Probiotic enrichment of copepod (Acanthocyclops sp.) towards improving fish survival, nutritional content, optimal growth and sustainability

Probiotic enrichment of copepod (Acanthocyclops sp.) towards improving fish survival, nutritional content, optimal growth and sustainability | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Probiotic enrichment of copepod (Acanthocyclops sp.) towards improving fish survival, nutritional content, optimal growth and sustainability Fazal Husain This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4674332/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The article illustrates a thorough examination of the importance of live feed culture in aquaculture, especially in nurseries. Live feed is essential for aquaculture's sustainable development and ensuring a steady supply of fry and fingerlings. After being washed in sterilized water, copepods were bio-enriched with probiotic bacterial isolates (KAF061, 124, & 135) and commercial probiotics. A phase-contrast microscopic analysis confirmed the bioenrichment of copepods. We assessed the nutritional composition of the live feed culture using proximate analysis, revealing a greater protein content in microalgae, copepods, rotifers, and artemia compared to commercial fish feed. Based on these findings, the probiotic-rich live feed culture has a lot of potential for improving the nutritional content of fish, mollusks, and crustaceans that are still larvae. This could lead to better growth and survival rates for fry and fingerlings. These findings have significant implications for long-term aquaculture practices in developing low-cost and ecologically acceptable live feed alternatives for growth and survival. Bioenrichment Live feed culture Acanthocyclops sp. Copepods Bacillus subtilis Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Aquaculture hatcheries and nurseries need live feed culture to support fry and fingerling survival, growth, and supply year-round. This is crucial because early hatched fish larvae need prepared nutrition. Finfish and shellfish hatcheries rely heavily on live feed culture because they have an immature digestive system and lack enzyme synthesis. Live feeds provide a healthy diet for fries and fingerlings. Previous research has demonstrated that live feed is essential for the growth and development of newborn fish fingerlings. Aquaculture hatcheries' newly hatched fish fry and fingerlings solely depend on live feeds of microalgae, copepods, rotifers, and Artemia [ 1 ]. Among its copepods, there is a superior zooplankton that has a minimal size and provides highly suitable nutrition for fish fry. Hatcheries use microalgae such as Nannochloropsis , Chlorella sp., Chaetoceros, Tetraselmis, Scenedesmus, Pavlova, Phaeodactylum, Skeletonema , and Thalassiosira to grow larval fish, mollusks, and crustaceans. Artemia to their well-established production and rearing methods in aquaculture, artemia and rotifers are the preferred zooplankton. Copepods are better for you than Artemia and rotifers ( Table 1 ) because they have more good things for you, like polyunsaturated fatty acids (PUFA) and especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) [ 2 ]. However, these lipids are scarce; therefore, enrichment is generally needed. Copepods are the favourite diet for fish fry and fingerlings, especially during early hunting, because their zig-zag movement and brief gliding phase provide visual stimulation and also clean rearing tanks by nibbling on algae and detritus [ 3 ]. Additionally, copepods reduce ecosystem acidification and global warming [ 4 ]. Rotifers are fish larvae's live feed because of their wide size range (100 to 300 mm), high fecundity, ability to grow in high population density, resistance to variable temperatures and salinities, ability to be supplemented with minimal nutritional supplements, and slow swimming movements that make them easy to hunt [ 5 ]. Aquaculture has used rotifers, Brachionus sp., as live feed since the 1970s [ 6 ]. The larvae of most marine fish, crabs, and mollusks filter water to feed. They need minimal prey size in their early stages [ 7 ]. Even though rotifers are resistant to abiotic factors [ 8 ], Birnavirus or fungal infections can stop them from producing a lot of fish. This means that they must be cultured all year, which takes more time and costs more money than seasonal outdoor rearing and often leads to complete culture losses [ 5 ]. Artemia is a popular