ABSTRACT Striped catfish (Pangasianodon hypophthalmus) is an important species of aquaculture in the Mekong River Delta (MRD) of Vietnam. However, it has been constrained by several obstacles, among them which are climate change and diseases. Plant extracts as a dietary supplement is regarded as the easiest and most efficient strategy to improve antioxidant activity while contributing to the stress mitigation. The study aimed to evaluate the effect of selected plant extracts medicated in feed on physiological haematological parameters, digestive enzyme activities and growth and stress responses of striped catfish exposed to environmental stressors (salinity, temperature and NO2-). The final aim would be a selection of plant extracts that have a positive effect on fish to reduce the use of antibiotics and avoid water environmental pollution. This doctoral dissertation was, therefore structured into four separate experiments. First, five plant extracts, 0.4% or 2% Euphorbia hirta (Eh), 0.2% or 1% Phyllanthus amarus (Pa), 0.4% or 2% Mimosa pudica (Mp), 0.2% or 1% Psidium guajava (Pg), and 0.4% or 2% Azadirachta indica (Ai), were investigated on haematology, digestive enzyme activities and growth throughout the duration of 60 days. These extracts were identified based on the promising and applicable findings regarding the immunity and antioxidant capacity of striped catfish reported (Nhu et al., 2019; Dao et al., 2020). P. hypophthalmus fingerlings' haematological indices and digestive enzyme activities were modified after sixty days of oral administration with Pg 0.2% or Pa 0.2% extracts, resulting in improved growth performance. Second, the effects of Psidium guajava L. (0.2%) - Pg0.2 and Phyllanthus amarus (0.5%) – Pa0.5 on haematology, thermal stress tolerance, enzymatic activities, and growth of striped catfish subjected to temperatures of 27°C, 31°C, and 35°C for 42 days were examined. Although haematological indicators were most significant at 35°C, they were not significantly different from results noted at 31°C on day 14 post-temperature challenge. The glucose concentration elevated on the third post-temperature challenge day subsequently decreased and remained constant at 35°C until the end of the trial, which was not significantly different compared to those at 27°C. After 42 days, the Pg0.2 and mix diets substantially lowered lipid peroxidation and increased catalase in the gills and liver. Digestive enzymes (trypsin, chymotrypsin, amylases, and pepsin) were accelerated by the Pg0.2 and mix treatments, and enzymatic activity improved from 31°C to 35°C. Overall, fish maintained at 31°C presented the most favorable growth performance, followed by those reared at 35°C, and there was no significant difference in survival rates among these treatments. Assuming the Mekong Delta's average water temperature remains below 35°C, feeding diets incorporating Pg0.2 or Mix (Pg0.2+Pa0.5) extracts strengthen fish health via haematology and oxidative stress resistance.
General introduction
The aquaculture of striped catfish (Pangasianodon hypophthalmus) in the Mekong River Delta, Vietnam, faces significant challenges, primarily due to climate change and disease, which are major concerns for fish farmers and researchers The region is particularly vulnerable to the impacts of global climate change, with the average temperature in Vietnam having risen by approximately 0.5-0.7°C in recent years, and projections indicating an increase of 2.3°C by the end of the 21st century (MONRE, 2009) This temperature rise poses a threat to the sustainable development of both fisheries and aquaculture As poikilothermic species, striped catfish are directly affected by temperature fluctuations, which can negatively influence their metabolic rates (Galloway and Kieffer, 2003), swimming performance (Hocutt, 1973), and immune functions (Hurst, 2007) These changes can reduce their ability to capture prey, increase disease susceptibility, and heighten mortality rates (Donaldson et al., 2008) Additionally, temperature shocks can hinder predator avoidance and alter recovery rates from exercise (Ward and Bonar, 2003; Suski et al., 2003).
2006), and disrupt homeostasis (Vanlandeghem et al., 2010)
The Mekong River Delta (MRD) is one of three significant low-lying areas in Vietnam projected to be affected by rising sea levels this century, according to Parry et al.
The striped catfish industry in Vietnam is at significant risk due to predicted sea-level rise, especially given the region's low elevation of less than 4.0 meters above mean sea level A forecasted 1-meter increase in sea level could transform approximately 1,000 km² of cultivated land into salt marshes and inundate 15,000 to 20,000 km² of the Mekong River Delta with seawater This increasing salinization of freshwater areas will adversely affect the local aquaculture sector, particularly the striped catfish culture, by altering both soil conditions and freshwater resources.
Intensive aquaculture ponds, when overfed and improperly managed, can result in the decomposition of organic matter, leading to the generation of toxic gases Under hypoxic and high-temperature conditions, harmful substances such as ammonia, nitrite, nitrate, carbon dioxide, and hydrogen sulfide are produced Notably, nitrite, a natural part of the nitrogen cycle, can accumulate to toxic levels, posing significant risks to aquatic animals.
In freshwater species, the formation of methaemoglobin due to the reaction of nitrite with hemoglobin is a significant factor leading to exceptionally low arterial oxygen levels Additionally, a decrease in blood oxygen affinity contributes to this issue While fish can tolerate relatively high levels of metHb at rest, reduced blood oxygen content restricts their activity These challenges have hindered fish farmers from converting the increased production yields from intensive farming methods into commercial profits.
The use of antibiotics and chemotherapeutics in aquaculture, particularly in Vietnamese striped catfish farming, has led to the emergence of drug-resistant bacteria and environmental pollution, with 17 different antibiotic compounds identified (Jian and Wu, 2004; Rico et al., 2013) To combat disease outbreaks and minimize economic losses, various therapeutic medicines are administered regularly, yet the increasing restrictions on pharmaceutical products due to their negative impacts on the environment and human health are prompting a shift in practices (Mckenzie et al., 2012) While vaccination is a potential solution, its high cost and limited effectiveness against single diseases pose challenges for widespread adoption among fish farmers (Harikrishnan et al., 2011) Consequently, researchers are focusing on developing natural nutritional supplements that enhance the growth, health, and immune response of farmed fish, utilizing cost-effective and non-toxic ingredients (Pandey et al., 2012).
In today's world, there is an increasing demand for environmentally friendly prophylactic and preventive solutions, leading to a growing interest in natural bio-active products that can boost the immune system and overall health of farmed animals Research indicates that plant-derived compounds, including phenolics, polyphenols, alkaloids, quinones, terpenoids, lectins, and polypeptides, serve as effective alternatives to antibiotics and synthetic additives.
2010) Phytochemicals have been shown to have antioxidant, antibacterial, antifungal, antidiabetic, anti-inflammatory, antiarthritic, and radioprotective properties (Nair et al.,
The use of plant extracts in aquaculture has gained global attention as a sustainable alternative to antibiotics, particularly in organic production In Vietnam, despite the rich diversity of wild plants and significant interest among aquaculture farmers in natural alternatives, the adoption of these bio-active products remains limited This is largely due to a lack of awareness about their availability and proven effectiveness in improving fish health.
The study by Nhu et al (2019) identified five types of plant extracts and their concentrations from an extensive screening of 20 plant species While the research primarily focused on the immune responses, it also highlighted the necessity to explore the physiological factors of striped catfish, including hematology, digestive enzymes, and stress responses Furthermore, the study examined the effects of specific extracts, such as Euphorbia hirta.
Research on the effects of Phyllanthus amarus, Mimosa pudica, Psidium guajava, and Azadirachta indica on striped catfish physiology is limited, yet these plants represent cost-effective and beneficial dietary components Our findings provide valuable biological insights for researchers and practitioners, emphasizing the potential of plant-based bioactive substances to enhance fish health Currently, there is a lack of information on herbal-derived products used in striped catfish farming, highlighting the need for comprehensive training for farmers on active constituents, proper usage, and dosage of plant extracts.
