VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE PHETPHAILIN SAISOMBUT EFFECTS OF MORINGA MORINGA OLEIFERA ON GROWTH AND ITS ANTIMICROBIAL ACTIVITIES AGAINST PATHOGENIC BACTERIA INFECTING G
Introduction
In recent years, Vietnam has seen the rise of intensive commercial farming models for traditional fish species, notably grass carp, which is favored in polyculture systems, particularly in the northern provinces Grass carp is known for its rapid growth, delicious meat, and high nutritional value, making it a popular choice among farmers However, the intensification of farming practices for this herbivorous species has led to significant challenges, including water pollution and the emergence of diseases, particularly bacterial infections The growth of intensive farming is often accompanied by complex disease issues and the unregulated use of medications, which poses difficulties for farmers and contributes to the increasing problem of antibiotic resistance.
In addressing the challenges of feed and disease control in grass carp culture, Moringa (Moringa oleifera) emerges as a promising solution due to its rich nutritional profile, which includes high protein content and essential minerals, vitamins, and phenolics Research indicates that the protein content of moringa leaves ranges from 19.7% to 27.7% of dry matter, making it a superior protein source compared to many other plants Studies have demonstrated the successful application of moringa leaf powder in the diets of various species, including laying chickens and tilapia, yielding positive results Furthermore, the use of moringa leaf meals as a plant protein source to replace fish meal in seabass diets has shown promise However, it is important to note that herbivorous species like grass carp and java barb may prefer fresh leaves over dried forms, which can significantly impact their ingestion, digestion, and growth Research has highlighted that the form of plant feed can influence digestibility, as evidenced by findings that dry forms of certain grasses can lead to poor digestibility in fish.
The growth performance of grass carp (Ctenopharyngodon idella) is positively influenced by the use of fresh grass, which is widely utilized in Asia without adverse effects on growth, providing significant benefits to local farmers Additionally, moringa leaves are rich in natural antioxidants and antibacterial compounds, including zeatin, quercetin, beta-sitosterol, caffeoylquinic acid, and kaempferol, enhancing their nutritional value (Anwar et al., 2007; Siddhuraju & Becker).
Moringa tree components, including leaves, roots, seeds, bark, fruit, flowers, and immature pods, exhibit various health benefits such as cardiac and circulatory stimulation, antitumor, hepatoprotective, antibacterial, and antifungal properties Research indicates that moringa has potential applications in aquaculture, serving as a source of antibacterial agents and immune system boosters However, improper processing may diminish the plant's medicinal properties, highlighting the need to assess the viability of using fresh moringa leaves as feed for herbivorous fish species.
1 Evaluate the anti-bacterial activities of moringa extracts against some pathogenic bacteria infecting grass carp.
2 Evaluation of the effects of moringa leaves on the growth and the ability to strengthen the immunity of grass carp with some types of bacteria.
Literature review
Origin of grass carp
Grass carp, a freshwater fish originally from China, is found across a vast range from the Pearl River basin in the south to the Heilongjiang River in the north, as well as in the delta of Vietnam (Adams et al., 2011) This species has been introduced to approximately 100 countries, including regions in Africa, Asia, Europe, and both North and South America (Chen et al., 2009) In Vietnam, grass carp populations are notably present in the Red River.
Figure 2.1 Herbiverous grass carp 2.1.2 Morphological characteristics
Grass carp, as described by Page & Burr (2011), have an elongated, cylindrical body with a rounded belly and lack ventral vertebrae Their length is approximately 3.6 to 4.3 times the width of their tail, and they feature a slightly short head with a wide, arched mouth.
Grass carp are characterized by a front without antennae, a longer and wider upper jaw compared to the lower jaw, and jaws that can nearly reach the eyes, which are positioned on either side of the head They lack tentacles, have short and sparse gill folds, and possess large, round scales The anus is located near the anal fin, with pale greenish-yellow hips, a dark back, and a light grayish-white belly (Schofield, 2005; Page).
Grass carp are commonly found in diverse aquatic environments, including ponds, lakes, and the deeper sections of large rivers Typically, they remain close to their habitats, only undertaking annual migrations for reproduction (Bain et al., 1990) Their preferred living depth ranges from 0 to 30 meters, and they thrive in clean water, often inhabiting the mid-levels of these water bodies.
Grass carp grows and develops normally in an environment with sanility of 0-8‰, adapts to temperatures from 13-32 0 C with the optimal level from 22 to
28 0 C, low oxygen threshold from 0.5-1 mg/l (Maceina & Shireman, 1979) Suitable pH ranges from 6-9 Living in pH below 5 or above 9.5 can result in weak or massive mortality (Bui, 2004)
Grass carp, classified as an omnivorous fish species by Schofield (2005), primarily consume plants Starting from three days old, when they reach a body length of about 7 mm, their diet includes rotifers, insect larvae, and algae As they grow to 2-3 cm, they begin to incorporate plant sprouts into their diet, while the proportion of rotifers decreases, and plankton crustaceans remain predominant Fish measuring 3-10 cm can crush superior plants and transition to young aquatic plants, particularly grasses Adult grass carp primarily feed on macroplants such as paddle weed, dog tail seaweed, shrimp seaweed, duckweed, water hyacinth, and various terrestrial vegetables They also consume leaves from plants like bamboo, cassava, and banana (Steinbronn et al., 2004; Steinbronn et al., 2005) The daily feed intake of grass carp ranges from 22.1% to 27.8% of their body weight.
