Ảnh hưởng của một số chiết xuất thảo dược lên các chỉ tiêu sinh lý cá tra (Pangasianodon hypophthalmus).Ảnh hưởng của một số chiết xuất thảo dược lên các chỉ tiêu sinh lý cá tra (Pangasianodon hypophthalmus).Ảnh hưởng của một số chiết xuất thảo dược lên các chỉ tiêu sinh lý cá tra (Pangasianodon hypophthalmus).Ảnh hưởng của một số chiết xuất thảo dược lên các chỉ tiêu sinh lý cá tra (Pangasianodon hypophthalmus).Ảnh hưởng của một số chiết xuất thảo dược lên các chỉ tiêu sinh lý cá tra (Pangasianodon hypophthalmus).Ảnh hưởng của một số chiết xuất thảo dược lên các chỉ tiêu sinh lý cá tra (Pangasianodon hypophthalmus).Ảnh hưởng của một số chiết xuất thảo dược lên các chỉ tiêu sinh lý cá tra (Pangasianodon hypophthalmus).Ảnh hưởng của một số chiết xuất thảo dược lên các chỉ tiêu sinh lý cá tra (Pangasianodon hypophthalmus).Ảnh hưởng của một số chiết xuất thảo dược lên các chỉ tiêu sinh lý cá tra (Pangasianodon hypophthalmus).Ảnh hưởng của một số chiết xuất thảo dược lên các chỉ tiêu sinh lý cá tra (Pangasianodon hypophthalmus).Ảnh hưởng của một số chiết xuất thảo dược lên các chỉ tiêu sinh lý cá tra (Pangasianodon hypophthalmus).Ảnh hưởng của một số chiết xuất thảo dược lên các chỉ tiêu sinh lý cá tra (Pangasianodon hypophthalmus).Ảnh hưởng của một số chiết xuất thảo dược lên các chỉ tiêu sinh lý cá tra (Pangasianodon hypophthalmus).Ảnh hưởng của một số chiết xuất thảo dược lên các chỉ tiêu sinh lý cá tra (Pangasianodon hypophthalmus).Ảnh hưởng của một số chiết xuất thảo dược lên các chỉ tiêu sinh lý cá tra (Pangasianodon hypophthalmus).Ảnh hưởng của một số chiết xuất thảo dược lên các chỉ tiêu sinh lý cá tra (Pangasianodon hypophthalmus).Ảnh hưởng của một số chiết xuất thảo dược lên các chỉ tiêu sinh lý cá tra (Pangasianodon hypophthalmus).Ảnh hưởng của một số chiết xuất thảo dược lên các chỉ tiêu sinh lý cá tra (Pangasianodon hypophthalmus).Ảnh hưởng của một số chiết xuất thảo dược lên các chỉ tiêu sinh lý cá tra (Pangasianodon hypophthalmus).Ảnh hưởng của một số chiết xuất thảo dược lên các chỉ tiêu sinh lý cá tra (Pangasianodon hypophthalmus).Ảnh hưởng của một số chiết xuất thảo dược lên các chỉ tiêu sinh lý cá tra (Pangasianodon hypophthalmus).Ảnh hưởng của một số chiết xuất thảo dược lên các chỉ tiêu sinh lý cá tra (Pangasianodon hypophthalmus).Ảnh hưởng của một số chiết xuất thảo dược lên các chỉ tiêu sinh lý cá tra (Pangasianodon hypophthalmus).
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 This region is particularly vulnerable to the impacts of global warming, with average temperatures in Vietnam having increased by approximately 0.5-0.7°C in recent years and projected to rise by 2.3°C by the end of the 21st century (MONRE, 2009) Such temperature changes threaten the sustainable development of fisheries and aquaculture, as fish are poikilothermic species, making temperature a critical factor influencing their life Fluctuating temperatures can negatively impact fish by altering metabolic rates (Galloway and Kieffer, 2003), impairing swimming performance (Hocutt, 1973), and compromising immune functions (Hurst, 2007), which can lead to reduced prey capture ability, increased disease susceptibility, and higher mortality rates (Donaldson et al., 2008) Additionally, temperature shocks can hinder predator avoidance and recovery from exercise (Ward and Bonar, 2003; Suski et al.).
2006), and disrupt homeostasis (Vanlandeghem et al., 2010)
The Mekong River Delta (MRD) is one of three significant low-lying areas in Vietnam anticipated 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, as the region's low-lying terrain, with elevations below 4.0 meters, makes it vulnerable A projected 1-meter increase in mean 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 salinization of freshwater areas will adversely affect the local aquaculture sector, particularly the striped catfish culture, by altering both soil conditions and freshwater availability.
Intensive pond farming, characterized by overfeeding and waste accumulation, can trigger the decomposition of organic matter, leading to the production of toxic gases Under hypoxic and high-temperature conditions, harmful substances such as ammonia, nitrite, nitrate, carbon dioxide, and hydrogen sulfide are significantly generated Notably, nitrite, a naturally occurring element of the nitrogen cycle, can reach harmful concentrations, posing serious toxicity risks to aquatic animals.
Methaemoglobin formation due to the reaction of nitrite with hemoglobin is a key factor leading to low arterial oxygen levels in freshwater fish, while decreased blood oxygen affinity also plays a role Although fish can tolerate high levels of metHb at rest, reduced blood oxygen content restricts their activity These challenges hinder 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 development of drug-resistant bacteria and environmental pollution, with 17 different antibiotic compounds identified (Jian and Wu, 2004; Rico et al., 2013) Although these pharmaceuticals are commonly used to prevent disease outbreaks and minimize economic losses, their negative impacts on the environment and human health have prompted stricter regulations (Mckenzie et al., 2012) Vaccination is being explored as an alternative, but high costs and limited efficacy against single diseases hinder widespread adoption (Harikrishnan et al., 2011) Consequently, researchers are focusing on natural ingredients to create alternative nutritional supplements that enhance the growth, health, and immune systems of farmed fish while minimizing toxicity (Pandey et al., 2012).