The objectives of the dissertation Error! Bookmark not defined
The study aims to identify plants that positively impact fish, promoting the production of biologically safe products and enhancing environmental sustainability It also recommends that fish farmers incorporate herbs in striped catfish farming to minimize antibiotic use and prevent water pollution.
The study aims to evaluate the impact of specific plant extracts incorporated into feed on the physiological hematological parameters, digestive enzyme activities, and growth and stress responses of striped catfish subjected to environmental stressors such as salinity, temperature, and nitrite (NO2-).
The main contents of the dissertation
A study investigated the impact of dietary supplementation with five plant extracts—Euphorbia hirta, Phyllanthus amarus, Mimosa pudica, Azadirachta indica, and Psidium guajava—on the hematological physiology, digestive enzyme activities, and growth performance of striped catfish fingerlings The findings revealed significant improvements in the health and growth metrics of the fish, highlighting the potential benefits of these natural supplements in aquaculture.
- Study on the effect of dietary supplementation of P amarus and P guajava on hematology physiology, digestive enzymes activities, oxidative stress and growth performance of striped catfish exposed to elevated temperatures
- Study on the effect of dietary supplementation of P amarus and P guajava on hematology physiology, digestive enzymes activities, oxidative stress and growth performance of striped catfish exposed to sublethal salinities
- Study on the effect of dietary supplementation of P amarus and P guajava on hematology physiology, digestive enzyme activities, oxidative stress and growth performance exposed to various nitrite concentrations
The hypotheses of the dissertation
The five chosen plant extracts exhibited varying dose-dependent effects, enhancing hematological parameters, digestive enzyme activities, and growth performance in striped catfish when incorporated into their diet.
Supplementation of P amarus and P guajava in diets for striped catfish effectively supported rapid growth performance, as elevated temperatures did not adversely affect hematological parameters, digestive enzymes, or oxidative stress levels.
Chronic exposure of striped catfish to increased salinity led to their gradual acclimatization without negatively impacting physiological functions or growth performance when fed with P amarus and P guajava diets.
Supplementation with the two specified extracts enhances the tolerance of striped catfish to elevated nitrite levels, resulting in improved physiological parameters and growth performance compared to those on a standard diet.
New findings of the dissertation
In five plant extract-based diets (Euphorbia hirta (Eh); Phyllanthus amarus (Pa);
Mimosa pudica, Psidium guajava, and Azadirachta indica, when incorporated into diets at varying doses and feeding durations, have shown the potential to enhance hematological parameters, enzymatic activity, and growth in P hypophthalmus over a 60-day period Evaluating fish preferences for these plant extract-based diets is crucial, as it may indicate increased aquafeed utilization, ultimately leading to improved fish growth performance.
The Pg0.2 and Mix diets enhance the health of P hypophthalmus by improving hematology profiles, digestive enzyme activity, and oxidative stress biomarkers, indicating their effectiveness in promoting fish health and reducing stress Notably, several hematological parameters, including RBCs, Hct, Hb, and glucose levels, remained stable at elevated temperatures (35°C) up to day 7, while oxidative stress indicators (LPO, CAT in gill and liver) showed no changes until day 14 Subsequently, the fish demonstrated recovery and adaptation to the experimental conditions.
In a salinity experiment, the liver and gills of fish experienced significant oxidative damage at elevated salinity levels, particularly at 20‰, while changes were minimal up to 10‰ This suggests that the species may be viable for farming in low-salt brackish environments Additionally, fish fed with Pg0.2-administered food, followed by a mixture of Pg0.2 and Pa0.5, demonstrated the most effective preservation of normal physiological functions, including haematology, digestion, and stress reduction.
Striped catfish are adversely affected by nitrite-induced stress at concentrations of 0.8 mM, which can lead to a reduction in red blood cells (RBCs), hemoglobin (Hb), and hematocrit (Hct), while increasing plasma glucose levels after 24 hours of exposure Prolonged exposure, ranging from 7 to 42 days, results in decreased digestive enzyme activities, ultimately impairing growth performance These detrimental effects are exacerbated with longer exposure times and higher nitrite concentrations.
To promote optimal growth and reduce stress in fish, nitrite concentrations should be kept below 0.8 mM in production systems Additionally, the Pg0.2 and Mix diets have been shown to enhance the health of P hypophthalmus by positively affecting hematology, digestive enzyme activity, and oxidative stress biomarkers.
Significant contributions of the dissertation
The dissertation highlighted that P amarus and P guajava enhance the health of catfish by positively influencing various physiological parameters Incorporating small amounts of these extracts into the daily diet significantly boosts the growth performance of striped catfish, even in challenging environmental conditions.
The study presents promising plant extract solutions that have the potential for large-scale application and further development, significantly reducing the reliance on antibiotics and chemicals in aquaculture, thereby promoting environmental sustainability Additionally, the insights gained from this research can serve educational purposes, act as a valuable reference, and lay the groundwork for future studies on plant extracts in various aquatic species.
The four primary contents of the dissertation are illustrated as follows:
The status and importance of aquaculture and fisheries
Fisheries and aquaculture significantly contribute to the domestic economy, particularly in developing countries In 2020, the sector's sale value was estimated at
In 2020, global markets saw participation from 225 countries, contributing to a total value of $424 billion, despite a 7% decline due to the Covid-19 pandemic The FAO (2022) reported a remarkable production of 214 metric tons of fish and algae, which included 178 million aquatic animals.
36 million algae Aquaculture experienced a 2.7% growth, below the 4.5% annual average growth over the previous decade (FAO, 2022)
Figure 2.1 Total fisheries and aquaculture production 2020 (FAO, 2022).
The COVID-19 pandemic has significantly impacted the global trade of aquatic products, leading to a projected 7.0% decline in export value, reaching US$ 151 billion in 2020 This downturn follows a 2.1% decrease in 2019, marking a notable decline from previous peak levels.
In 2020, trade volumes were projected to drop by 10.1% across all regions, reflecting the challenges faced during the pandemic However, as fishing and aquaculture resumed and international markets reopened, trade rebounded significantly in 2021, resulting in a 12% increase in the overall value of global aquatic product exports compared to the previous year Despite this recovery, trade volume growth remained limited due to cautious aquaculture planning and persistent logistical challenges.
Figure 2.2 World fisheries and aquaculture production, utilization and trade
Figure 2.3 World production of striped catfish (thousand tons) (FAO, 2022)
Figure 2.4 Farming area and production of striped catfish in Mekong Delta (2015-2021;
Farming area (ha) Production (thousand tonnes)
In 2020, Asia emerged as the leading producer of aquatic animal products, contributing to 70% of global production, with China holding a significant 35% share as the top exporter Following China, Norway and Vietnam also played key roles in exports, while the European Union stood out as the largest importer of these products.
In 2020, Asia significantly shaped global aquaculture, contributing 91.6% of aquatic species and algae The total production of air-breathing fish reached 6.2 million metric tons, representing 12.6% of the global output, although this marked a slight decrease from 2019 due to reduced supply in Vietnam Three fish families dominated this production, with Pangasiidae (striped catfish) accounting for 47%, Clariidae (North African catfish) for 26.5%, and Channidae (snakehead) for 10.5%, collectively making up 83.9% of the total Notably, striped catfish remains one of the top 10 aquaculture species worldwide, consistently showing annual production growth.