To produce 1 kg of fish, 40 kg of fresh plants are required Grass carp primarily consume rice bran, corn, and cassava as supplemental feed, but they can also be raised on industrial feed However, a diet high in starch can lead to slower growth rates and increased fat content in the fish (Ni & Wang, 1999).
Grass carp are large fish, with the heaviest individuals weighing between 35-40 kg, while the average commercial size is 3-5 kg per fish Under optimal conditions, grass carp exhibit faster growth rates compared to other fish species In pond environments, they can reach 1 kg after the first year and 2-3 kg after two years in temperate regions, with growth potentially reaching 4-5 kg in tropical areas (Ni & Wang, 1999).
2.1.6 Biochemical characteristics of grass carp meat
Carp meat is highly regarded for its delicious flavor and nutritional value, comprising 74% water, 17.4% protein, 5.8% lipids, and 1.5% minerals, with the remaining 1.3% consisting of vitamins and mineral salts (Khalid & Naeem, 2018).
Research from 2018 indicates that feeding grass carp (Ctenopharyngodon idella) with grass can promote muscle growth and development by stimulating muscle fiber hypertrophy and significantly enhancing the expression of collagen type I alpha 1 (CoL1As) This diet not only improves muscle characteristics but also enhances flesh quality by increasing collagen production while reducing fat accumulation and moisture in the muscle Consequently, scientists have been exploring various alternative feed items to improve flesh quality and meet diverse consumer demands, with the production of crisp fish, particularly crisp grass carp, serving as a notable example.
2.1.7 The situation of grass carp farming in the world
Grass carp is a significant species in freshwater aquaculture, with 39 countries reporting its farming to the FAO (2002) However, only eight countries—Bangladesh, China, Taiwan, Egypt, India, Iran, Laos, and Malaysia—have achieved an output exceeding 1,000 tons Notably, China stands out as the world's largest producer, contributing 3,419,593 tons of grass carp.
In 2002, global production of farmed grass carp reached 3,572,825 tons, a remarkable increase of over 339 times from just 10,527 tons in 1950, representing 15.6% of total freshwater aquaculture production From 1993 to 2002, the average annual growth rate of farmed grass carp production was 10.1% worldwide, with China at 9.9% Notably, production growth outside of China during this period was significantly higher, averaging 17.8% per year.
Between 2001 and 2002, the growth of grass carp production slowed, with an increase of only 3.3% in both China and globally Woynarovich et al (2010) reported that world production reached 4,159,918 tons, representing over 16% of freshwater cultured fish production During the decade from 1993 to 2002, production levels varied significantly across many countries In India, grass carp production was approximately 13,000 tons in 1993 (Shen et al., 2019) By 2002, the global value of grass carp farming was estimated at $2.92 billion, reflecting the annual growth rate from 1993.
2.1.8 Status of grass carp farming in Vietnam
Grass carp is a key species in freshwater fish farming in Vietnam, commonly raised in ponds, lakes, and especially in cages or rafts along rivers and streams in mountainous provinces By 1995, the number of cages for grass carp farming had reached tens of thousands, with significant developments in provinces like Tuyen Quang, Son La, Ninh Binh, Hoa Binh, and Ha Noi, where each province hosts hundreds to thousands of cages In certain areas, grass carp has become the dominant species in aquaculture systems This fish is favored for its rapid growth and adaptability to simple farming conditions, requiring relatively low-quality feed.
Some diseases on grass carp
In recent years, many farming households have adopted high-yield grass models for intensive grass carp farming, achieving significant economic efficiency However, this high-intensity approach, characterized by increased stocking density and waste discharge, has created unfavorable conditions that contribute to disease outbreaks and fish mortality Current research indicates that grass carp production is increasingly challenged by viral and bacterial diseases.
2.2.1 Disease caused by virus on grass carp
Reovirus disease in grass carp, is one of the most serious infectious diseases, causing high mortality (Jiang, 2009)
Reovirus, characterized by its RNA nucleic acid structure and asymmetrical 20-sided cube shape, measures approximately 60-80 nm and contains 92 capsomers (Chen & Jiang, 1983) The optimal growth temperature for this virus ranges from 25-28℃ (Jiang, 2009) It is known to cause hemorrhagic disease primarily in grass carp, but can also affect black carp and catfish The pathogens originate from infected fish and virus-carrying aquatic animals, with the virus spreading through water or via infected aquatic plants such as duckweeds and water grass The disease transmission is exacerbated by untreated water sources that harbor the virus, facilitating its movement between water bodies Acute infections predominantly affect fry and fingerlings under 10 cm, although fry and adult fish can also be susceptible.
