energy reserves stored in the body tissues may result in reduced growth in stressed fish. Catecholamines deplete glycogen reserves in fish liver and muscle (Kandeepan 2014).
To meet the increased energy request of stressed animals, glycogen, due its accessibility for energy production, is speedily catabolized leading to the huge losses of this energy reserves. In this study, it was noted that the exposure of fish to heat stress resulted in a decrease in glycogen levels in the liver. Reduction in glycogen content of liver observed in the present study supports this view. Catecholamines deplete glycogen reserves in fish. Therefore, the observed glycogenolysis in the liver in this study after exposure to stress could possibly have been caused by a stress induced increase in circulating catecholamine. The depletion in glycogen stores should be Stress induced breakdown of carbohydrate pool, supplies the growing energy requirement to meet the stress condition in general accompanied by an increase in glucose content as observed in this study (Kandeepan 2014). The depletion in glycogen supplies should be supplemented by an increase in glucose content as perceived in this study. The abrupt decline in liver glycogen in the heat stress group resulted in the hyperglycemia of the blood. This consistent decrease in glycogen reserves suggests that glycogenesis was impaired. Murugaian (2008) also observed that the glycogen content in the liver and muscle decreased with increasing temperatures and with more exposure periods of thermal stress. Because of the stress, the fish makes suitable adjustments for which the stored energy is utilized. This may be the reason for the decreased amount of glycogen content (Wu et al. 2015). The glycogen content was observed in the decreasing order with time under heat stress. Because of the stress, the fish makes suitable adjustments for which the stored energy is utilized. This may be the reason for the decreased amount of glycogen content.
Complement system (C3) is an important component of the immune defense of fish. It plays an important role in the immune defense against the inflammation and the bacterial invasion. C3 is the key component of both classical and lectin pathways responsible for various immune effector functions (Holland and Lambris 2002). Previous studies show that the C3 mRNA expression level can increase significantly in response to environmental stressors (Qi et al. 2011). In addition, the complement activity can be considered as an essential indicator of immune-competence in fish under the exposure to stressors. In this study the levels of C3 increased gradually, suggesting that the complement system was activated by thermal stress. Complement, an important component of the innate immune system, is comprised of about 35 individual proteins. Activation of complement results in the generation of activated protein fragments that play a role in microbial killing, phagocytosis, inflammatory reactions, immune complex clearance, and antibody production (Qi et al. 2011). Fish seem to possess activation pathways like those in mammals, and the fish supplement proteins identified thus far show various homologies to their mammalian counterparts. Because information about supplement proteins, complement receptors and regulatory proteins in fish is far from complete, it is uncertain whether all the supplement functions that have been known in mammals also occur in fish. However, it has been clearly demonstrated that fish complement can lyse foreign cells and opsonize foreign organisms for destruction by phagocytes. There are also signs that supplement fragments participate in inflammatory responses. Fish possess several isoforms of numerous complement proteins, such as C3 and factor B. It has been assumed that the function of this multiplicity in complement proteins helps to expand their innate immune recognition capacity and response. Understanding the functions of complement in fish and the roles the individual proteins, including the various isoforms, play in host defense, is important not only for understanding the evolution of this system but also for the development of new strategies in fish health management (Holland and Lambris 2002).
4.4. Effect of heat stress on Liver Anti-Oxidant Enzymes
Superoxide dismutase (SOD) is considered to play a key role in the first step of the enzymatic anti-oxidative defense system (Dagoudo et al. 2021). SOD activity helps the organism to control the intracellular steady status level of superoxide (O2-) which is a relatively strong oxidizing agent. In the present study, there was significant increase in SOD activity in the liver of the juvenile catfish subjected to heat stress under high temperature of 350C. This demonstrates that SOD provides effective protection to cells against heat shock in yellow catfish. Lushchak and Bagnyukova (2006) demonstrated that SOD activity significantly increased in the brain, liver and kidney after 12 h exposure to high temperature (350C) in goldfish (Carassius auratus). Parihar et al. (1996) found that SOD activity in the gills of the freshwater catfish (Heteropneustes fossilis) increased significantly at 32 0C and 370C after 1–4 h compared with the control (250C). It can be inferred from these results that fish can protect their cells from ROS damage by increasing SOD activity during heat stress (Wang et al. 2016).
Catalase (CAT) is one of the most efficient antioxidant enzymes found in cells. It catalyzes the reaction by which hydrogen peroxide is decomposed to water and oxygen. Hydrogen peroxide is a hypothetically dangerous species because of its ability to easily cross biological membranes and high stability. CAT has the ability to eliminate hydrogen peroxide (H2O2), which helps to counteract the influence of oxidative stress (Duan et al. 2015). In this present study, there was a significant increase in the CAT activity in the heat stress group. The significant increase in the activity of CAT in liver could have been for protection against lipid hydro peroxides and H2O2 in the fish liver under heat stress. The increased rates of mitochondrial respiration caused by stress could have enhanced the formation of ROS, and hence increased the initiation of SOD and CAT at translational and transcriptional levels against oxidative damage (Wang et al. 2017). Madeira et al (2013) also demonstrated that Catalase activity significantly increased in L. ramada, subjected under thermal stress. The antioxidant enzymes are the first intracellular defense against oxidative stress and they regulate redox-dependent signaling, which is indispensable for innate immunity (Dixon et al. 2012).
Malondialdehyde (MDA) is a cytotoxic end-product of lipid peroxidation, so the MDA content indicates the extent of oxidative damage (Mancino et al. 2011). In this study a significant increment in the MDA concentrations was observed at 6, 12, 24 and 48 hours after high temperature stress showing that heat stress caused oxidative stress, which probably led to injury of liver cells. These results further suggest that heat stress worsens oxidative stress by generating ROS. This occurrence could be due to the capacity of high temperature to denature and damage antioxidant enzymes (Abele and Puntarulo 2004), reduce antioxidant defenses and disturb physiological homeostasis (Stress et al. 2005), leading to lipid peroxidation injury in the liver of the juvenile yellow cat fish (Parihar et al. 1996).
Lysozyme enzyme is a critical innate immunity component in maintaining immune defense system to prevent bacterial infection by destroying the peptidoglycan layer of predominant gram-positive bacteria and some gram-negative bacteria. Lysozyme activity is stimulated to improve the immune defense by inducing alterations in immune-regulatory functions when pathogenic bacteria and various stress-induced substances attack the fish (Kong et al. 2012). In this present study, the lysosome activity increased after increased significantly after 6 hours up to 96 h in the heat stress group. Ndong et al. (2007), demonstrated that lysozyme activity increased significantly when Tilapia fish (Oreochromis mossambicus) transferred to 31°C and 35°C for over 48–96 h. Also Guardiola et al. (2015) reported a substantial increase in the lysozyme activity of gilthead seabream, (Sparus aurata) exposed to arsenic, cadmium, and mercury.
Alkaline phosphatase (AKP) is a vital enzyme which performs a significant role in phosphorus metabolism in the fish organs and tissues. In this study, the activities of AKP significantly increased in the heat stress group, indicating that the increased level of AKP might result from the disturbances in both physiological and functional mechanisms under heat stress. Thus, taken together, heat stress can exhibit an adverse effect on the innate immunity by inducing oxidative damage to macromolecules and disrupt the physiology by regulating the activities of metabolic and immune-related enzymes (Cheng et al. 2017).