Vietnam's aquaculture, especially in striped catfish production, plays a crucial role in the global market, solidifying its status as a leading exporter Since 2014, Vietnam has emerged as the third-largest exporter of aquatic products, particularly excelling in farmed striped catfish In 2020, the country exported aquatic products worth approximately US$ 8.5 billion, accounting for 5.6% of the global total (FAO, 2022) A significant trend in Vietnam's striped catfish sector is the adoption of quality standards like ASC and Global GAP, which align with international commodity trade requirements.
Striped catfish farming is primarily concentrated in 10 provinces and cities in the Mekong River Delta (MRD), including An Giang, Dong Thap, Tien Giang, Can Tho, Vinh Long, Ben Tre, Hau Giang, Soc Trang, Tra Vinh, and Kien Giang, along with Tay Ninh and Quang Nam Can Tho, An Giang, and Dong Thap are the leading regions, contributing over 75% of the nation’s striped catfish production (Hien, 2020) The industry emphasizes sustainable production by effectively managing raw material sources As reported by VASEP (2022), the striped catfish farming area in the MRD was approximately 5,700 hectares in 2020, yielding 1.553 million tons In 2021, the farming area slightly decreased to 5,400 hectares, resulting in a production of 1.484 million tons, reflecting a 4.5% decline from the previous year.
In 2020, the demand for striped catfish saw a decline due to the impact of COVID-19; however, consumption experienced a slight rebound in 2021 Viet Nam aimed to expand its striped catfish market to 138 countries by 2020, focusing on key regions such as China, Hong Kong, the United States, and ASEAN.
In 2021, the striped catfish export turnover reached over $1.6 billion, marking a 10% increase from the previous year, with the EU, UK, Mexico, Brazil, and Colombia as key markets However, exports to China faced a significant decline due to long-standing restrictive import policies The striped catfish sector has struggled in recent years, particularly during the 2020-2021 period, experiencing stagnation in production, processing, and export chains.
Figure 2.5 Export value of striped catfish in the period 2015-2021 (VASEP, 2022)
Striped catfish farming is a crucial industry in Vietnam, playing a significant role in the national government's policy goals However, the sector faces major challenges that threaten its long-term sustainable growth, including issues related to diseases, chemical usage, sustainable development, and seed quality Additionally, the industry must prepare for severe future challenges posed by climate change, such as saline intrusion and rising temperatures.
Climate changes and impacts on aquaculture and fisheries
Climate change arises from both natural processes and human activities that alter the atmosphere's composition Long-term documentation shows that fluctuations in climate occur over extended periods (UNFCCC, 2003) The increase in hazardous gases, including CO2, N2O, and CH4, has significantly contributed to this phenomenon (IPCC, 2013) The consequences of climate change, such as rising temperatures, droughts, sea-level increases, and variations in seasonal precipitation, profoundly impact various sectors, particularly agriculture and aquaculture (USEPA, 2012).
Vietnam ranks 27th globally in terms of vulnerability to climate change impacts on the fisheries sector The Mekong River Delta (MRD), a low-lying area in southern Vietnam, spans 3.96 million hectares and contributes to 65% of the country's aquaculture production Saline intrusion affects between 1.2 and 1.6 million hectares of its coastal regions, with the salinity-affected area during the dry season potentially reaching 2.4 million hectares, or about 65-70% of the total MRD area Forecasts indicate that the MRD will be among the top three regions worldwide most severely affected by climate change, owing to its unique geographic and natural characteristics.
Since 1906, global temperatures have increased by 0.74°C and are projected to rise by an additional 0.2°C per decade over the next 20 years, according to the IPCC (2007) In Vietnam, coastal regions are expected to experience temperature increases of 1.1 to 1.5°C, while interior areas may see a rise of 1.8 to 2.5°C.
2050 and 2070 (UNFCCC, 2003) Over the next century, temperatures are predicted to rise by 1 - 4°C, with the highest temperature in the lower Middle East (MRD) reaching
By 2050, the Mekong River Delta (MRD) is projected to experience peak dry season temperatures of 32°C, with an increase of 2°C anticipated between 2030 and 2040 (Tuan & Suppakorn, 2011) Climate change significantly affects aquaculture and fisheries, particularly impacting striped catfish nurseries and grow-out systems, which are vulnerable to extreme rainfall and temperature fluctuations This change has notable consequences, including an increase in parasite infections in striped catfish.
Drought and rising temperatures increase evaporation, reducing freshwater availability in rivers and bays, while tidal changes affect salinity in estuarine regions (USEPA, 2012) Global sea levels are rising at an accelerating pace, posing significant risks to low-lying coastal areas, with floods potentially impacting around 20 million people in Southeast Asia if sea levels increase by 40 cm (Barange and Perry, 2009) Projections indicate that sea levels could rise by 33 cm by 2050 and up to 1.0 m by 2100 (UNFCCC, 2003).
A 2009 forecast predicted that sea levels would rise by 25 cm by 2030 and 75 cm by the end of the 21st century The MRD region, characterized by its topography of less than 2 meters above sea level, is particularly vulnerable to the impacts of rising sea levels (VNU, 2019).
A study by Nicholls (2006) predicts that approximately 1,000 km² of cultivated land in the Mekong River Delta (MRD) may transform into salt marshland if sea levels rise by 1 meter (Parry, 2007) Research conducted by Anh et al (2014) assessed the impact of rising sea levels on striped catfish farming under three scenarios (+30, +50, and +75 cm) Their findings indicate that even a moderate rise of +50 cm could lead to a 3-meter flood level, adversely affecting aquaculture operations in An Giang and Dong Thap provinces, as well as Can Tho city.
Research indicates that adaptive strategies are essential for addressing the impacts of climate change on the sector The genetic resources available in the MRD can aid in developing selective breeding programs for strains resistant to climate change However, salinity intrusion is limiting the expansion of striped catfish farms in downstream areas, and rising river salinity is likely to increase operational costs due to the need for salinity-tolerant seeds and longer culture periods (Anh et al., 2018).
Striped catfish farmers are adopting advanced technologies, including chemical treatments, aeration, and effective water quality management, to mitigate the negative effects of climate change and salinity on their farming practices.
Mechanism of stress on fish
A fish's ability to adapt to environmental changes relies on its effective stress response, energy reallocation to defense mechanisms, and behavioral adjustments to mitigate risks This process is governed by two key neuroendocrine pathways: the hypothalamic-pituitary-interrenal (HPI) axis, which triggers a rapid stress response that increases heart rate and blood flow to essential tissues, and the brain-sympathetic–chromaffin cell (BSC) axis, which reorganizes energy by enhancing catabolic processes and suppressing energy-intensive activities like immune responses.
Stress can be beneficial, as an acute stress response effectively triggers physiological and behavioral changes that help individuals adapt to environmental challenges and maintain homeostasis However, when stress becomes chronic and unavoidable, the resulting physiological and behavioral adjustments may lead to negative outcomes (Barton, 2002).
The stress response in fish is activated at various organizational levels—cell, tissue, organ, and organism—triggered by the interpretation of environmental stimuli in the hypothalamus and telencephalon This response is regulated through the activity of monoaminergic neurotransmitters, including dopamine, noradrenaline, and serotonin, which play crucial roles in managing the stress state (Schreck and Tort, 2016; Gorissen and Flik, 2016; Ìverli et al., 2005).
The brain orchestrates a comprehensive stress response through two main neuroendocrine pathways: the brain-sympathetic-chromaffin cell (BSC) axis, which stimulates chromaffin cells in the anterior kidney to release catecholamines into the bloodstream, and the more complex hypothalamic-pituitary-interrenal (HPI) axis These pathways enable catecholamine-induced responses that provide energetic substrates for muscle and tissue use, facilitating behavioral reactions, while cortisol mediates genomic changes via glucocorticoid and mineralocorticoid receptors to mobilize and redistribute energy substrates for effective adaptation.