2.2.2.1 Red spot disease on grass carp
A hydrophila is a bacterial pathogen responsible for an infectious disease primarily affecting grass carp over one year old This disease can occur year-round, but it is most prevalent during the months of March to May and August to October, particularly when water temperatures range between 25-30°C (Bui, 2004).
Disease outbreaks of A hydrophila are primarily linked to stress factors Key contributors include elevated water temperatures in organic-rich environments, high-density fish farming, physical stress during capture or transport, and poor-quality seed sources.
Signs of fish illness include a lack of appetite and floating on the water's surface The skin may darken and lose its silver luster, while scales become dry and flaky Additionally, red hemorrhagic spots can appear on the skin, fins, operculum, and around the mouth, often accompanied by deep sores that emit a foul odor Other symptoms include a red and swollen anus, an enlarged abdomen, and damaged fin rays that are torn or truncated, along with hemorrhagic and fibrous gills (Bui, 2004).
Internal indicators of liver distress include a pale, swollen, and necrotic liver, accompanied by dark black bile The kidneys appear soft, while the gonads and abdominal area show signs of hemorrhage Additionally, the intestines may be devoid of feed, potentially exhibiting flatulence or necrotic hemorrhage The abdominal cavity is often filled with foul-smelling mucus (Bui, 2004).
2.2.2.2 Disease caused by Flavobacterium columnare in grass carp
The disease primarily affects grass carp during the colder months of winter and spring, manifesting across all sizes of the fish Its symptoms can often be mistaken for those of a fungal infection.
Diseased fish exhibit distinct symptoms, including white skin, worn tails, and torn fin rays, along with pale gills Additionally, signs such as hemorrhagic gill bases and a red, swollen anus may be present Internal examination reveals swollen kidneys, a swollen spleen that may appear hemorrhagic or pale, and a necrotic liver, while the intestines are often devoid of feed (Tien et al., 2012).
Pathogens: Some species in the trichodinidae family such as T centrostrigata, T domerguei, T nigra, Trichodina clavodonta
Pathological signs of disease in fish include slimy, slightly milky fins that are more visible in water than on land Affected fish exhibit gray skin, itchiness, and often float on the surface In severe cases, the disease can lead to dense attachments on the fins and gills, destroying gill silks and causing suffocation Seriously ill fish may carry excessive slime and appear white and silver They swim disoriented, eventually flipping onto their backs and sinking to the bottom of the pond, leading to death (Martins et al., 2015).
Ichthyophthirius multifiliis, commonly known as white-tailed spot disease, is a highly dangerous parasitic infection affecting freshwater fish in both wild and aquaculture settings This obligate protozoan parasite attaches to the skin, fins, and gills of its host, leading to the formation of numerous visible white spots, each representing an encapsulated parasite Infected fish exhibit symptoms such as pale skin and skin ulcers due to tissue damage, particularly in the gills, which impairs their respiratory function and oxygen intake Outbreaks are most prevalent in fingerlings and juveniles, especially when water temperatures drop below 25°C; however, under poor environmental conditions, the disease can affect fish at all life stages across various temperature ranges.
Lernae disease affects fish year-round, predominantly during their juvenile stage These parasites can attach to various parts of the fish's body, where they feed on nutrients, leading to hemorrhagic wounds This results in weakened and emaciated fish, increasing their vulnerability to other opportunistic pathogens.
The disease is prevalent throughout the year, particularly in spring and autumn Trumpet worms attach themselves to white tufts that can be mistaken for fungus, adhering to the skin, fins, and gills of fish, which impacts their respiration and development These parasites do not extract nutrients from their host but instead absorb them from the surrounding environment, often thriving in areas rich in organic matter and high culture density with poor environmental control.
Mycosis fungoides is the most prevalent disease affecting grass carp, typically emerging during the winter and winter-spring across all developmental stages Infected fish exhibit grayish-white patches on their skin and damaged tissues, which later develop into white cotton-like tufts as thin hyphae form One end of the mycelium adheres to the damaged tissue while the other end disperses in the water This fungal infection spreads rapidly, leading to significant harm, including symptoms such as itchiness, erratic swimming, and disorientation, ultimately resulting in the fish becoming weak, unresponsive, or dead Histopathological analysis reveals swift destruction of the epidermis, characterized by necrotic tissue and a mild inflammatory response.
Antibiotics and antibiotic resistance in aquaculture
Antibiotics play a crucial role in animal husbandry across various countries, serving three primary functions: disease prevention, treatment, and growth promotion These antibiotics can be classified as natural, semi-synthetic, or fully synthetic, and they are designed to minimize harm to the animals.
Antibiotics are classified into two main categories: bacteriostatic agents, which inhibit the growth of bacteria or fungi, including rifamycins and quinolones, and bactericidal agents, which actively kill these microorganisms, such as Erythromycin, Spiramycin, Oxytetracycline, and Sulphonamides However, the use of antibiotics can have detrimental effects, as they may eliminate beneficial gut bacteria when mixed with feed or harm beneficial environmental bacteria when added to pond water (Walsh, 2003).