In today's world, there is a growing demand for environmentally friendly prophylactic and preventive solutions, leading to an increased interest in natural bio-active products that can boost the immune system and overall health of farmed animals Plant-derived compounds, including phenolics, polyphenols, alkaloids, quinones, terpenoids, lectins, and polypeptides, have proven to be 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 Despite the rich diversity of wild plants in Vietnam and the growing interest among aquaculture farmers in natural alternatives, the adoption of bio-active products remains limited This is primarily due to a lack of awareness about these products and insufficient evidence of their effectiveness in improving fish health.
The study identified five types of plant extracts and their concentrations based on screening research involving 20 plant species (Nhu et al., 2019) It is essential to explore the physiological aspects of striped catfish, including hematology, digestive enzymes, and stress responses Furthermore, the effects of specific extracts, such as Euphorbia hirta, warrant investigation.
Research on the effects of Phyllanthus amarus, Mimosa pudica, Psidium guajava, and Azadirachta indica on the physiology of striped catfish is limited, despite their potential as cost-effective dietary components Our findings provide valuable biological insights and emphasize the availability of plant-based bioactive substances that can enhance fish health The current use of drugs and herbal extracts in striped catfish farming reveals a lack of information on herbal-derived products among farmers Therefore, it is crucial to implement training programs for farmers to improve their knowledge of active ingredients, appropriate usage, and dosing of plant extract products.
The objectives of the dissertation Error! Bookmark not defined
The primary objective of this study is to identify plants that positively impact fish health, thereby promoting the production of biologically safe products and enhancing environmental sustainability Additionally, the study aims to encourage fish farmers to incorporate herbs in striped catfish farming, which can help minimize antibiotic use and prevent water pollution.
This 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 activity, and growth performance of striped catfish fingerlings The findings revealed significant improvements in growth metrics and digestive efficiency, highlighting the potential benefits of these natural supplements in aquaculture practices.
- 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, leading to enhancements in hematological parameters, digestive enzyme activities, and growth performance in striped catfish when incorporated into their diet.
Supplementation with P amarus and P guajava-based diets at elevated temperatures did not adversely affect hematological parameters, digestive enzymes, or oxidative stress levels, leading to improved growth performance in striped catfish.
Chronic exposure of striped catfish to increased salinity led to gradual acclimatization without negatively impacting physiological functions or growth performance when fed P amarus and P guajava diets.
Supplementing striped catfish diets with the specified extracts enhances their tolerance to elevated nitrite levels, resulting in improved physiological parameters and growth performance compared to a basal 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 extracts, when incorporated into fish diets at varying doses and durations, have shown potential to positively influence hematology, enzymatic activity, and the growth of P hypophthalmus Specifically, diets containing 0.2% extracts of Psidium guajava or other plant sources over a 60-day period may enhance fish growth performance Therefore, assessing fish preferences for these plant extract-based diets is crucial, as it could lead to increased aquafeed consumption and improved growth outcomes.
The Pg0.2 and Mix diets enhance the health of P hypophthalmus by improving hematology profiles, digestive enzyme activity, and oxidative stress biomarkers These diets are effective for promoting fish health and reducing stress Key hematological parameters, such as RBCs, Hct, Hb, and glucose, remained stable at elevated temperatures (35°C) until day 7, while oxidative stress indicators (LPO, CAT in gill and liver) showed no significant changes until day 14 Subsequently, the fish adapted to the experimental conditions and demonstrated recovery.
In a study on salinity effects, the liver and gills of fish exhibited significant oxidative damage at elevated salinity levels, particularly at 20‰, while changes were minimal at 10‰ This suggests that the species could be viable for farming in low-salt brackish environments Additionally, a diet supplemented with Pg0.2, combined with Mixture (Pg0.2:Pa0.5), proved most effective in maintaining the fish's normal physiological functions, including haematology, digestion, and stress management.
Striped catfish experience significant stress when exposed to nitrite concentrations of 0.8 mM, leading to a reduction in red blood cells (RBCs), hemoglobin (Hb), and hematocrit (Hct), along with an increase in plasma glucose levels after just 24 hours Prolonged exposure to high nitrite levels, particularly between 7 to 42 days, further decreases digestive enzyme activities, adversely affecting the fish's growth performance These negative effects intensify with longer exposure and higher nitrite concentrations.
To ensure optimal growth performance and reduce stress in fish, production systems should avoid nitrite concentrations of 0.8 mM or higher The Pg0.2 and Mix diets have been shown to enhance the health of P hypophthalmus by positively influencing hematology, digestive enzyme activity, and oxidative stress biomarkers.
Significant contributions of the dissertation
The dissertation highlights the scientific contributions of P amarus and P guajava in enhancing the health of catfish by 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 can be further developed for large-scale application in aquaculture, significantly reducing the reliance on antibiotics and chemicals, thus promoting environmental sustainability Additionally, the research findings serve as a valuable educational resource and a foundation 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 a participation from 225 countries, with a total value of $424 billion, despite a 7% decline due to the Covid-19 pandemic The FAO (2022) reported a record production of 214 metric tons of fish and algae, which included 178 million aquatic animals.