The activation of corticotropin-releasing factor (CRF) neurons triggers the release of CRF into the anterior pituitary, stimulating the secretion of adrenocorticotropic hormone (ACTH) and subsequently cortisol from the interrenal cells in the anterior kidney Stress hormones, including catecholamines and cortisol, facilitate various physiological responses to help fish cope with stressors, such as increased blood glucose levels, enhanced cardiac output, and altered immune function However, chronic stress can lead to detrimental effects on fish health, including impaired immune response, growth, and reproductive success, due to the reallocation of energy substrates.
The impact of continuous stressors on fish populations varies with their severity, leading to significant repercussions (Barton, 2002) Adaptive responses, such as maintaining homeostasis, help fish cope with stressors However, systemic alterations can occur when stress becomes overwhelming, hindering the fish's ability to adapt and negatively affecting their health, performance, growth, reproduction, disease resistance, and behavior (Barton, 2002).
Methodologies for assessing stress responses in fish encompass a range of endpoints, including measurements of whole body or organ weights such as condition factor, hepatosomatic index, and gonadosomatic index Biochemical analyses, including plasma cortisol, corticosterone, glucose levels, tissue damage enzymes, and heat shock proteins, are also utilized Additionally, researchers examine immunological responses, gene expression patterns, fish steroids, and both macroscopic and microscopic morphology to gain insights into fish stress responses.
The diencephalon orchestrates the body's primary stress response to elevated temperatures by releasing catecholamines, primarily adrenaline and noradrenaline, through the brain-sympathetic-chromaffin cell (BSC) axis, and cortisol via the hypothalamic-pituitary-interrenal (HPI) axis In the short term, these hormones increase cardiovascular activity, such as heart rate and blood pressure, while mobilizing energy sources like glucose and lactate Concurrently, catecholamines promote the production of heat shock proteins (HSPs) and antioxidant enzymes to mitigate oxidative damage As cortisol is released, it inhibits HSP formation and serves as a negative feedback regulator for future cortisol release Over the long term, this stress response leads to reduced energy allocation for essential biological functions, including immunity, growth, and reproduction.
Effects of environmental factors on fish
2.4.1 Effect of temperature on fish
2.4.1.1 Effects of temperature on hematological parameters of fish
Temperature is a critical environmental factor influencing aquatic animals, particularly poikilotherms, which are highly sensitive to temperature fluctuations (Fry, 1971) Climate change, with its rising temperatures, poses complex challenges, as the range of environmental temperatures that aquatic animals endure significantly impacts their well-being While most fish can acclimate to minor temperature changes, substantial shifts can adversely affect their growth and survival rates, leading to detrimental physiological responses, especially in ectotherms that struggle to regulate their body temperature (Kemp, 2009; Wright & Tobin, 2011).
Temperature significantly affects the metabolic rates of aquatic animals, influencing their energy balance, behavior, and reproduction, particularly in ectotherms like fish As ectotherms, fish adjust their body temperatures according to their environment, leading to a doubling of metabolic activity with every 10°C increase within their normal tolerance range While slight temperature increases can enhance growth by boosting energy production, lower temperatures generally hinder performance due to alterations in mitochondrial structure Most tropical fish thrive in temperatures between 25 to 32°C, but species-specific adaptations to temperature fluctuations vary widely Each aquatic species has a defined temperature tolerance range influenced by factors such as maturation, genetics, and environmental conditions Laboratory studies indicate that excessive temperature increases can harm growth and may even be lethal, with the critical point of decline being unique to each species Additionally, research shows that vaccinated fish are adversely affected by low temperatures, suffering mortality rates similar to non-vaccinated fish due to a lack of antibodies.
Hematology is a crucial area of study in invertebrates, particularly fish, to assess their health and physiological conditions Key hematological parameters, including red blood cell (RBC) counts, hemoglobin (Hb) levels, and hematocrit (Hct), are essential for evaluating oxygen-carrying capacity (Houston, 1997) Secondary indices, known as Wintrobe indices, such as mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC), help classify anemia (Gabriel et al., 2015) Additionally, white blood cell (WBC) counts and their differential counts provide insights into the innate immune status of animals, especially under stress, with the neutrophil-to-lymphocyte ratio serving as a useful stress indicator (Van Rijn & Reina, 2010) Fish physiologists emphasize hematological assessments as sensitive biomarkers for monitoring physiological and pathological changes (Oluyemi et al., 2008; Patra et al., 2014), making these evaluations routine in fish farms to ensure stock health (Haghighi & Rohani, 2013) Factors such as environmental stress (Hickey, 1976) and malnutrition (Casillas & Smith) further impact these hematological parameters.
1977), gender (Collazos et al., 1998), fish size (Garcia et al., 1992), seasonal changes and breeding efficiency all contribute to variations in fish hematological characteristics (Cech and Wohlschlang, 1981)
Temperature significantly affects fish health, serving as a critical environmental stressor that alters hematological indices (Portz et al., 2006; Barton, 2002) Elevated temperatures can enhance the blood's oxygen-carrying capacity, improving oxygen delivery during increased metabolic rates (Carvalho & Fernandes, 2006; Zarejabad et al., 2010) Changes in hemoglobin (Hb) concentration are vital for maintaining oxygen delivery across varying environmental conditions (Brauner & Wang, 1997) The impact of temperature stress on hematology is linked to reduced oxygen solubility in water, necessitating adaptations in fish hematological traits (Cech & Brauner, 2011) Additionally, opercular beat rates are used to measure fish stress responses, with increased opercular activity indicating higher oxygen demand (Dalla Valle et al., 2003) Given that water holds significantly less oxygen than air and diffuses more slowly, temperature increases can lower HbO2 affinity, requiring fish to adjust to decreased oxygen solubility and heightened oxygen demands (Graham, 1990; Salama & Nikinma, 1990).
Extreme temperatures significantly reduce hematological parameters in fish, leading to decreased opercular movements and lethargy (Kapila et al., 2002; Hrubec et al., 2000) To cope with rapid temperature fluctuations exceeding 10°C, fish must enhance oxygen absorption at the gills and improve circulatory distribution to meet tissue oxygen demands Despite these adjustments, cardiac function may not limit metabolic rates until reaching critical thermal maxima, while reduced blood oxygen binding capacity can further impact oxygen consumption (Gollock et al., 2006) A decline in hemoglobin (Hb) and red blood cell (RBC) quantity and quality results in diminished oxygen transport, affecting metabolism beyond mere oxygen delivery (Gross et al., 1996) Additionally, increased RBCs and Hb levels correlate with decreased mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC), indicating poikilo-anisocytosis, with these changes intensifying under prolonged thermal stress during acclimatization (Ahmad et al.).
Temperature acclimation in fish involves changes in haematological parameters, characterized by an initial period of thermal stress followed by gradual recovery A fish is deemed well-acclimated when it stabilizes its haematological parameters between initial and elevated temperature conditions (Maricondi-Massari et al., 1998) Ectothermic animals, like fish, demonstrate acclimation responses to prolonged temperature changes, which may involve enzyme modifications that help mitigate the effects of temperature on metabolism (Hazel & Prosser).
Temperature significantly influences the biochemical and physiological functions of aquatic animals, enhancing growth rates and reducing maturation periods up to a certain threshold However, when temperatures exceed optimal ranges, they can adversely affect health by increasing metabolic rates and oxygen consumption The metabolism of non-photosynthetic organisms stabilizes at intermediate levels depending on the species Temperature acclimation helps mitigate the effects of environmental fluctuations on ectothermic species Historical stressors, such as temperature variations, have driven the evolution of physiological mechanisms in aquatic creatures to cope with these changes Fish, in particular, are highly sensitive to ambient temperatures and the rate of temperature change, experiencing severe stress when relocated to different habitats.