aquaculture live feed because of its availability and high salinity tolerance. We grow Artemia salina, A. sinica, A. persimilis, A. urmiana , and A. tibetiana for their easy storage and hatching. Artemia's nutritional content needs enrichment, but technology has solved this problem. Live feeding with arthritis causes issues such as unpredictable supply, poor nutrition, shifting pricing, and cyst shortages. Growers may need to adapt pricing or fish larvae diets. Despite these drawbacks, artemia remains popular due to its many advantages. In order to tackle these issues, scholars are now investigating copepods as prospective substitutes for live-feed aquaculture. Artemia, like rotifers, necessitates vital elements such as fatty acids, minerals, and vitamins; nonetheless, contemporary enrichment technologies have facilitated its utilization. Despite Artemia's widespread use, its limitations have prompted research into copepods. Copepods exhibit a range of life phases, including eggs, nauplius, copepodite, and maturity, which enables them to meet diverse size needs within aquatic settings. Copepods have the ability to provide viable solutions to the challenges associated with the practice of live-feeding Artemia. Probiotics are beneficial bacteria that protect host animals from dangerous microbial infections, stimulate quick development, and boost activity. Microbial community expansion in hatcheries is a major cause of large mortalities, as well as uneven survival and growth. In hatchery cultures, probiotics have replaced antibiotics and their microbial control hazards. Note that probiotic strains have varied health advantages. Aquaculture uses live foods such as Artemia and rotifers, pellet food, direct water addition, and injection to provide probiotics. Effective probiotic distribution reduces illness onset, treatment, and prevention costs. By enriching copepods with probiotic bacteria and directly inoculating them into the target animals' digestive tracts, probiotics and copepods can enhance aquatic fish rearing by providing better nutritional content. This study cultivates copepods inside and enriches them with probiotic bacteria. 2. Materials and Methods 2.1. Sample collection We collected freshwater zooplankton samples from multiple ponds and stagnant water bodies in and around the Cauvery River in Tamil Nadu, India ( Table 2 ). Prior to transportation to the laboratory for further processing, the samples were filtered using a 70 µm mesh. The collection took place during the morning hours, around 6 a.m., before the surface water temperature began to rise. 2.2. Probiotic Bacillus subtilis KAF061, Bacillus cereus KAF124, Bacillus thuringiensis KAF135, and a commercial probiotic called Bacillus subtilis were used in this study to find possible probiotic strains that would help increase the number of copepods. Husain et al. [9] revealed the powerful probiotics KAF124 and KAF135. B. subtilis , a commercially available probiotic, served as a positive control in this investigation. 2.3. Morphology & Anatomy of Acanthocyclops sp . Witty [10] described the morphological analysis using stereomicroscopy. Copepods were the most numerous groups, which were isolated zooplanktons, followed by rotifers, Cladocera, protozoa, and Ostracoda ( Table 3 ). 2.4. Copepod analysis in water samples: isolation, identification, and hemolysis Nylon mesh with a 300 µm hole size was used to isolate the copepods from surface water samples. An inverted microscope at 40x magnification evaluated the samples after 24 h of stabilization. Copepods were the most prevalent zooplankton and had sophisticated eyes, sexual dimorphism, colouring, and viable female egg sacs [11]. For research purposes, copepods were mass-produced in the lab. Husain et al. (2022) provided instructions for the hemolysis test, a method for identifying potentially harmful microorganisms on the outside and inside of copepods. We homogenized the copepods and plated them on nutrient agar at 37 °C for 24 h. We formed separate colonies on blood agar plates and examined blood cell lysis in cultures after 72 h [12]. 2.5. Probiotic survival at varied temperatures with commercial fish feed To investigate the shelf life, we blended autoclaved commercial fish feed (0.5 g) with KAF061, KAF124, and KAF135 (6.5 x 107 CFU/mL) probiotic isolates and incubated them at 4, 27, and 37 °C. After 5, 10, 15, 20, 30, and 45 days, we tested the bacterial isolate's survival using plate counts at different storage temperatures. We quantified the logarithmic colony-forming unit (CFU) values [13]. 