Recent advancements have led to the development and introduction of numerous antibiotics to address various human needs In aquaculture and veterinary medicine, antibiotics are commonly utilized for disease treatment, prevention, and growth promotion (Teuber, 2001) However, developed nations, including the US, Canada, Switzerland, and EU member countries, are increasingly moving towards minimizing or significantly reducing antibiotic use in aquaculture (Lillehaug et al.).
In developing countries, approximately 90% of seafood production relies on common antibiotics to prevent and control pathogens However, this extensive use of antibiotics is contributing to the rising emergence of antibiotic-resistant bacterial strains.
Antibiotic resistance arises from the inappropriate use of antibiotics, including overuse, underdosing, and self-medication, which create favorable conditions for microorganisms to develop, mutate, and spread resistance Additionally, using treatment drugs that are unsuitable for specific bacteria, viruses, and parasites, along with incorrect dosages and timing, further exacerbates the issue.
2.3.2 Harmful effects of overuse of antibiotics
The routine use of low-dose antibiotics in animal feed and water to combat harmful bacteria, lower feed expenses, enhance nutrient absorption, and promote rapid growth was previously considered effective However, numerous countries worldwide have since restricted, regulated, or prohibited their use due to the adverse effects associated with antibiotics.
The transmission of antibiotic-resistant bacteria to humans and terrestrial animals poses a significant public health risk (Shariff, 1998) Thayumanavan et al (2003) highlighted the growing threat of antibiotic-resistant strains of A hydrophila in fish and shrimp, which endangers human health Furthermore, the rising use of antibiotics in aquaculture is likely to escalate costs in the industry.
Therefore, it is important to reduce the cause of the disease and reduce the use of drugs to a minimum (Gudding et al., 1999)
Improper antibiotic use in livestock can lead to the development of drug-resistant superbugs, disrupting the gastrointestinal microbial balance and causing drug resistance (FAO, 2019) This antibiotic resistance not only harms livestock directly but also poses risks to human health through contact with farmers and environmental waste Many farming operations fail to adhere to the recommended withdrawal times for antibiotics, resulting in antibiotic residues in food These residues significantly impact consumer health and contribute to the body's resistance to antibiotics (Teuber, 2001).
Table 2.1 List of chemicals and antibiotics banned from use in the production and trading of aquatic animals
No Name of chemicals, antibiotics
6 Dapsone 18 Gentian Violet (Crystal violet)
2.3.3 Measures to limit antibiotic use
To ensure the safety of aquaculture, farmers must adhere to essential antibiotic usage guidelines, as highlighted by Bui (2004) This includes accurately identifying infections and the specific pathogenic bacteria involved, followed by appropriate isolation and treatment measures.
Crustaceans are susceptible to various diseases, including White Spot Disease (WSSV), Monodon Baculovirus (MBV), Yellow Head Disease (YHD) in shrimp, and Reovirus Hemorrhagic Disease in grass carp Selecting the appropriate antibiotic is crucial; it should effectively diffuse to the affected organ or be actively transported through it Proper dosage, indications, and timing are essential for treatment Combining multiple antibiotics is not advisable, as it can lead to the development of drug-resistant bacterial strains, increased toxicity, and complications for livestock.
Organic acids, enzymes, antibody-rich bioproducts, and various plant-based products are viable alternatives to antibiotics, garnering significant interest for both human and animal health Herbal medicine is increasingly recognized as a safe alternative to synthetic drugs in the pharmaceutical industry Since 2010, over 50 medicinal plants have been researched and utilized in aquaculture, demonstrating their effectiveness in promoting growth, enhancing immunity, and combating bacterial infections in farmed shrimp and fish Many herbs possess antimicrobial properties due to compounds like tannins, citrals, phenols, and quinones Notable herbs with natural antibiotic effects include garlic, oval leaves, black pot, earthworm, licorice, cinnamon, radial lotus, betel nut, tangerine, and moringa.
Effects of some herbs
Garlic: Garlic is grown in many parts of the world, such as China, India,
Garlic, a powerful superfood, contains 149 Kcal per 100g and is rich in vitamins B1, B2, B3, B5, B6, C, carotene, and essential minerals like calcium, iron, magnesium, phosphorus, potassium, sodium, manganese, and zinc Its active compounds, Allicin, Alliin, and Ajoene, exhibit strong antibiotic properties, surpassing penicillin in effectiveness Research published in the British Journal of Cancer (March 1993) highlights garlic's ability to inhibit various bacteria and viruses, as well as its potential in preventing cancerous tumor growth Additionally, garlic supports cardiovascular health by preventing platelet aggregation, regulating blood sugar, lowering cholesterol, and reducing the risk of hypertension and stroke Recent studies also reveal its efficacy in deworming and combating intestinal parasites, making it beneficial in aquaculture for treating fish inflammatory bowel disease and preventing shrimp intestinal diseases.