2.4.1.2 Effects of temperature on digestive enzymes activities of fish
Temperature significantly influences the secretion of digestive fluids, gastrointestinal motility, and the rates of digestion and absorption in fish The performance of digestive enzymes can be optimized by temperature and species-specific pH variations in the gut Due to the diverse habitats, dietary habits, and physiological characteristics of fish, temperature effects are complex and vary by species The impact of temperature is also affected by the duration, frequency, and rate of exposure; while acute temperature changes can adversely affect fish physiology, gradual long-term alterations may facilitate acclimation, reflected in changes to metabolic and digestive enzyme profiles.
Temperature significantly influences intestinal transit time and absorption rates in fish Cooler water temperatures can hinder nutritional digestibility by slowing down the digestive process, extending gut transit time, and reducing gastrointestinal evacuation rates.
Temperature can significantly influence the gastrointestinal tract by affecting the fatty acid content in intestinal mucosa, altering gut acidity levels, and modifying intestinal pH Higher temperatures may lower pH levels, while also impacting the transportation processes of amino acids due to changes in lipid solubility Additionally, temperature variations can affect the composition of intestinal bacteria and enhance metabolic rates in response to feeding.
Temperature variations can significantly influence enzyme-metabolite interactions in two primary ways First, the bonds stabilizing these interactions can be easily modified by environmental thermal energy, which may either hinder or enhance complex formation depending on temperature-induced changes in enzyme structure Second, temperature fluctuations can alter enzyme structure itself, thereby impacting the affinities between enzymes and metabolites (Hochachka & Somero, 1973).
Most fish possess similar primary digestive enzymes, including proteolytic enzymes like trypsin and carboxypeptidases, carbohydrate enzymes such as maltase and amylase, lipolytic enzymes like lipase, and phosphatases like alkaline phosphatase (Bakke et al., 2010) Digestive proteases, including trypsin, chymotrypsin, carboxypeptidases, and aminopeptidases, are primarily produced in the digestive gland, with additional production occurring in the well-developed gland of Leiblen found in muricid gastropods (Andrews & Thorogood, 2005).
Pepsin, a key digestive enzyme, is one of the three primary proteases in the digestive system, alongside chymotrypsin and trypsin Trypsin, found in various isoforms within the pyloric caeca and intestine, may influence genetic diversity in proteins An increase in trypsin secretion into the lumen leads to reduced trypsin-specific activity in the pyloric caecal tissue In fish, trypsin is essential for activating other pancreatic proteases, such as chymotrypsin These proteolytic enzymes break down protein structures into smaller components, facilitating efficient nutrient absorption in the intestines during digestion Consequently, they are vital for food digestion and energy storage, playing a crucial role in fish growth and development.
Carbohydrates play a lesser role in the digestion of carnivorous fish compared to herbivorous and omnivorous species, primarily due to their diet being predominantly protein-based Research indicates that amylase is responsible for breaking down polysaccharides, while optimal protease activity in various fish species occurs at temperatures between 30 to 60°C The temperature within the fish gut is directly related to the surrounding environment, with pepsin exhibiting peak performance at 30°C Investigating fish digestive secretions enhances our understanding of nutritional physiology and addresses dietary challenges Therefore, studies on digestive enzyme activity are crucial for elucidating fish nutritive physiology and resolving nutritional issues, including meal suitability and overall nutritional capacity.
Enzyme activity, crucial for understanding cumulative conversion, is significantly influenced by temperature and the feed's composition (Tijskens et al., 2001) Fish acclimated to varying temperatures exhibit different enzyme activity levels, affecting amino acid absorption and transportation due to temperature-induced changes in enzyme affinity (Sunde et al., 2004) Additionally, research by Gelman et al (2008) indicates that genetic factors determine enzyme temperature adaptation, leading to phenotypic changes Variations in enzyme structure, substrate affinity, activation energy, and the secretion and synthesis rates of isozymes, which catalyze the same reactions with differing efficiencies at various temperatures, further contribute to these disparities.
Effects of plant extracts on fish
2.5.1 Effects of plant extracts on hematological parameters of fish
Research by Kolawole et al (2011) highlights that evaluating the impact of medicinal plants on hematological and biochemical parameters in experimental animals is a key method for distinguishing appropriate from inappropriate prescriptions Hematological changes often serve as the earliest detectable responses to environmental shifts, reflecting the blood profile in fish and playing a crucial role in innate immune protection and immunological function (Ballarin et al., 2004) Numerous aquaculture studies have demonstrated that various medicinal plants can significantly enhance these hematological parameters.
Plant extracts have a significant impact on the hematology profiles of various fish species Research by Dügenci et al (2003) highlighted the immunostimulatory effects of medicinal plant extracts, including mistletoe, nettle, and ginger, on rainbow trout, with ginger extract notably enhancing phagocytosis and extracellular burst activity in blood leukocytes This led to a substantial increase in total leukocyte counts, indicating improved overall resistance Additionally, Gabriel et al (2019) found that dietary Aloe vera polysaccharides positively influenced the hematological indices of African catfish fingerlings, promoting erythropoiesis and enhancing oxygen-carrying capacity, thereby strengthening their defense mechanisms against physiological stress These beneficial effects are linked to the rich composition of vitamins and minerals in Aloe vera, such as β-carotene, vitamins C, E, B12, riboflavin, thiamine, folic acid, and essential and nonessential amino acids.
Aloe vera supplementation in fish leads to increased leukocyte levels and enhanced resistance to low pH, suggesting its potential to promote leucopoiesis and improve stress resilience Research on Zingiber officinale's impact on the immune response of Asian sea bass (Lates calcarifer) revealed that adding 10 g/kg of the plant extract stimulated erythropoiesis and lymphopoiesis, resulting in higher RBC, WBC, hemoglobin, and hematocrit levels, thereby enhancing oxygen transport and stress resistance This effect may be linked to antioxidants in plant extracts that mitigate oxidant-induced hemolysis Additionally, compounds such as polyphenols, alkaloids, glycosides, and sugar reduction could contribute to the observed increase in white blood cells.
Flavonoids derived from plant extracts have the potential to regulate interferon, contributing to nonspecific cellular immunity by acting as biocatalysts in the production of white blood cells (WBC) Additionally, these compounds help reduce red blood cell (RBC) hemolysis and protect biological membranes from oxidative damage caused by free radicals.
Research on Myrmecodia tuberosa in striped catfish has shown that it contains flavonoids, which can serve as antioxidants to protect heme iron and enhance erythropoiesis.
Several therapeutic plant extracts have been found to have no significant impact on hematological parameters in tilapia species, suggesting that the effects of medicinal plants on these indices may only be evident under specific stress conditions The exact mechanisms behind these effects are still unclear, but they may involve interference with erythropoiesis, haemosynthesis, and osmoregulation, or the promotion of red blood cell destruction in hematopoietic organs Therefore, in aquaculture, it is crucial to optimize herbal extracts not only based on growth and feed efficiency but also on hematological parameters Evaluating hematological and biochemical features in fish is essential for understanding their normal, diseased, and toxicological states (Jenkins et al., 2003).