36 million algae Aquaculture experienced a 2.7% growth, below the 4.5% annual average growth over the previous decade (FAO, 2022)
Figure 2.1 Total fisheries and aquaculture production 2020 (FAO, 2022).
The COVID-19 pandemic has significantly impacted the global trade of aquatic products, leading to a projected 7.0% decline in export value, which reached US$ 151 billion in 2020 This downturn followed a 2.1% decrease in 2019, highlighting ongoing challenges in the industry.
In 2020, global trade volumes experienced a significant decline of 10.1%, affecting all regions However, as fishing and aquaculture resumed and international markets reopened, trade saw a robust recovery in 2021, with the overall value of global aquatic product exports rising by 12% compared to the previous year Despite this growth in value, trade volume expansion remained limited due to cautious planning regarding aquaculture supplies 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 was the leading producer of aquatic animal products, contributing to 70% of global production, with China dominating the market at a 35% share Following China, Norway and Vietnam also emerged as significant exporters, while the European Union held the title of the largest importer in the world.
In 2020, Asia played a crucial role in aquaculture, contributing 91.6% of the world's aquatic species and algae The global output of air-breathing fish reached 6.2 million metric tons, representing 12.6% of total production, though this marked a slight decline from 2019 due to reduced supply in Vietnam Notably, three fish families dominated air-breathing finfish production, with Pangasiidae, Clariidae, and Channidae accounting for 83.9% of the total, sharing 47%, 26.5%, and 10.5% of production, respectively Striped catfish, a key species in aquaculture, consistently ranks among the top 10 globally produced species, showcasing continuous annual production growth.
Vietnam plays a crucial role in global aquaculture, particularly in the production of striped catfish, which significantly contributes to the country's overall aquaculture output Since 2014, Vietnam has emerged as the third-largest exporter of aquatic products, leading the world in the production and export of farmed striped catfish In 2020, the nation exported approximately US$ 8.5 billion worth of aquatic products, accounting for 5.6% of the global total (FAO, 2022) A notable trend in Vietnam's striped catfish sector is the adoption of quality standards such as ASC and Global GAP, aligning with international commodity trade requirements.
Striped catfish farming is predominantly 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 city, An Giang, and Dong Thap are the leading areas, contributing over 75% of the nation's total striped catfish production (Hien, 2020) The industry effectively manages raw material sources to ensure sustainable production According to VASEP (2022), the striped catfish farming area in the MRD was approximately 5,700 hectares in 2020, yielding a total production of 1.553 million tons In 2021, the farming area 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 fell significantly due to the impact of COVID-19, but there was a slight recovery in consumption in 2021 Viet Nam aims to expand its striped catfish exports to 138 markets, including key regions such as China, Hong Kong, the United States, and ASEAN countries.
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 identified as the primary 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, aligning with national government policy goals Challenges to sustainable growth, including disease management, chemical usage, sustainable practices, and seed quality, pose significant threats to the sector's expansion (Phuong and Oanh, 2010) Additionally, the industry faces severe future challenges due to climate change, particularly from saline intrusion and elevated 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, leading to significant environmental shifts (UNFCCC, 2003) The increase in harmful gases like CO2, N2O, and CH4 has intensified these changes (IPCC, 2013) As a result, climate change manifests through rising temperatures, droughts, sea-level rise, and variations in seasonal precipitation, profoundly impacting various sectors, particularly agriculture and aquaculture (USEPA, 2012).
Vietnam ranks 27th in vulnerability to climate change impacts on its fisheries sector, with the Mekong River Delta (MRD) being particularly affected Covering 3.96 million hectares, the MRD contributes to 65% of the country's aquaculture production Saline intrusion threatens 1.2 to 1.6 million hectares in coastal areas, and during the dry season, salinity levels can exceed 4‰ across 2.4 million hectares, which is about 65-70% of the total MRD area Forecasts indicate that the MRD will be among the top three regions globally affected by climate change due to its unique geographic and natural characteristics.
Since 1906, global temperatures have increased by 0.74°C, with projections indicating a further rise of 0.2°C per decade over the next 20 years (IPCC, 2007) In Vietnam, coastal regions are expected to experience temperature increases ranging from 1.1 to 1.5°C, while interior areas may see rises between 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 reaching 32°C, with an increase of 2°C anticipated between 2030 and 2040 Climate change poses significant threats to aquaculture and fisheries, especially affecting striped catfish nurseries and grow-out systems, which are vulnerable to extreme rainfall and temperature fluctuations This rising temperature can lead to an increased incidence of parasite infections in striped catfish, highlighting the urgent need for adaptive strategies in aquaculture practices.
Drought and rising temperatures lead to increased evaporation, reducing freshwater availability in rivers and bays, while tidal changes affect estuarine salinity (USEPA, 2012) Barange and Perry (2009) note that global sea levels are rising at an accelerating pace, threatening extensive low-lying coastal regions If sea levels increase by 40 cm, around 20 million people in Southeast Asia could be impacted by flooding Projections indicate that sea levels may rise by 33 cm by 2050 and up to 1.0 m by 2100 (UNFCCC, 2003).
A 2009 forecast predicted a sea level rise of 25 cm by 2030 and 75 cm by the end of the 21st century Given that most of the MRD region has an elevation of less than 2 meters above sea level, it is particularly vulnerable to the impacts of rising sea levels (VNU, 2019).
A study by Nicholls (2006) indicates that around 1,000 km² of cultivated land in the MRD could transform into salt marshland if sea levels rise by 1 meter Research by Anh et al (2014) assessed the effects of rising sea levels in three scenarios (+30, +50, and +75 cm) on striped catfish farming Their findings revealed that even a moderate sea level increase of 50 cm could lead to significant flooding, adversely affecting farms in An Giang and Dong Thap provinces, as well as Can Tho city, by causing water to disperse downstream.