Litsea cubeba, an herbal plant found in various Asian countries, including Vietnam, is primarily cultivated in the Northwest region This plant is rich in antibacterial properties and is widely used in traditional medicine to treat conditions such as headaches, depression, and muscle aches, with crushed leaves also utilized for skin healing (Chen et al., 2013) Research indicates that Litsea cubeba has significant potential in aquaculture, promoting growth and enhancing nonspecific immunity in carp A study by Thị Trang et al (2017) identified nine strains of endophytic actinomycetes in the tangerine plant that exhibit antagonistic effects against pathogenic strains A hydrophila GL14, A caviae HD60, and S agalactiae HY10, which affect tilapia and grass carp.
Moringa (Moringa oleifera)
Moringa (Moringa oleifera) is scientifically known as Moringa oleifera
Lam Common name is Horse Radish Tree
The taxonomy of Moringa is:
Figure 2.2 The moringa fractions used in this study
Moringa (Moringa oleifera) is a versatile plant native to Asia, particularly India and Sri Lanka, recognized for its numerous benefits across food, pharmaceutical, and industrial sectors This fast-growing, drought-tolerant perennial can reach heights of up to 4 meters and typically blooms within its first year of planting.
Fresh Moringa leaves are a nutritious food source, while powdered dried leaves can be stored for extended periods without losing their high nutritional value These leaves are rich in vitamin A, contain calcium comparable to milk, and provide more iron than spinach Additionally, they offer as much vitamin C as oranges and nearly the same amount of potassium as bananas.
Moringa is rich in sulfur, which is essential for the optimal growth and activity of rumen microbes (Brisibe et al., 2009) Its mineral composition significantly contributes to its nutritional, medicinal, and therapeutic benefits (Al-Kharusi et al., 2009).
Dried Moringa leaves are rich in essential nutrients, particularly copper (Cu) and zinc (Zn), which play significant roles in enhancing the immune system (Anwar et al., 2007) The zinc content in these leaves is notably high at 25.5 mg/kg, highlighting its importance in the diets of both animals and humans (Barminas et al., 1998) Zinc is crucial for the synthesis of DNA, RNA, and insulin, as well as for the function and structure of various enzymes (Brisibe et al., 2009) Additionally, it is vital for cell reproduction and growth, especially in sperm cells, and is recognized for its anti-viral, antibacterial, anti-fungal, and anti-cancer properties (Brisibe et al., 2009).
Moringa pods are a nutritious food source, offering 32 kilocalories of energy per 100 grams They are rich in essential nutrients, providing 1.2 grams of fiber, 9 milligrams of calcium, 26 milligrams of phosphorus, and 1.5 milligrams of iron Additionally, they contain 0.05 milligrams of vitamin B, 0.6 milligrams of niacin, and a significant amount of vitamin C at 262 milligrams.
Seeds the oil obtained from freshly pressed seeds are used as cooking oil.
Tuan et al (2021) and Rahman et al (2009) reported that moringa showed potential in aquaculture as a source of antibacterial sustain and immunity stimulants
2.5.3 Important substances in moringa leaves
Moringa is recognized for its impressive protein profile, which includes essential nutrients such as ascorbic acid, flavonoids, phenolics, carotenoids, and various compounds like estrogenic and beta-sitosterol It is rich in minerals including iron, calcium, phosphorus, and copper, as well as vitamins A, B, C, alpha-tocopherol, and riboflavin Additionally, moringa provides essential amino acids such as methionine, cystine, tryptophan, and lysine nitrile, along with unique compounds like mustard oil glycosides and thiocarbamate glycosides Notably, thiocarbamate compounds, including Niazinin A, Niazinin B, Niaziminin A, and Niaziminin B, are rare in nature and contribute to the plant's nutritional value (Mahmood et al., 2010; Stadtlander & Becker, 2017).
Table 2.2 The nutrient composition of Moringa leaves, powder, seeds and pods
Note All values are in 100 g per plant material
(Gopalakrishnan et al., 2016; Rajbhar et al., 2018)
Table 2.3 Chemical constituents in dried moringa leaves
Total energy (Kcal/kg of DM) 3,911
Moringa oleifera, an herbal tree cultivated in 80 countries, thrives particularly in Africa and Asia, with notable growth in Vietnam's provinces like Da Nang and Khanh Hoa This versatile tree is extensively utilized in pharmaceuticals, cosmetics, nutritional beverages, and functional foods In many developing nations, Moringa serves as a vital medicinal resource for treating serious diseases and common ailments Every part of the Moringa tree—flowers, leaves, seeds, and roots—contains active compounds with potent antibacterial and antifungal properties, such as Anthonine and Isothiocyanate Additionally, Moringa leaves are rich in essential nutrients, including protein, amino acids, trace minerals, and vitamins.