2.5.2 Effects of plant extracts on digestive enzymes activities and growth of fish
Research has demonstrated that plants significantly impact aquatic animals, leading to increased growth rates and enhanced digestive enzyme activities in fish Studies have indicated that various plant extracts can stimulate appetite and promote weight gain in cultured fish (Harikrishnan et al., 2011) Notably, the supplementation of garlic (Allium sativum) has shown growth-stimulating effects in Nile tilapia (Oreochromis niloticus) (Shalaby et al.).
In a study by Punitha et al (2008), grouper fish (Ephinephelus tauvina) that were fed a diet supplemented with a blend of methanolic herb extracts, including Bermuda grass (Cynodon dactylon), long pepper (Piper longum), stonebreaker (Phyllanthus niruri), coat buttons (Tridax procumbens), and ginger (Zingiber officinale), exhibited a 41% increase in weight compared to those on a control diet Additionally, research by Talpur et al (2013) highlighted that plant extracts, such as ginger, enhance nutrient digestibility and availability, resulting in improved feed conversion rates and higher protein synthesis in Asian sea bass (Lates calcarifer).
Medicinal plant products, including flavonoids, alkaloids, and essential oils, are utilized in finfish and shrimp larviculture for their growth-promoting and immunostimulant properties (Sivaram et al., 2004) Various therapeutic plant extracts have been found to enhance growth, feed consumption, and survival rates in aquatic species The positive effects on growth performance and feed efficiency stem from the immune-nutritional components of these extracts, particularly polysaccharides, which are believed to possess prebiotic properties that improve nutritional digestibility and gastrointestinal health (Zahran et al., 2014) Additionally, herbs such as rosemary, thyme, and fenugreek have been shown to boost digestion in fish by increasing bile production and stimulating pancreatic enzyme release (Heidarieh et al., 2013).
Research indicates that herbal extracts influence fish growth in a dose-dependent manner, with growth increasing until an optimum inclusion level is reached, after which it declines with higher extract levels Excessive concentrations of anti-nutritional components, such as saponins and tannins, along with toxic substances and allergic reactions, can hinder fish development and growth This explains why certain herbal extracts, including Moringa oleifera, Eucalyptus citrodora, and Capsicum frutescens, among others, may have detrimental or negligible effects on fish growth and feed utilization.
Numerous studies have explored the role of herbs as appetizers and growth enhancers in aquatic animals Lee and Gao (2012) highlight that herbs initiate feeding through taste, influencing eating habits, digestive fluid secretion, and overall feed intake Feed additives are crucial for stimulating digestive secretions such as saliva and digestive enzymes Additionally, olfactory components in feed enhance growth by encouraging increased feed consumption (Adams, 2005) For example, Harada (1990) demonstrated garlic's olfactory stimulation effects, revealing its significant food-attracting properties in species like the oriental weather loach and Japanese amberjack Similar findings were reported by Lee and Gao (2012) across various aquatic species, including Pelodiscus sinensis and Cyprinus carpio, where allicin, a garlic compound, boosted feed intake Further microbiome and proteome studies are necessary to assess the effects of intensive aquaculture on intestinal digestive enzymes and microorganisms Table 2.3 provides a summary of research on the influence of different herbal extracts on digestive enzyme activity in cultured fish.
Mucus is essential for protecting the gastric wall from stomach acid, and its absence increases the risk of ulcers Flavonoids enhance cytoprotection by boosting the mucosal content of prostaglandins and mucus Additionally, flavonoids promote anti-obesity effects by regulating noradrenaline in the sympathetic nervous system, which enhances thermogenesis and fat burning, while also limiting adipocyte growth and fat accumulation Compounds such as caffeic acid, chlorogenic acid, catechin, epigallocatechin gallate, and quercetin exhibit thermogenic properties, aid in fat oxidation, reduce appetite, and inhibit digestive enzymes related to carbohydrate and lipid absorption Quercetin, in particular, has been shown to inhibit carbohydrate digestion and regulate postprandial blood glucose levels Furthermore, phenolics in lentil extracts, including p-hydroxybenzoic acid and kaempferol, effectively inhibit lipase and α-glycosidase, contributing to the regulation of blood glucose and obesity.
Table 2.2 Effects of some herbal extracts on hematological parameters of fish under culture
Products Concentration Duration Hematological parameters Species References
Aegle marmelos Acetone extracts 1g/kg 45 days Hct (>) O mossambicus Immanuel et al., 2009
Allium sativum Crude extracts 10, 20, 30 & 40 g/kg 90 days RBC (>), Hb (>), Hct () O niloticus Shalaby et al., 2006
Allium sativum Crude extract 10, 20 & 30 g/kg 2 weeks RBC (>), Hct (>), MCV (>), Hb (>),
Oncorhynchus mykiss Farahi et al, 2010
Allium sativum Crude extract 0, 0.5 &1 % 4 weeks RBC (>), WBC (>) O niloticus ×Oreochromis aureus Fall and Ndong, 2011
Aloe vera Crude powder 0.5, 1, 2 & 4%/kg 60 days RBC (>), Hct (>), Hb (>), MCV (>),
Aloe vera Crude powder 0.1, 0.5 & 1% 60 days RBC (>), Hct (>), MCV (>), Hb (>),
Cyprinus carpio Alishahi and Abdy, 2013
Aloe vera Crude extract 1% 2 weeks RBC (=), Hct (=), MCV (=), Hb (=),
Oncorhynchus mykiss Haghighi et al., 2014
Aloe vera Polysaccharides 0.5%, 1% and 2%, 60 days RBC (>), Hct (>), MCV (=), Hb (>),
Clarias gariepinus Gabriel et al., 2019
500, 1000, 2000 & 3000 mg/kg 45 days RBC (>), WBC (>), Hb (>), MCV
(>), MCH (>), MCHC (>) O mossambicus Prasad and Mukthiraj,
Apium graveolens Methanolic extract 0.1%, 0.5% &1% 45 days Hb (), RBC (), Hb (>), Hct (>), RBC (=) Clarias macrocephalus × C gariepinus Panase et al., 2018
Euphorbia hirta Crude powder 0, 5, 10, 20, 25 and 50 g extract/kg 50 days RBC (>), WBC (>), Hb (>), C carpio Pratheepa and Sukumaran,
50, 100, 200, 250 mg/kg 119 days RBC (>), Hct (>), Hb (>), MCV (>),
Magnifera indica Crude powder 0, 1, 5 &10 g/kg 60 days WBC (>), RBC (>), Hb (>) Labeo rohita Sahu et al., 2007
Mangifera indica Crude extract 5, 1.0, 2.0, 4.0 and 8.0 g/kg 56 days RBC (>), WBC (>) and Hb (>), Hct
Oreochromis niloticus Obaroh et al., 2014
Nasturtium nasturtium Crude extract 0.1%&1% 21 days RBC (=), Hct (=), MCV (=), Hb (>),
Crude seed extract 3 %/kg 30 days WBC (>) Oreochromis niloticus Elkamel and Mosaad, 2012
Ocimum sanctum Crude powder 2.5 & 5% 30 days RBC (>), WBC (>) Clarias batrachus Nahak and Sahu, 2014
Crude powder 0.5 g 60 days WBC (>), RBC (>), and Hb (>) Mystus montanus Kumar et al., 2014
Notes: (>) Significantly increased; (), Hb (>), Hct (>), MCV (), Hb (>), Hct (>), MCV (), Hb (=), Hct (>) Oreochromis mossambicus Gültepe et al., 2014
Sargassum wightii Crude extract 1%, 2% & 3% 60 days Hb (>), Hct (>) Labeo rohita Gora, 2018
Thymus vulgaris Crude powder 1%/kg 60 days WBC (>), RBC (>), Hb (=), Hct (>) Oreochromis mossambicus Gurkan et al., 2015
Tilia tomentosa Crude powder 0.01, 0.05 & 0.1% 45 days Hb (=), Hct (=), RBC (=), MCH (=),
MCV (=), MCHC (=) C carpio Almabrok et al., 2018
Lyophilized Extracts 0.1, 0.5 & 1%/kg 60 days Hct (>), Hb (=),
Quezada-Rodríguez and Fajer-Ávila, 2016
Aqueous extracts 0.1, 1%, 2% WBC (>) Oncorhynchus mykiss Dügenci et al., 2003
Zingber officinale Acetone extracts 1g/kg 45 days Hct (>) Oreochromis mossambicus Immanuel et al., 2009
Zingiber officinale Crude powder 1% 12 weeks Hb (>), Hct (>),
RBC (>), WBC (>) O mykiss Haghighi and Rohani,
An aqueous extract of Tournefortia paniculata Cham leaves, rich in phenolic compounds, has shown the ability to inhibit α-amylase and α-glycosidase enzymes both before and after exposure to gastric fluid simulation In Wistar rats on a high-calorie diet, this extract led to reductions in weight, food intake, liver fat, glucose, and serum triglycerides The findings suggest that the inhibitors in P guajava leaves maintain their activity even after passing through simulated stomach juice, indicating potential for in vivo effectiveness The phenolic compounds in P guajava leaves may complex with digestive enzymes, contributing to their inhibitory effects, which could offer new treatment options for obesity and type 2 diabetes by acting in the small intestine Additionally, a study on the ethanolic extract of katuk (Sauropus androgynous) revealed that a 1% supplementation improved appetite and growth in grouper fish, while higher concentrations (2.5% and 5%) resulted in lower growth rates These results emphasize the importance of appropriate dosing to achieve desired effects and highlight the need for further research to chemically characterize these extracts for quantifying active molecules and establishing optimal doses.