Research indicates that adaptive strategies for the sector are feasible in light of predicted climate change impacts The genetic resources currently cultivated in the Mekong River Delta (MRD) can aid in developing selective breeding programs for climate change-resistant strains However, salinity intrusion has limited the scale of striped catfish farms in downstream areas, and rising river salinity may 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 applications, aeration, and effective water quality management, to mitigate the adverse effects of climate change and salinity challenges on their farming practices.
Mechanism of stress on fish
A fish's ability to adapt to environmental changes hinges on its effective stress response, energy reallocation for defense, and behavioral adjustments to mitigate risks (Barton, 2002) This process is governed by two key neuroendocrine pathways: the hypothalamic-pituitary-interrenal (HPI) axis, which triggers a rapid stress response by increasing heart and respiratory rates to supply glucose to essential tissues, primarily mediated by adrenaline, and the brain-sympathetic–chromaffin cell (BSC) axis, which reorganizes energy by enhancing catabolic processes, supplying glucose, converting fatty acids for energy, and suppressing costly activities like immune responses, with plasmatic cortisol levels playing a crucial role (Wendelaar Bonga, 1997).
Stress isn't inherently negative; in fact, an acute stress response can effectively prompt necessary physiological and behavioral changes to adapt to environmental challenges and uphold homeostasis However, when stress becomes chronic and unavoidable, the resulting physiological and behavioral adaptations may become detrimental (Barton, 2002).
In response to stressors, various organizational levels, including cells, tissues, organs, and entire organisms, initiate specific processes The stress response begins with the interpretation of environmental stimuli, both internal and external, primarily occurring in the hypothalamus and telencephalon of fish brains This response is regulated by the control and organization of the stress state through the activity of monoaminergic neurotransmitters such as dopamine, noradrenaline, and serotonin.
The brain orchestrates a comprehensive stress response through two primary neuroendocrine pathways: the brain-sympathetic-chromaffin cell axis (BSC), which directly stimulates chromaffin cells in the anterior kidney to release catecholamines into the bloodstream, and the more intricate hypothalamic-pituitary-interrenal (HPI) axis These pathways trigger catecholamine responses that provide energy substrates for muscle and other tissues, enabling behavioral reactions, while cortisol-mediated changes, primarily through glucocorticoid and mineralocorticoid receptors, mobilize and reallocate energy resources to support 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), which in turn promotes cortisol production from the interrenal cells of the anterior kidney Stress hormones like catecholamines and cortisol play crucial roles in various tissues, aiding the body in coping with stressors by increasing blood glucose levels, enhancing cardiac output, and suppressing immune function However, chronic stress can lead to detrimental effects on fish, including impaired immune response, growth issues, and reduced reproductive success due to the reallocation of energy substrates.
The impact of continuous stressors on fish populations varies based on severity, leading to significant repercussions (Barton, 2002) Adaptive responses, such as maintaining homeostasis, help fish cope with stressors However, systemic alterations can occur as tertiary responses, which hinder the fish's ability to adapt and negatively affect their health, performance, growth, reproduction, disease resistance, and behavior (Barton, 2002).
Assessing stress responses in fish involves various methodologies, including measurements of body and organ weights such as condition factor, hepatosomatic index, and gonadosomatic index Biochemical analyses are also crucial, focusing on plasma cortisol, corticosterone, glucose, tissue damage enzymes, and heat shock proteins Additionally, evaluating immunological responses, gene expression patterns, fish steroids, and both macroscopic and microscopic morphology contributes to a comprehensive understanding of fish stress responses.
The diencephalon orchestrates the 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 boost cardiovascular activity, increasing heart rate and blood pressure while mobilizing energy sources like glucose and lactate Concurrently, catecholamine release promotes 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, the 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 crucial environmental factor influencing aquatic animals, particularly poikilotherms, which are highly sensitive to temperature fluctuations (Fry, 1971) Climate change, characterized by rising temperatures, poses complex challenges, as the environmental temperature range significantly impacts these species While many fish can acclimate to moderate changes, substantial temperature shifts can adversely affect their growth and survival rates, leading to negative physiological responses, particularly for ectotherms with limited body temperature regulation capabilities (Kemp, 2009; Wright & Tobin, 2011).
Temperature significantly affects the metabolic rate of aquatic animals, influencing their energy balance and behaviors such as locomotion, feeding, and reproduction, particularly in ectothermic species like fish Ectotherms experience body temperature changes based on their environment, with metabolic rates typically doubling for every 10°C increase within their normal tolerance range While slight temperature increases can enhance growth by providing more energy, 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 ambient temperature tolerance influenced by factors like maturation, genetics, and environmental conditions Laboratory studies indicate that excessive temperature increases can be detrimental to growth and even lethal The specific temperature at which development declines varies by species, and research shows that vaccinated fish are significantly affected by low temperatures, suffering mortality rates similar to non-vaccinated fish due to a lack of antibodies.