C, group B and β-Carotene (Ho, 2000; Van Chi & Hop, 1999), and many compounds not found in other plants (Tarafder, 1983) In medicine, active ingredients extracted from moringa are used to prepare antibacterial, antifungal, cholesterol-lowering, phospholipids, triglycerides in the blood, and to produce functional foods against malnutrition
Table 2.4 Nutritional properties of moringa leaf powder
Nutrients volume Mineral (mg/100g) Volume
(Mensah et al., 2012). Besides, the high content of minerals in moringa is promising as a good feed source for livestock and poultry (Afuang et al., 2003; Yaméogo et al.,
2011) A study by Mensah et al (2012) on nutrition and minerals of moringa leaf powder found that the nutritional components including vitamins, essential
Moringa leaves are rich in 18 amino acids and various phytochemicals, making them an excellent source of nutrients This nutrient density positions moringa leaves as a beneficial supplemental food that can enhance human health Table 2.3 presents the nutritional value and mineral content of moringa leaf powder.
Moringa seeds exhibit significant antibacterial properties, primarily due to the presence of four (alpha-L-Rhamnosyloxy) benzyl isothiocyanates, which demonstrate the strongest antibiotic activity among the extracted compounds These isothiocyanates effectively inhibit the growth of various pathogenic bacteria and fungi, with a minimum inhibitory concentration of 56 µg/ml for Bacillus subtilis and 40 µg/ml for Mycobacterium phlei (Mensah et al.).
Moringa seeds exhibit significant antibacterial activity, particularly against pathogens such as S typhii, V cholerae, and E coli, as highlighted in studies by Fuglie (1999) and Peter et al (2011) Additionally, research by Aiyegoro et al (2010) suggests that moringa products may serve as effective natural antimicrobials for managing bacteria responsible for water-borne diseases.
Duckweed
Duckweed, scientifically known as Spirodela polyrrhiza (L.) Schleid, belongs to the Lemnaceae family and is a naturally occurring aquatic plant renowned for its high nutritional value This plant is rich in protein, containing approximately 20-40%, and also provides essential amino acids, along with a fiber content of about 4-6%.
Duckweed is a nutrient-rich aquatic plant that offers a complete composition of free fatty acids, proteins, fats, and essential minerals Its high levels of essential amino acids make it particularly beneficial for fish growth, making it an ideal choice for aquaculture (Leng et al., 1995).
Duckweed, small floating aquatic plants belonging to the Lemnaceae family, thrive in nutrient-rich fresh or brackish waters, often forming dense mats These monocotyledons are frequently confused with algae and grow optimally in water temperatures ranging from 6℃ to 33°C To survive low temperatures, many duckweed species produce a turion, allowing the plant to sink and remain dormant at the bottom of lagoons until warmer conditions trigger growth.
Figure 2.3 Duckweed (Spirodela polyrrhiza) 2.6.1 Important elements
The study of natural water sources where small duckweed, large duckweed, and water eggs thrive revealed that the temperature ranged from 22.08 to 31.55 ℃, with a pH level between 7.83 and 11.27 Additionally, the conductivity measured between 182.7 and 206.0 µS/cm, while turbidity levels varied from 7.90 to 30.07 NTU Total nitrogen (TKN) concentrations were found to be between 0.167 and 1.556 mg/l, and total phosphorus (TP) levels ranged from 0.004 to 0.250 mg/l (Panumas et al., 2017).
Azolla requires various nutrients for optimal growth, similar to other plants While it obtains nitrogen from water-green algae, it often lacks sufficient phosphorus, which is crucial for nucleic acid formation, cell division, and nitrogen fixation.
Light: The growth rate of duckweed is maximally increased at a light intensity of 49,000 lux, but the need and tolerance of light intensity depends on the type of Azolla
Water is an important factor for growth Duckweed will grow well in shallow water or moist soil Most grow in water level 15-30 cm (Panumas et al., 2017)
Table 2.5 Chemical composition of duckweed
Materials and methods
Location of the study
The experiment was carried out from October 2020 to August 2021 at the Faculty of Fisheries, Viet Nam National University of Agriculture.
Materials of the study
Moringa seeds sourced from Thailand were utilized for crude oil extraction and to assess their antibacterial properties Concurrently, Moringa trees were cultivated at the Faculty of Fisheries to ensure a continuous supply of fresh leaves for experimental purposes.
Grass carp: A total of 200 fishes with the same size (2.3±0.2 g) were purchased from the Research Institute for Aquaculture No 1
Types of infectious bacteria: 02 A hydrophilla and F columnare are provided by Aquatic Phathology Lab – VNUA, isolated and identified from grass carp sampling in Northen Viet Nam
The 120L plastic tanks were equipped with dark nets to prevent fish from jumping out An air pump system, along with rackets, siphon tubes, and feed boxes, was installed and connected to each tank Additionally, water quality test kits for NH3/NH4 were provided to ensure optimal conditions.