Table 2.3 Effects of some herbal extracts on digestive enzyme activities of fish under culture
Notes: (>) Significantly increased; () O niloticus Zahran et al.,
Amylase (>) C carpio Mohamed et al., 2018
Amylase (>) O mykiss Awad et al
Amylase (=) C carpio Almabrok et al., 2018 guar meal , canola meal, soybean meal and cottonseed meal
Powder Amylase (=) Labeo rohita Iqbal et al.,
2016 c) Effects of plant extracts on oxidative stress of fish
Oxidative stress is an inherent aspect of aerobic life, arising from the production of reactive oxygen species (ROS) like superoxide, hydroxyl radicals, and peroxyl radicals In healthy aerobic organisms, there exists a balance between ROS generation and protective mechanisms against them However, an increase in ROS production can lead to cellular damage or even death, resulting in a state known as oxidative stress.
Reactive oxygen species (ROS) are naturally produced during the body's metabolism and can have both positive and negative effects depending on their concentration At low levels, ROS serve as important signaling molecules that regulate cell division (apoptosis), activate transcription factors such as NFkB and p38 MAP kinase for immune and anti-inflammatory gene expression, and modulate the expression of genes that encode antioxidant enzymes However, when present in high quantities, ROS can lead to oxidative damage, causing DNA mutations, protein denaturation, and lipid oxidation.
81 EFFECTS OF PLANT EXTRACTS ON SELECTED HAEMATOLOGICAL PARAMETERS, DIGESTIVE ENZYMES, AND GROWTH PERFORMANCE
Introduction
The rapid growth of the striped catfish, Pangasianodon hypophthalmus (Sauvage,
The intensification of farming systems in the Mekong Delta, Vietnam, has led to a rise in stress-related diseases and mortality among fish, significantly compromising their immune systems This has resulted in frequent disease outbreaks, including bacillary necrosis of Pangasius (Phan et al., 2009).
(BNP) and motile Aeromonas septicaemia (MAS), caused by Edwardsiella ictaluri and
Aeromonas hydrophila, respectively, commonly occur in farmed P hypophthalmus
Aquaculture diseases pose a significant threat to the industry, leading to substantial economic losses (Crumlish et al., 2010) Research indicates that various stressors, including environmental degradation, agricultural runoff, poor management practices, high stocking densities, and low-quality seeds, heighten the vulnerability of fish stocks to infectious diseases (Phuong et al., 2007) These stressors can disrupt metabolic processes (Santos et al., 2010), degrade the quality of fish fillets (Jittinandana et al., 2003), increase susceptibility to disorders (Wu et al., 2013), and in severe cases, lead to high mortality rates (McKenzie et al., 2012).
Efforts to address mortality in aquaculture have included the use of various pharmaceuticals, with a study by Rico et al (2013) indicating that all P hypophthalmus farmers in Vietnam utilized 17 different types, such as penicillin and tetracyclines However, the environmental and health repercussions, including pollution and antibiotic-resistant bacteria, have led to increasing restrictions on these products (Andrieu et al., 2015) Additionally, while vaccination is being explored as a preventive measure against disease outbreaks, the high cost of commercial vaccines and their specificity to individual infections limit their accessibility for fish farmers (Triet et al., 2019).
Researchers are increasingly integrating natural components into nutritional supplement formulas to improve the health, growth, and immunity of fish, utilizing cost-effective and non-toxic sources essential for sustainable aquaculture (Gabriel, 2019; Gupta et al., 2021) In this field, phytoconstituents serve as immune stimulants and anti-stress agents (Chakraborty and Hancz, 2011) Various plants contain active compounds such as flavonoids, alkaloids, phenolics, steroids, terpenoids, and essential oils, which offer a diverse range of physiological benefits (Ghosh et al., 2019).
Haematology profiles are vital indicators of fish health and metabolism, with blood being the primary tissue used to assess their health status (Fazio, 2019) These profiles provide insight into fish biological responses to external conditions, reflecting their homeostasis (Shahjahan et al., 2018) An imbalance in dietary supplements can negatively affect fish health and increase disease susceptibility, highlighting the importance of proper nutritional practices to maintain a healthy environment and reduce disease outbreaks (Kiron, 2012) Additionally, measuring digestive enzymes is essential for understanding digestion mechanisms and how fish adapt to dietary changes in response to their environment (Sunde et al., 2004; Uys and Hecht, 1987).
Vietnam boasts a rich diversity of medicinal herbs across its various ecological zones, which have been utilized to enhance the immunity and health of P hypophthalmus Notably, Phyllanthus amarus Schumach is recognized as a key antioxidant in this context, as highlighted by Dao et al (2020).
& Thonn (Pa) extract, followed by extracts of Psidium guajava L (Pg), Euphorbia hirta
This study investigates the effectiveness of five plant extracts, including L (Eh) and Mimosa pudica L (Mp), on the haematology, enzymatic activities, and growth performance of P hypophthalmus Notably, Pa extracts demonstrated the highest antibacterial properties against Aeromonas hydrophila, indicated by a low minimum inhibitory concentration (MIC) Additionally, extracts from Azadirachta indica (Ai), Eh, and Pa significantly enhanced the expression of pro-inflammatory, antiviral, and adaptive immune cytokines in striped catfish cells Despite the potential benefits of these bioactive compounds, farmers remain largely unaware of their efficacy in fish and their ability to improve aquatic species' health The findings aim to promote environmentally friendly biological products in aquaculture, reducing reliance on chemically synthesized antimicrobial treatments commonly used in intensive fish farming systems.