2.6. Mass Culture of Copepods using Spirulina sp . and Chlorella sp . as feed: Cultivation and feeding strategies We used Spirulina sp. for copepod mass culture, and we cultivated pure Spirulina sp. culture in Zarrouk's medium [14] with the following contents: (g/L) NaHCO3, 16.8; K2HPO4 0.5; NaNO3, 2.5; K2SO4, 1.0; NaCl, 1.0; MgSO4-7H20, 0.2; CaCl2, 0.04; FeSO4-7H2O, 0.01; EDTA, 0.08; Solution A5, (g/L) H3BO3, 2.86; MnCl2-4H2O, 1.81; ZnSO4-7H2O, 0.222; CuSO4 5—1 ml/L; Solution B6, (mg/L, NH4VO3, 22.96; KCr (SO4)2 - 12H2O, 192.0; NiSO4-6H2O, 44.8; Na2PO4-2H2O, 17.94; TiOSO4H2SO4 - 8H2O, 61.1; and Co(NO3)2 - 6H2O, 43.98) optimized for copepod feed. The culture broth was incubated at 27 °C with a 10 mg/L inoculWe measured the optical density (OD) of the culture broth at 450 nm every 24 h for 10 days. every 24 h for 10 days. We produced the growth curve using the OD values of the broth culture. Similarly, copepod mass culture used Chlorella sp. as food. A BG11 medium [15] was used to grow Chlorella sp. It had 1.5 g of NaNO3, 0.04 g of K2HPO4∙2H2O, 0.075 g of MgSO4∙7H2O, 0.036 g of CaCl2∙2H2O, 0.006 g of citric acid, 0.02 g of Na2CO3, 0.006 g of ferric ammonium citrate, 0.001 g of Na-EDTA, and 1 mL of trace metal A5. The trace metal A5 solution had 2.86 H3BO3, 1.81 MnCl2 ∙ 4H2O, 0.22 ZnSO4∙7H2O, 0.39 NaMoO4∙2H2O, 0.079 CuSO4∙5H2O, and 0.05 CoCl2∙6H2O (g/L). The solution was incubated at 27 °C with a 10 mg/L inoculum in BG11 media. We measured the optical density (OD) of the Chlorella sp. culture broth at 450 nm every 24 h for 10 days. We plotted the growth curve using the OD values of the broth culture. We used Whatman No. 1 paper to isolate the cultures of Spirulina sp. and Chlorella sp. To make it more suitable for copepods, we diluted it in sterile water. We mixed Spirulina sp. and Chlorella sp. to feed copepods. Once we collected the pure cultures of Spirulina sp. and Chlorella sp., we diluted each culture to 10 mg/L and blended them at a 1:1 ratio. 2.7. Copepod culture, media composition and optimisation We optimized the culture media composition of copepods in 20 L culture tanks using various diets. Group 1: water alone (10 L) (W), Group 2: water + fish feces matter (0.5 g/L) (W+F), Group 3: water (10 L) + Chlorella sp. (10 mg/L) (W+S+C) + Spirulina sp. (10 mg/L), Group 4: water + Spirulina sp. + fish feces, Group 5: water + Chlorella sp.+ fish feces, and Group 6: water + Spirulina sp. + Chlorella sp. + fish feces (W+S+C+F). I inoculated each tank with 25 copepods. We aerated the tank for 1 hour at 8 a.m. and 7 p.m. during the experiment. The light and dark conditions lasted 12 h. Room temperature (27 ± 1 °C). We manually counted copepods under a stereomicroscope from the culture broth every 5 days [16]. 2.8. Optimization and visualization of copepod bio enrichment with probiotic bacteria Copepods were washed in sterilized water (1.5 mL) in groups of 6 to optimize bio-enrichment. Before adding the probiotic culture, they were kept at room temperature for 12 h. We cultured probiotic bacteria KAF061, 124, 135 and commercial probiotics in nutritional broth for 12 h, and then cleaned it twice with sterilized water. Approximately 3 µL of bacterial strains (≈2.4 × 107 CFU/mL) were introduced and maintained at room temperature. We visualized the bioenriched copepods using a phase-contrast microscope (Nikon, Inverted Microscope, Japan) after 0, 30, 60, 90, and 180 min and took images. 2.9. Statistical analysis The investigations in this study were performed in triplicate and analysed using GraphPad Prism 8 software's one-way analysis of variance (ANOVA) and student's t-test. The dataset was presented as mean and standard deviation (SD) values for triplicate measurements. We evaluated the statistical significance at three different levels: P = 0.05, P = 0.01, and P = 0.001. 3. Results 3.1. Copepod isolation, identification, and safety evaluation Multiple features were detected, including sexual dimorphism, with smaller males 0.8–1.0 mm shorter than females 1.0–1.4 mm. The medial setae were the longest, and the lateral seta was located along the caudal ramus, one-third of the posterior end. All copepods lack complex eyes. Males have two geniculate initial antennae, whereas females have straight ones that seldom reach the genital segment. The initial endopod segment of the fourth leg was long and broad, with a smooth inner border. Finally, the caudal rami base displayed no development ( Fig.1 ). Copepod microorganisms were analysed to determine their biosafety. Copepods yielded 36 bacterial strains. These strains included 12 Gram-positive cocci, 23 Gram-positive rods, and 1 Gram-negative rod. All isolates showed gamma (γ) hemolysis, ( Table 4 ). 