Hematology is a crucial field of study in invertebrates, including fish, to assess their health and physiological state Key hematological parameters such as red blood cell (RBC) counts, hemoglobin (Hb), and hematocrit (Hct) are essential for evaluating oxygen-carrying capacity (Houston, 1997) Secondary indices, known as Wintrobe indices, help classify anemia, with calculations for mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) derived from these primary measurements (Gabriel et al., 2015) Additionally, white blood cell (WBC) counts and their differential counts, including lymphocytes and neutrophils, provide insights into stress conditions, with the neutrophil-to-lymphocyte ratio serving as a valuable stress indicator in invertebrates (Van Rijn & Reina, 2010) Hematological parameters are widely recognized as sensitive biomarkers for monitoring physiological and pathological changes in fish (Oluyemi et al., 2008; Patra et al., 2014), and are routinely assessed in fish farms to ensure stock health (Haghighi & Rohani, 2013), as they can be affected by environmental stress (Hickey, 1976) and malnutrition (Casillas & Smith).
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 impacts fish health, acting as a stressor that alters hematological indices (Portz et al., 2006; Barton, 2002) Elevated temperatures enhance blood oxygen-carrying capacity, facilitating oxygen delivery as metabolic rates increase (Carvalho & Fernandes, 2006; Zarejabad et al., 2010) Changes in hemoglobin (Hb) concentration are vital for sustaining oxygen delivery across varying environmental conditions (Brauner & Wang, 1997) The reduction of oxygen solubility in water due to temperature stress necessitates adaptations in fish hematology (Cech & Brauner, 2011) Additionally, the opercular beat rate serves as an indicator of stress response, with increased opercular activity correlating to heightened oxygen demand (Dalla Valle et al., 2003) Given that water holds significantly less oxygen than air and diffuses more slowly, fish must adjust to decreased HbO2 affinity as temperatures rise, compensating for lower oxygen availability and increased metabolic needs (Graham, 1990; Salama & Nikinma, 1990).
Extreme temperature fluctuations significantly impact hematological parameters in fish, leading to reduced opercular movements and lethargy (Kapila et al., 2002; Hrubec et al., 2000) To cope with rapid temperature changes exceeding 10°C, fish must enhance oxygen uptake at the gills and improve circulatory distribution to meet tissue oxygen demands Cardiac function is unlikely to limit metabolic rates until reaching critical thermal maxima, although decreasing blood oxygen binding capacity may affect oxygen consumption (Gollock et al., 2006) A reduction in hemoglobin (Hb) and red blood cell (RBC) quantity and quality impairs oxygen transport, impacting metabolism beyond mere oxygen delivery (Gross et al., 1996) Additionally, increases in RBCs and Hb correlate with decreased mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC), contributing to poikilo-anisocytosis These changes are closely related to the severity of thermal stress and are particularly pronounced during prolonged exposure to high temperatures during acclimatization (Ahmad et al., 2006).
Temperature acclimation in fish involves changes in haematological parameters, characterized by an initial phase of thermal stress followed by gradual recovery A fish is deemed well-acclimated when it maintains stable haematological levels between initial and elevated temperature conditions (Maricondi-Massari et al., 1998) Ectothermic animals demonstrate acclimation responses to prolonged temperature variations, often involving enzyme modifications that help mitigate temperature's impact on metabolism (Hazel & Prosser).
Temperature significantly influences the biochemical and physiological functions of aquatic animals, enhancing growth rates and shortening maturation periods up to a certain point However, temperatures beyond the optimal range can harm their health by increasing metabolic rates and oxygen consumption Non-photosynthetic organisms may stabilize their metabolism at intermediate levels, while temperature acclimation helps ectothermic species cope with environmental fluctuations The natural environment can present historical stressors like temperature, leading to the evolution of physiological mechanisms for stress tolerance Aquatic creatures, particularly fish, are highly sensitive to both extreme 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 efficiency of digestion and absorption in fish Optimal performance of digestive enzymes may require specific pH levels that vary with temperature and species Given the diverse habitats and physiological traits of fish, the impact of temperature is both complex and species-specific Factors such as the duration and rate of temperature change also play crucial roles; while acute temperature fluctuations can adversely affect fish physiology, gradual long-term changes may facilitate acclimation, resulting in altered 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 digestion, prolonging gut transit time, and reducing gastrointestinal evacuation rates (Nakagawa, 2018; Das, 2018; Mazumder).
Temperature significantly influences the gastrointestinal tract by affecting the fatty acid content of lipids in the intestinal mucosa, altering gut acidity levels, and impacting intestinal pH Higher temperatures can lower pH, while cooler temperatures may have the opposite effect Additionally, temperature changes can modify the transportation processes of amino acids across intestinal membranes due to variations in lipid solubility Furthermore, temperature affects the composition of intestinal bacteria and enhances metabolic rates in response to feeding.
Temperature variations can significantly influence enzyme-metabolite interactions in two primary ways First, the bonds stabilizing these interactions possess low energy levels, making them susceptible to alterations from environmental thermal energy, which can either hinder or promote complex formation without changes in enzyme structure Second, temperature fluctuations may also impact the structure of enzymes, thereby affecting their affinities for 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 (lipase), and alkaline phosphatases (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 in the pyloric caeca and intestine, can influence genetic diversity in proteins An increase in trypsin secretion into the lumen leads to a decrease in its 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 proteins into smaller peptides and amino acids, facilitating nutrient absorption in the intestines, thereby playing a critical role in digestion and energy storage Additionally, as protein intake is vital for growth, proteases are essential for fish development.
Carbohydrates play a lesser role in the digestion of carnivorous fish compared to herbivorous and omnivorous species, which primarily rely on carbohydrate-rich diets The enzyme amylase facilitates the breakdown of polysaccharides by targeting 1,4-α-D-glucosidic linkages Optimal protease activity in various fish species occurs at temperatures between 30 to 60°C, with pepsin demonstrating peak performance at 30°C Research into fish digestive secretions enhances our understanding of nutritional physiology and addresses dietary challenges By studying digestive enzyme activity, we can gain insights into fish nutritive physiology, aiding in the evaluation of dietary suitability and the overall nutritional capacity of fish.