NO2, pH, DO from SERA company (Germany) were perchased
Machinery include: Machinery for PCR assessment and bacterial culture: refrigerators, incubators, drying cabinets, cold centrifuges, vortex machines, Real-time PCR machines
Instruments include: Micropipettes of all kinds, tips of all kinds, PCR tubes, Eppendorf tubes, gloves, specialized surgical kits, protective equipment such as slides, cage plates
Environment - Chemicals - Materials include: DNA extraction kit, nutrient medium BHB, BHI, NA, Gram staining solutions (gentians violet, lugol, alcohol 90%, alkaline fuchsin), alcohol 100%, alcohol 70%, biochemical test kit
Methodologies and experimental design
3.3.1 Experiment 1: Evaluation of the antibacterial ability of moringa to some pathogenic bacteria
The extracts from moringa were prepared
The kernel, sourced from Thailand, was obtained by removing the seed shell It was then pressed using a MISHIO oil pressing machine (MK-39) without the use of chemicals or solvents The extracted liquid was stored in a sealed bottle at a temperature of 2-4°C and should be used within a week for optimal freshness.
Dried leaves: there are the dried leaves obtained from formula 3 Use a blender machine the dried leaves with 10g of dried moringa leaves + 400ml of distilled water
Fresh moringa leaves, bark, and roots were sourced from the Faculty of Fisheries at Vietnam National University of Agriculture A blender was used to process the materials, combining 200g of fresh moringa leaves, bark, and roots with 400ml of distilled water for each product.
Pathogenic bacterial fish samples were collected from aquaculture farms and transported to the Vietnam National University of Agriculture in plastic bags containing 50 L of dechlorinated tap water with an oxygen supply Prior to bacterial collection, tissues from diseased fish were disinfected with 70% alcohol Bacteria were isolated and cultured from the affected fish, with A hydrophila obtained from the kidney and cultured on Rimler Short Agar, while F columnare was isolated from lesion sites and gills, cultured on Cytophaga agar All bacterial strains underwent re-isolation three times and were classified using PCR analysis with specific primer pairs and gene kits as per the manufacturer's guidelines The bacteria were subsequently stored at -80°C for future analysis.
3.3.1.2 Method of culture and isolation of pathogenic bacteria on grass carp
Fish exhibiting hemorrhagic symptoms on their fins, skin, and tail were suspected to be infected with A hydrophila Conversely, fish showing worn-out gill rays, white gills, and white spots on the skin, along with dry and damaged fins, were suspected of being infected with F columnare.
Fish samples exhibiting typical symptoms were rapidly diagnosed by staining fresh samples from affected areas and internal organs, including the kidneys and liver Culture sampling targeted the anterior kidney, spleen, and liver for suspected A hydrophila infections, while lesions on gills and skin were sampled for F columnare The culture process employed Rhimler Shotts (RS) culture for A hydrophila and Cytophaga Agar culture for F columnare, with agar plates incubated at 28℃ for 24-48 hours Following incubation, colonies with characteristic features, as outlined by Dung et al (2012), were sampled and stained with Gram stain to assess bacterial morphology.
3.3.1.3 Methods for screening antibacterial activity
Utilize various components of the moringa plant, including dried leaves, fresh leaves, oil-pressed seeds, and roots, to assess their antibacterial properties After pressing 20 mL of the seeds mixed with talc (Dimethyl sulfoxide), the solution is placed on filter paper and applied to agar plates inoculated with bacteria Incubate the plates at 28-30 ℃ for 24-48 hours to evaluate the results and determine the inhibitory effects on each pathogen.
The antibacterial activity of extracts against pathogenic bacteria A hydrophila and F columnare was assessed using the agar diffusion method (Dhanasekaran et al., 2012) A micro pipet was employed to apply 20 µL of each extract onto discs, which were then incubated at room temperature for 24 hours to observe bacterial growth and measure the zones of inhibition (Kafur & BASHEER, 2011) Due to the high lipid content, dimethyl sulfoxide was added to the extracts in 50:50 and 20:80 volume ratios, following the methodology outlined by Rahman et al (2009) The resulting inhibition zones were compared to those of standard antibiotics.
Florfenicol with the concentration of 20 mg/L All the tests were carried out with
Figure 3.2 Perform screening antibacterial activity test from Moringa products 3.3.2 LD50 determination of A hydrophila
Grass carp, averaging 4.82±0.44 g per fish, were sourced from the Research Institute for Aquaculture No 1 and acclimated in an 800 L pond for one month Prior to infection, 15 randomly selected fish underwent clinical assessments to check for abnormal symptoms and lesions The selected fish were cultured to isolate bacteria, ensuring that the experimental subjects were free from pathogens, exhibited no abnormal pathological manifestations, and showed no bacterial presence after culture.