3.2. Probiotic bacterial culture in fish feed at different storage temperatures and time intervals At varied storage temperatures (4, 27, and 37 °C), probiotic bacterial culture (KAF061, 124, 135 & commercial probiotic) with sterile fish feed was tested for its viability at 0, 5, 10, 15, 20, 30, and 45 days. KAF061 had the highest CFU (5.85 log 10 ) after 45 days at 4 °C ( Fig. 2a ), comparable to commercial probiotics (5.9 log 10 ). After 45 days at 4 °C, KAF124 and KAF135 had log 10 5.42 and 5.14, respectively ( Fig. 2b & c ). All isolates had the greatest log10 values at 4 °C, not 27 or 37 °C ( Fig. 2a, b, c, d ). 3.3. Analysing different cultural media for copepod growth with microalgae and fish feces The six culture media sets were created utilising two microalgae ( Spirulina sp . & Chlorella sp . ) and fish faeces to determine the best copepod growth medium. The copepods spent 30 days at room temperature. The growth rate was much greater (P<0.01) in group 5 (W + F + C), with 190 ± 5.5 individuals on day 30. Group 3 (W + S + C) included 182.3 ± 4.6 people, followed by group 6 (W + F +S + C) with 171.3 ± 5.2 individuals ( Fig 3 ). 3.4. Impact of probiotic microorganisms on copepod gastrointestinal system To improve the bioenrichment study, 200 copepods were separated into four groups of 50. These groups were incubated with probiotic microorganisms. Group I was cultured with KAF061, Group II with KAF124, Group III with KAF135, and Group IV with a commercial probiotic. The copepods were collected at 0, 30, 60, 90, 120, and 180 minutes and inspected using a phase-contrast microscope. The copepod's stomach tract was empty at 0 minutes. The copepod's intestines were partially filled with probiotics for 30 and 60 minutes. Probiotics filled the gastrointestinal system to capacity after 90 minutes. As the copepod digested, the gastrointestinal tract shrank ( Fig 4 ). 4. Discussion Aquaculture has a multitude of benefits that have substantial global significance. The aforementioned factors include enhanced nutritional composition, heightened market demand, rapid growth rates, diverse flavor profiles, an effective feed conversion ratio, local employment prospects, the possibility of substantial financial gains, foreign currency generation, and simplified upkeep requirements. During the first phases of fish fingerling and crustacean larval development, the presence of phytoplankton and zooplankton is of utmost importance. According to Samat et al. [17], these organisms play a critical role in serving as the fundamental foundation of the diet and a valuable reservoir of vital nutrients. Consequently, it is essential for hatcheries to engage in the cultivation and preservation of a sufficient quantity of phytoplankton and zooplankton. This is critical to safeguarding the well-being and optimal growth of juvenile fish, shielding them from possible outbreaks of infectious diseases and malnutrition disorders. The Food and Agriculture Organization [18] has highlighted the impracticality and potential introduction of environmental illnesses associated with relying on wild monospecies procurement. To tackle this issue, it is usual practice to cultivate microorganisms such as Chlorella sp. and Spirulina sp., which possess a substantial protein content for zooplankton growth and are good feed for fish fry and fingerlings. People frequently use these microorganisms to produce fish fry and fingerlings, as well as for human and veterinary applications. Due to their high protein and pigment content, they are considered special cyanobacterial species and are used as space food supplements. Moreover, studies [19, 20] have proven that Spirulina sp.'s pigment, C-Phycocyanin, has a high antioxidant value and prolongs cataract progression in animal models. Moreover, copepods, which are diminutive crustaceans, demonstrate abundant proliferation in many aquatic habitats, presenting considerable diversity in terms of size, species composition, and nutritional content. It is advantageous to ensure a consistent and ample provision of copepods during the initial developmental phases of fish larvae due to their essential role as a nutrient