Enzyme activity, crucial for understanding cumulative conversion, is significantly influenced by temperature and the feed's composition (Tijskens et al., 2001) Fish acclimated to different temperatures exhibit varying enzyme activity levels, affecting amino acid absorption and transport due to changes in enzyme affinity for substrates (Sunde et al., 2004) Additionally, Gelman et al (2008) highlighted that genetic factors determine enzyme temperature adaptation, leading to phenotypic changes Variations in enzyme structure, substrate affinity, activation energy, and the rate of isozyme secretion and synthesis further contribute to differences in catalytic efficiency across temperatures.
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 the importance of evaluating the impact of medicinal plants on hematological and biochemical parameters in experimental animals to distinguish between effective and ineffective treatments Hematological changes often serve as the earliest and most quantifiable responses to environmental shifts, acting as key indicators of fish blood profiles and playing a crucial role in innate immune protection and immunological function (Ballarin et al., 2004) Numerous aquaculture studies have effectively demonstrated that various medicinal plants can significantly improve these hematological parameters.
Plant extracts have a significant impact on the hematological profile of various fish species Research by Dügenci et al (2003) revealed that medicinal plant extracts like mistletoe, nettle, and ginger enhance the immune response in rainbow trout, with ginger extract notably increasing phagocytosis and extracellular burst activity in blood leukocytes This boost in leukocyte counts indicates 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 their oxygen-carrying capacity, which strengthens their defense against physiological stress These beneficial effects are linked to the rich content of vitamins and minerals in Aloe vera, including β-carotene, vitamins C, E, B12, riboflavin, thiamine, folic acid, and essential and nonessential amino acids.
Aloe vera supplementation in fish has been linked to increased leukocyte levels and enhanced resistance to low pH, suggesting its potential to stimulate leucopoiesis and improve stress resilience Research on Zingiber officinale's impact on the immune response of Asian sea bass (Lates calcarifer) indicated that a 10 g/kg inclusion of the plant extract led to increased erythropoiesis and lymphopoiesis, resulting in higher levels of RBC, WBC, Hb, and Hct, which collectively enhance oxygen transport and stress resistance These benefits may stem from the antioxidants present in plant extracts that reduce oxidant-induced hemolysis, as well as the influence of polyphenols, alkaloids, glycosides, and sugar reduction on white blood cell production.
Flavonoids derived from plant extracts can regulate interferon and play a crucial role in nonspecific cellular immunity by acting as biocatalysts for white blood cell production Additionally, they contribute to reducing red blood cell hemolysis and protect biological membranes from oxidative damage caused by free radicals.
Myrmecodia tuberosa, studied in relation to striped catfish, has been found to contain flavonoids, which may serve as antioxidants that help preserve heme iron and support erythropoiesis.
Research indicates that many therapeutic plant extracts do not significantly impact hematological parameters in tilapia unless the fish are exposed to specific stressors The mechanisms behind these effects remain unclear, but they may involve interference with erythropoiesis, haemosynthesis, and osmoregulation, or increased destruction of red blood cells in hematopoietic organs Therefore, in aquaculture, it is essential to optimize the use of herbal extracts not only for growth and feed efficiency but also for monitoring hematological parameters Evaluating hematological and biochemical characteristics in fish is crucial for understanding their health status, disease, and toxicological responses (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 activity in fish Notably, various plant extracts have been shown to stimulate appetite and promote weight gain in cultured fish (Harikrishnan et al., 2011) Specifically, the supplementation of garlic (Allium sativum) has been identified as a growth-stimulating agent for Nile tilapia (Oreochromis niloticus) (Shalaby et al.).
A study by Punitha et al (2008) revealed that grouper (Ephinephelus tauvina) 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), achieved a 41% increase in weight compared to those on a control diet Furthermore, research by Talpur et al (2013) indicated that plant extracts, particularly ginger, enhance nutrient digestibility and availability, resulting in improved feed conversion and increased protein synthesis in Asian sea bass (Lates calcarifer).
Medicinal plant products, rich in flavonoids, alkaloids, phenolics, and other bioactive compounds, serve as growth promoters and immunostimulants in finfish and shrimp larviculture Research indicates that various therapeutic plant extracts enhance growth, feed consumption, and survival rates in aquatic species due to their immune-nutritional components, particularly polysaccharides These complex carbohydrates are believed to possess prebiotic properties, improving nutritional digestibility and gastrointestinal health Additionally, herbs like rosemary, thyme, and fenugreek have been shown to boost digestion in fish by increasing bile production and stimulating the pancreas to release digestive enzymes.
Research indicates that herbal extracts impact fish growth in a dose-dependent manner, with growth increasing until an optimal inclusion level is reached, after which it declines High concentrations of anti-nutritional components, such as saponins and tannins, along with toxic substances and excessive dosages, can hinder fish development and lead to poor growth or no growth at all This explains why certain herbal extracts, including Moringa oleifera, Eucalyptus citrodora, and others, may negatively affect fish growth and feed utilization.
Numerous studies have examined the role of herbs as appetizers and growth enhancers in aquatic animals According to Lee and Gao (2012), 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, enzymes, bile, and mucus Additionally, olfactory components in feed serve as feeding enhancers, increasing fish feed consumption (Adams, 2005) For example, Harada (1990) found that garlic effectively stimulates olfaction, promoting appetite in species like the oriental weather loach and Japanese amberjack Lee and Gao (2012) observed similar effects across various aquatic species, including Pelodiscus sinensis and Cyprinus carpio The active compound allicin in garlic has been shown to boost fish feed consumption Further research into microbiomes and proteomes is necessary to understand the effects of intensive aquaculture on intestinal digestive enzymes and microorganisms Table 2.3 provides a summary of research on the effects of different herbal extracts on digestive enzyme activity in cultured fish.