Material and Method
3.2.1 Plant extract and feed preparation
Fresh samples of Eh (leaves and twigs), Pa (twigs and leaves), Pg (leaves), Mp (twigs and leaves), and Ai (leaves) were manually collected near Can Tho city, Vietnam The plant identities were verified, and the samples were processed at the College of Natural Sciences, Can Tho University After washing with sterile distilled water and removing damaged parts, the plant materials were sun-dried for several days and then dried at 60°C The fine powder was stored at room temperature, and to create an ethanolic extract, 100 g of the dried powder was immersed in 800 mL of 96% ethanol for 24 hours The samples were then decanted, and excess solvent was evaporated using a rotary evaporator under low pressure (Nhu et al., 2019).
The study focused on formulating fish diets enriched with plant extracts, ensuring that all experimental diets were iso-lipidic, iso-proteic, and iso-energetic The control diet served as a baseline without plant extract supplementation, while the feed's composition included 3.21% fiber, 10.58% ash, 30% crude protein, 6.66% crude fat, and 4.41 kcal/g of energy Mixture 1 comprised sterilized rice bran, cassava, soybean meal, and fishmeal, while Mixture 2 contained butylated hydroxytoluene (BHT), vitamins, and minerals, all thoroughly blended with varying concentrations of plant extracts The final mixture was extruded using a mini-extrusion machine, followed by pelletizing, air-drying, grinding, and sieving to achieve a 2 mm pellet size The pellets were then stored at -20°C in labeled polythene bags for future use.
Table 3.1 Experimental feed ingredients and formulation
Ingredients (100 g of feed) Basal diet Supplementary plant extract diet
Carboxymethyl cellulose (CMC) (g) 0.50 0.50 0.50 0.50 0.50 Butylated hydroxytoluene (BHT) (g) 0.02 0.02 0.02 0.02 0.02
The premix formulation consists of 1% attractant, 0.03% vitamin C, 0.5% CMC, along with a blend of vitamins and minerals Key ingredients such as fishmeal, soybean meal, cassava, and rice bran are weighed, mixed, and sterilized at 110°C for 10 minutes, although the sterilization process may extend to 60-90 minutes due to the cooling phase to 70°C, effectively eliminating bacteria and enhancing fish digestibility This crucial step in feed processing involves thoroughly mixing BHT, the premix, and CMC with a cooked powder (prepared by cooking for 30 minutes and stirring) before combining it with the sterilized mixture and fish oil The resulting experimental feeds are then dried at 60°C for approximately 24 hours and stored at -20°C for future use, while the control diet is prepared using the same method without the addition of plant extract.
3.2.2 Experimental fish acclimation, facilities, and feeding management
P hypophthalmus fingerlings (14.1±0.46 g/fish) were sourced from a hatchery in
In a controlled study, fish from Can Tho city were transported in oxygenated bags and acclimated to 2 m³ experimental tanks with well-aerated water and natural light cycles They were fed a basal diet twice daily until satiation, amounting to 3-5% of their body weight After a two-week acclimation period, 2,475 fish were divided into 33 fiberglass tanks, each containing 300 liters of water, with a stocking density of 75 fish per tank Various plant extracts were administered in two doses: Eh (0.4% and 2.0%), Pa (0.2% and 1.0%), Pg (0.4% and 2.0%), Mp (0.4% and 0.2%), and Ai (0.4% and 2.0%).
During the 60-day experiment, all tanks were consistently supplied with well-aerated filtered water, and fish were manually fed twice daily until satiated, accounting for 3-5% of their body weight To minimize fouling from feed residues, daily siphoning was conducted, and 30% of the water was replaced weekly with fresh, dechlorinated water Water quality parameters, including dissolved oxygen (3.95 to 4.90 mg/L), pH (7.41 to 7.63), and temperature (28.28 to 29.15°C), were monitored bi-weekly using a pH instrument and an Oxy Guard device, all falling within the optimal range for P hypophthalmus The experiment adhered to Vietnam's legislation on animal protection and welfare.
On days 30 and 60 of the experiment, three fish from each replicate were sampled, starting with an initial collection of 30 fish To reduce stress, a cold moist cloth was applied to the head of each fish during handling Blood samples were quickly obtained from the caudal peduncle vein using heparin-coated syringes, with at least 300 µL drawn and stored in labeled tubes One portion of the blood was used for haematological analysis, where it was diluted with Natt and Herrick's solution and red blood cells (RBCs) were counted using a Neubauer haemocytometer Hematocrit (Hct) and blood samples were centrifuged for plasma analysis, and hemoglobin (Hb) levels were quantified using Drabkin's solution and a spectrophotometer Mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) were calculated from the obtained data White blood cells (WBCs) were counted by smearing blood on slides, staining them, and examining under a microscope Additionally, blood was centrifuged to separate the supernatant for glucose analysis, which was performed using a standardized assay.
To prepare for sampling, fish were fasted for 48 hours to ensure their stomachs were empty Following blood collection, the fish were euthanized with ice and dissected to extract the stomach and intestine for enzyme assays, specifically pepsin and amylase from the stomach and amylase, trypsin, and chymotrypsin from the intestine A scalpel was utilized to remove any remaining gut and stomach contents, with all manipulations performed on ice to preserve enzymatic activity The stomach and intestine were then cut longitudinally, rinsed with distilled water, blotted dry with filter paper, and placed in labeled 1.5-mL tubes, which were refrigerated at -80°C until homogenization After defrosting, the isolated stomachs and intestines were weighed and homogenized for further analysis.
A KH2PO4/NaCl buffer with a pH of 6.9 was prepared, and samples were centrifuged for 30 minutes at 4,200 g, with the supernatant collected for enzymatic activity analysis Pepsin activity was quantified using the Worthington and Manual (1982) method, which involved mixing 100 μL of the sample with bovine hemoglobin (Sigma-Aldrich) as a substrate in 1N HCl Trichloroacetic acid (TCA; Sigma-Aldrich) was then added to the reaction solution, and each sample was centrifuged at 4,000 g for 10 minutes at 4°C to measure pepsin activity at 280 nm.
Trypsin activity was measured by mixing 15 μL of the sample with a 0.1 M BApNA solution and phosphate buffer at pH 8.2, followed by optical density assessment at 407 nm after 5 minutes (Tseng et al., 1982) Chymotrypsin activity was evaluated using 50 μL of the sample with BTEE and buffer at pH 7.8, with measurements taken at 256 nm (Worthington and Manual, 1982) Amylase activity was calibrated using maltose, quantified at 540 nm (Bernfeld, 1951) Total protein content was analyzed using diluted homogenates and bovine serum albumin for calibration (Bradford, 1976) Enzyme activity was expressed in units per milligram of protein (U/min/mg protein).
3.2.5 Growth performance and survival rate
At 30-day intervals of the experiment (days 30 and 60), weight gain (WG), daily weight gain (DWG), and survival rate (SR) were assessed All fish were gathered from the corresponding tank and weighed with a digital balance and the number of fish was recorded to determine the survival rate Fish were gently returned to respective tanks after measurement Growth indicators were identified based on the formula: weight gain (WG, g) = (Wf −Wi); daily weight gain (DWG, g/day) = (Wf −Wi)/t; and survival rate
(%) = No of fish harvested × 100/No of fish stocked Wi and Wf are initial and final weight (g), respectively, and t is the duration of the experiment (days)
Statistical analyses were conducted using SPSS software, version 20 (IBM Corp., Armonk, NY) The Levene test assessed the homogeneity of variance among groups, while a one-way analysis of variance (ANOVA) and Duncan's multiple range test were utilized to identify treatment differences at each sample interval A significance level of p0.05) Additionally, a dose-dependent increase in hematocrit (Hct) was observed in all groups receiving a diet enriched with 0.2% Pg, with significant differences from the basal diet noted on both day 30 and day 60, yielding values of 30.6±19% and 31.2±24%, respectively (p