The study focused on the virulence and pathogenicity of the A hydrophila (AH-TC-HD) strain in grass carps Prior to experimentation, bacteria were recovered from glycerol storage on TSA culture medium, followed by Gram staining and biochemical analysis The bacteria were cultured in BHIB medium at 28℃ for 24 hours, and their density was measured using a spectrophotometer at 600 nm (OD600), alongside colony counts on agar plates To confirm bacterial toxicity, injections were performed based on methods from previous studies (Hoai et al., 2019; Nguyen et al., 2016; Tuan, 2017) Bacteria were diluted to concentrations of 1x10² to 1x10⁸ CFU/ml, with physiological saline (0.9% NaCl) as the control for fish injections The LD50 experiment involved seven concentrations across seven tanks, with a total of 315 fish, using 15 fish per concentration in three replicates The experiment was conducted from May 13 to May 26, 2021.
The LD50 (Lethal Dose) value is calculated using the formula established by Reed & Muench in 1938, expressed as LD50 = 10 (a-x), where 'a' represents the concentration at which 50% of the fish are alive and 50% are dead The variable 'x' is determined by the equation x = (Pa-50)/(Pa-Pu).
Pa, Pu is the upper and lower ratio of the 50% lethal dose
3.3.3 Experiment 2: Evaluation of the effect of moringa leaves on the growth of grass carp
3.3.3.1 Steps for preparing a Feed preparation
Experiments were conducted with four different feed formulations Each feed formulation was performed with three replicates in each experiment
- Treatment 1 (Ctrl): is industrial feed (AQUAGREEN) Industrial feed with 30% crude protein, 5% lipid was used as control diet
Treatment 2 (FDW) utilized 100% fresh Duckweed, which is an ideal green feed for the biological needs of grass carp (Pípalová, 2003; Van Dyke & Sutton, 1977) The Duckweed was locally sourced and thoroughly washed with Potassium permanganate (KMnO4) to eliminate parasites and prevent disease transmission to the fish It was then stored in a composite tank filled with clean water until it was ready for use.
Dried moringa leaves (DML) were prepared by drying fresh leaves sourced from the department of fisheries in a hot air oven at 50℃ for 10 hours, after which they were stored in a plastic bag During the feeding trial, fish were given the dried moringa leaves directly, without any mixing This study aims to compare the effects of dried moringa leaves versus fresh moringa leaves on fish Prior to the experiment, the dry matter content of all diets was measured to accurately calculate the feed amounts based on dry matter, ensuring that only the edible portions of the plant were selected for fish preparation.
- Treatment 4 (FML): is Fresh Moringa leaves are grown at the faculty of fisheries to feed the fish daily b Aquaria preparation
Experimental tanks: were washed, cleaned, and disinfected with Chlorine for 30 minutes with a dosage of 200-300 ppm Tanks were dried for 1-2 days before supplying treated water to set up experiment
Fish preparation and experimental set up total 200 grass carp with the same size (2.3±0.2 g) were purchased from the Research Institute for Aquaculture No
1, then adapted and trained to eat plant materials for more than 20 days before assigning into the experiment Fish were equally and randomly distributed into
The study utilized 12 round tanks, each with a volume of 120 liters (80 cm in diameter and 40 cm in height), arranged in three replicates An air pump was employed to ensure optimal oxygen levels, maintaining a temperature range of 25 to 28°C Fish were fed three times daily at 8 AM, 1 PM, and 5 PM, with a total feed amount of 3% of their live body weight, calculated based on dry matter Prior to the experiment, feed samples were analyzed for chemical composition following AOAC (1990) standards to determine daily feed amounts and protein productivity Daily siphoning was conducted to remove fish feces, and water was added to maintain consistent volume All tanks were aerated to keep dissolved oxygen levels above 5.0 mg/L, while other water parameters, including pH and ammonia, remained undetectable as per Sera kit tests (Germany) Fish weight and length were measured weekly throughout the 70-day experiment.
Figure 3.3.Tanks used in the experiment
Fish growth was assessed by monitoring weight and length every 10 days, leading to adjustments in feed quantity The parameters for larval rearing were evaluated using methods from El-Hawarry et al (2016) Body weight gain was calculated using a specific formula.
BWG = Final body weight – Initial body weight
Specific growth rate: the initial live weight and final live weight were used to calculate and specific growth rate for the period of the trials using the following equations
SGR=(InWt-InWini) t ×100 Feed conversion ratio (FCR) which is essentially a measure of the feed utilization for growth, is calculated as:
Body weight gain Survival rate was determined according to the following formula:
Survival (%) = number of surviving animals initial number of animals × 100
Protein efficiency ratio (PER) = Body protein (g)
3.3.4 Experiment 3: Evaluation of the effect of moringa leaves on disease resistance in grass carp
3.3.4.1 Challenge test design a Experimental design
The tanks were initially cleaned with soap and subsequently treated with chlorine at a concentration of 1 ppt for three days before being dried Following this process, each tank was filled with 120 liters of fresh water All tanks were then connected to a continuous air pump system operating 24 hours a day for the experiment.
Statistical analysis
The analysis of variance (ANOVA) was conducted using the Completely Randomized Design (CRD) method, and differences in means were assessed through Duncan's New Multiple Range Test (DMRT) with SPSS version 16.0 and Excel 2016 Statistical significance was determined at a p-value of 0.05 (P