Mucus serves to protect the gastric wall from stomach acid, and its absence increases the risk of ulcers Flavonoids enhance this protective effect by boosting the mucosal levels of prostaglandins and mucus Additionally, flavonoids aid in anti-obesity activities by regulating the sympathetic nervous system and promoting thermogenesis and fat burning, while also limiting the growth of adipocytes to control body weight Compounds like caffeic and chlorogenic acid, catechin, epigallocatechin gallate, and quercetin contribute to fat oxidation, appetite reduction, and modulation of obesity-related hormones, while inhibiting digestive enzymes that absorb carbohydrates and lipids Quercetin, in particular, has been shown to inhibit carbohydrate digestion and manage postprandial glucose levels Moreover, phenolics found in lentil extracts, including p-hydroxybenzoic acid and kaempferol, effectively inhibit lipase and α-glycosidase, supporting blood glucose regulation and combating 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, effectively inhibited α-amylase and α-glycosidase enzymes, demonstrating resilience even after exposure to gastric fluid simulation When administered to Wistar rats on a high-calorie diet, it led to reductions in weight, food intake, liver fat, glucose, and serum triglycerides Similarly, phenolic compounds in P guajava leaves showed potential in blocking digestive enzymes, offering a promising avenue for treating obesity and type 2 diabetes by acting in the small intestine rather than the central nervous system Additionally, a study by Samad et al revealed that a 1% ethanolic katuk extract in grouper diets improved appetite and growth, while higher concentrations (2.5% and 5%) resulted in decreased growth, underscoring the necessity for precise dosing to achieve desired outcomes and the importance of further research to quantify active molecules and establish 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) such as superoxide, hydroxyl radical, and peroxyl radical A healthy aerobic organism maintains a balance between ROS generation and protective mechanisms However, an increase in ROS can lead to cellular damage or death, resulting in 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 play crucial roles in regulating cell division and apoptosis, activating transcription factors like NFkB and p38 MAP kinase for immune and anti-inflammatory gene expression, and modulating the production of antioxidant enzymes However, excessive ROS can lead to oxidative damage, resulting in 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 practices in the Mekong Delta, Vietnam, since 1878 has led to a rise in stress-related diseases and mortality among fish This increased stress has weakened their immune systems, resulting 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 subpar seed quality, heighten the vulnerability of fish stocks to infectious diseases (Phuong et al., 2007) These stressors can disrupt metabolic processes (Santos et al., 2010), result in lower quality fillets (Jittinandana et al., 2003), increase susceptibility to disorders (Wu et al., 2013), and in severe instances, 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) revealing that 100% of P hypophthalmus farmers in Vietnam utilized 17 different types of drugs, such as penicillin and tetracyclines However, the environmental and health repercussions, including pollution and antibiotic resistance, have led to increasing restrictions on pharmaceutical use (Andrieu et al., 2015) Additionally, while vaccination is being explored as a method to prevent disease outbreaks, the high cost of commercial vaccines and their specificity to single infections limit their accessibility for fish farmers (Triet et al., 2019).
Researchers are increasingly incorporating natural components into nutritional supplements for fish, focusing on enhancing health, growth, and immunity while ensuring sustainability in aquaculture The use of phytoconstituents serves as immune stimulants and anti-stressors, with various plants containing active substances like flavonoids, alkaloids, phenolics, steroids, terpenoids, and essential oils, which offer a broad range of physiological benefits.
Haematology profiles are vital indicators of fish health and metabolism, with blood serving as the primary tissue for assessing health status in vertebrates, including fish (Fazio, 2019) This analysis provides insights into fish biological responses to external conditions, which are essential for maintaining 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 ensure a safe cultural environment and reduce disease outbreaks (Kiron, 2012) Additionally, measuring digestive enzymes is crucial for understanding digestion mechanisms and how fish adapt to dietary changes in response to their external environments (Sunde et al., 2004), as well as for exploring key aspects of nutritional physiology (Uys and Hecht, 1987).
Vietnam boasts a rich diversity of medicinal herbs across its varied ecological zones, with many botanicals utilized to enhance the immunity and overall health of P hypophthalmus Notably, Phyllanthus amarus Schumach has been identified as a key antioxidant, according to Dao et al (2020).
& Thonn (Pa) extract, followed by extracts of Psidium guajava L (Pg), Euphorbia hirta
This study evaluates the antibacterial properties and health benefits of various plant extracts, specifically focusing on L (Eh) and Mimosa pudica L (Mp), with Pa extracts demonstrating the highest in vitro antibacterial activity against Aeromonas hydrophila Additionally, extracts from Azadirachta indica (Ai), Eh, and Pa significantly enhanced the expression of immune-related cytokines in striped catfish cells Despite these benefits, many farmers remain unaware of the bioactive compounds in these plants and their potential to improve the haematological and digestive enzyme activities in fish, particularly P hypophthalmus This research aims to objectively assess the impact of five plant extracts on the haematology, enzymatic activities, and growth performance of P hypophthalmus, ultimately supporting sustainable aquaculture practices by promoting environmentally friendly alternatives to chemical therapeutics in fish farming.
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 confirmed, and the samples were processed at the College of Natural Sciences, Can Tho University, where they were extracted using ethanol 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 dried powder was stored at room temperature, and to create an ethanolic extract, 100 g of the powder was soaked in 800 mL of 96% ethanol for 24 hours The mixture was then decanted, and excess solvent was evaporated using a rotary evaporator under low pressure (Nhu et al., 2019).
Experimental fish diets were formulated with and without plant extract supplementation, featuring a biochemical composition of 3.21% fiber, 10.58% ash, 30% crude protein, 6.66% crude fat, and 4.41 kcal/g of energy Mixture 1 included sterilized rice bran, cassava, soybean meal, and fishmeal, while Mixture 2 combined butylated hydroxytoluene (BHT), vitamins, and minerals with varying concentrations of plant extracts These mixtures were blended with fish oil and processed using a mini-extrusion machine to create a homogeneous mixture The resulting pellets were air-dried, ground, and sieved to a size of 2 mm before being 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 fish feed formulation includes a premix consisting of 1% attractant, 0.03% vitamin C, 0.5% CMC, and 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 machine's temperature reduction to 70°C, effectively eliminating bacteria and enhancing fish digestibility This step is crucial in feed processing BHT, the premix, and CMC are thoroughly combined with a cooked powder (cooked for 30 minutes and stirred well) before being mixed with the sterilized mixture and fish oil The 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 steps but without the 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 acclimated in well-aerated 2 m³ experimental tanks under natural lighting conditions and fed a basal diet twice daily until satiation, constituting 3-5% of their body weight After a two-week acclimation period, 2,475 fish were distributed into 33 fiberglass tanks, each containing 300 liters of water, with a stocking density of 75 fish per tank across 11 treatments in triplicate Various plant extracts were administered in specific doses: Eh at 0.4% and 2.0%, Pa at 0.2% and 1.0%, Pg at 0.4% and 2.0%, Mp at 0.4% and 0.2%, and Ai at 0.4% and 2.0%.
During the 60-day experiment, all tanks were continuously supplied with well-aerated filtered water, and fish were manually fed twice daily until satiated, consuming 3-5% of their body weight To prevent fouling from feed residues, daily siphoning was conducted, and 30% of the water was exchanged weekly with fresh, dechlorinated water Water quality was monitored twice a week, measuring pH values with a Mettler Toledo SG2 instrument, and dissolved oxygen and temperature were assessed using an Oxy Guard H04PP The recorded 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), remained 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 during handling, a cold moist cloth was applied to each fish's head (Snellgrove and Alexander, 2011) Blood samples were collected from the caudal peduncle vein within three minutes using heparin-coated syringes (Becker et al., 2012), with at least 300 µL drawn and stored in labeled tubes One part of the blood was used for haematological analysis, diluted with Natt and Herrick's solution (1:200) and counted for red blood cells (RBCs) using a Neubauer haemocytometer at 40× magnification (Natt and Herrick, 1952) Hematocrit (Hct) was determined via microcentrifugation for six minutes at 12,000 rpm (Larsen and Snieszko, 1961), while hemoglobin (Hb) levels were quantified using Drabkin's solution and a spectrophotometer (Zijlstra et al., 1983) Mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) were calculated from the respective formulas (Ware, 2020) White blood cells (WBCs) were counted on stained smears under a microscope (Hrubec et al., 2000), and a portion of the blood was centrifuged at 4°C for later glucose analysis, quantified using a standardized assay (Huggett and Nixon, 1957).
Fish were fasted for 48 hours before sampling to ensure their stomachs were empty After blood collection, the fish were euthanized with ice and dissected to obtain the stomach and intestine for enzyme assays A scalpel was used to remove gut and stomach remnants, and all manipulations were performed on ice to preserve enzymatic activity The stomach and intestine were cut longitudinally, rinsed with distilled water, blotted dry, and transferred to labeled 1.5-mL tubes, which were stored at -80°C until homogenization After defrosting, the stomachs and intestines were isolated, 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 to isolate the supernatant 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) in 1N HCl Trichloroacetic acid (TCA; Sigma-Aldrich) was added to the reaction solution, followed by centrifugation at 4,000 g for 10 minutes at 4°C to measure pepsin activity at 280 nm.
Trypsin activity was assessed by mixing 15 μL of the sample with a 0.1 M BApNA solution and phosphate buffer at pH 8.2, followed by measuring the optical density at 407 nm after 5 minutes Chymotrypsin activity was evaluated using 50 μL of the sample with BTEE and buffer at pH 7.8, with absorbance measured at 256 nm Amylase activity was quantified through a calibration curve established with maltose, measured at 540 nm Total protein content was determined using diluted homogenates and bovine serum albumin for calibration 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, to assess the homogeneity of variance among groups with the Levene test A one-way ANOVA and Duncan's multiple range test were utilized to evaluate differences between treatments at each sample interval, with significance set at a p-value of less than 0.05 Results were reported as means and standard deviations of the mean (SEM).
Results
3.3.1 Effects of plant extracts on haematological parameters
The treatment with Mp2.0 resulted in the highest red blood cell (RBC) density on both day 30 and day 60, measuring 3.18±0.09×10^6 cells/mm^3 and 3.70±0.28×10^6 cells/mm^3, respectively, although no significant differences were observed among treatments (p>0.05) Additionally, a dose-dependent increase in hematocrit (Hct) was noted in all groups receiving a diet enriched with 0.2% Pg, with values significantly differing from the basal diet (p0.05), compared to a shorter 14-day period (p0.05) After 24 hours of exposure, a notable decline in red blood cell (RBC) count was recorded in the nitrite groups, with recovery evident by day 3 However, the RBC count remained lowest in the 0.8 mM nitrite group after 14 days of exposure (p