source, encompassing vital fatty acids, vitamins, and minerals. This distinguishes copepods from alternative live feeds, such as rotifer and artemia [17]. Scholars have focused their attention on improving copepod nauplii's cultural conditions and nutritional makeup in order to use them as live feed and/or vectors [21]. Live feeding helps newborn finfish and shellfish survive, grow quickly, establish strong innate immunity, and avoid nutritional abnormalities [22]. Copepods' high nutritional content, digestibility, size, coloration, and zig-zag movements make them easier for fry and fingerlings to hunt [23]. In many hatcheries and estuaries, fish, prawns, and mollusc fingerlings rely solely on copepods. Small and large hatcheries, nursery management systems, and ornamental fish farms supply copepods for their fry and fingerlings. The present study concludes that copepod enrichment is very essential in fish hatcheries to enhance fry and fingerling survival, rapid growth, and immunity. Copepod-supplied halibut fish fingerlings had greater survival, metamorphosis, colour, and eye development than Artemia and rotifers [24], proving copepods are healthier diets for fish larvae [25]. Copepods, collected from the Cauvery River in Tamil Nadu, India, were the dominant zooplankton in the specific natural environment, indicating their adaptability, optimal environmental condition, nutrition availability, etc. Interestingly, Mitsuka and Henry [26] reported similar findings in the Paranapanema River in Brazil. The composition and abundance of zooplankton populations are complex due to various biotic and abiotic interactions in lacustrine and lotic ecosystems. The teardrop body shape and large egg sacks identify the female copepods. Acanthocyclops sp. is considered the most prevalent freshwater species based on morphological traits [27]. Unlike other proteinaceous marine primary producers, microalgae, notably Chlorella sp. and Spirulina sp., have rich amino acid profiles, pigments, vitamins, and minerals [28]. They provide 60% digestible proteins, amino acids, beta-carotene, vitamins, and minerals [29]. Chlorella sp. inhibits superoxide dismutase and lipid peroxidation better [30]. This study utilized fish poop as a crucial additional food source for Chlorella sp. and Spirulina sp., revealing significant growth, consistent with previous findings [31]. Copepods prefer microalgae and fish feces owing to their nutritional needs and the variety of carbon and nitrogen sources [32]. Copepods containing microalgae improve fish survival, growth, fecundity, and nutritional storage [32]. Techniques such as mono- or poly-microalgal culture boost output, but species-specific needs vary. This led to the creation of a copepod growth medium utilizing Chlorella sp., Spirulina sp., and fish feces. In tanks containing Chlorella sp. and fish feces, growth was faster than in other media. Chlorella sp. has always been a better supplement for fish fingerlings in terms of survival, growth, innate immunity, and hematological markers [33]. The findings show that Chlorella sp. outperforms Spirulina sp. and the copepod Acanthocyclops sp. Both Chlorella sp. and Spirulina sp. contain single-cell protein, antioxidant enzymes, minerals, and vitamins; however, Chlorella sp. is the superior choice due to its smaller size, round shape, greenish color, higher fat content, and calorie count [34]. Previous research [35] examined the impacts of chicken, cow, and pig dung on phytoplankton, zooplankton, and Tilapia rendalli juveniles, observing that chicken manure enhanced phytoplankton. In fish-manure-treated tanks, copepod development was faster than in the control tank. Traditional outdoor copepod culture uses animal dung, soybean meal, and NPK to nourish the phytoplankton, copepods' main food supply [35]. Studies have shown that recirculation systems and probiotics can minimize mass mortalities in intense batch cultures [36]. Copepods do not need nutrient enrichment for growth and reproduction, unlike rotifers and artemia [32]. In this work, copepods were bioenriched with probiotics to boost aquaculture output for rapid growth, fry and fingerling survival, and disease resistance. This bioenrichment method delivers the indigenous beneficial bacteria directly to young fish's digestive systems through copepods, which are both a vector and a preferred food source [9]. Probiotic treatment increases a fish's ultimate body weight, body length, and specific growth rate (SGR), especially while eating [37]. Research on feeding native probiotics to young copepods to help them stay healthy and live longer is lacking [17], but more research is necessary to make aquaculture sustainable. Previous research by Husain et al. [9] examined the probiotic properties of KAF061, 124, and 135. This research examined probiotic viability at 4, 27, and 37 °C for optimum storage up to 45 days. Probiotics survived best at 4 °C, compared to 27 and 37 °C. We observed the least decline in survival at 27 and 37 °C, indicating their potential for growth at 27 °C over extended storage. Ashraf et al. [38] found comparable probiotic preservation results. Most commercial probiotics are stored and shipped at 27 °C. These findings demonstrate that commercial feed formulas can store the discovered probiotic at all temperatures analysed in this investigation. Copepods' bacterial composition before probiotic supplementation lacked 𝛼 and β hemolytic pathogens. Copepod probiotic enrichment lasted 180 min with 30-min intervals. Microscopy and bacterial cell count revealed that probiotic absorption peaked after 90 minutes. These findings align with the earlier research by Sun et al. [39], which confirmed that the gut size of copepods decreased after 90 minutes of probiotic enrichment, possibly due to digestion or forceful release. Due to their nutritional richness and simplicity of handling, copepods are the most popular live food [40]. The research found that probiotic-enriched copepods helped fish fingerlings survive and develop, making them effective carriers for antibiotics, vaccinations, growth supplements, and probiotics. Conclusion The study concluded that copepods are the best life feed for fry and fingerling growth and survival. The bioenrichment of indigenous probiotics with copepods further enhance their growth, survival, and disease resistance. Storage of probiotics with feed at 4 °C shows maximum survival in feed. Declarations Acknowledgement The author thanks Bharathidasan University for financial support, especially the URF fellowship (FH) and University Instrumentation Centre for facility support. Funding This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. Data availability The data that support the findings of this study are available from the corresponding author FH. References Mæhre, H. K., Hamre, K., & Elvevoll, E. O. (2013). 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J., Bell, J. G., Luizi, F. S., Gara, B., Bromage, N. R., & Sargent, J. R. (1999). Natural copepods are superior to enriched artemia nauplii as feed for halibut larvae (Hippoglossus hippoglossus) in terms of survival, pigmentation and retinal morphology: relation to dietary essential fatty acids. J Nutr , 129(6), 1186-94. doi:10.1093/jn/129.6.1186. Santhanam, P., Muralisankar, T., Devi, K. N., Krishnaveni, N., Divya, M., Gowthami, A., et al. (2023). Algae materials. In K. Arunkumar, A. Arun, R. Raja, & R. Palaniappan (Eds.), Algae Materials (pp. 435-449). Academic Press. doi:10.1016/b978-0-443-18816-9.00001-0. Mitsuka, P. M., & Henry, R. (2002). The fate of copepod populations in the Paranapanema River (Sao Paulo, Brazil), downstream from the Jurumirim Dam. Brazilian Archives of Biology and Technology , 45(4), 479-490. doi:Doi 10.1590/S1516-89132002000600012. Alekseev, V. R., Miracle, M. R., & Vicente, E. (2020). Redescription of Acanthocyclops vernalis (Fischer, 1853) and Acanthocyclops robustus (Sars, 1863) from neotypes, with special reference to their distinction from Acanthocyclops americanus (Marsh, 1892) and its invasion of Eurasia. Limnetica , 40(1), 57-78. doi:10.23818/limn.40.05. Macias-Sancho, J., Poersch, L. H., Bauer, W., Romano, L. A., Wasielesky, W., & Tesser, M. B. (2014). Fishmeal substitution with Arthrospira (in a practical diet for: Effects on growth and immunological parameters. Aquaculture , 426, 120-125. doi:10.1016/j.aquaculture.2014.01.028. Capelli, B., & Cysewski, G. R. (2010). Potential health benefits of spirulina microalgae*. Nutrafoods , 9(2), 19-26. doi:10.1007/bf03223332. Bengwayan, P. T., Laygo, J. C., Pacio, A. E., Poyaoan, J. L. Z., Rebugio, J. F., & Yuson, A. L. L. (2010). A comparative study on the antioxidant property of Chlorella (Chlorella sp.) tablet and glutathione tablet. E-International Scientific Research Journal , 2(1), 25-35. Juanita, U.-R., Dennis, A. H., & Michael, R. R. (1998). Analysis of copepod fecal pellet carbon using a high temperature combustion method. Marine Ecology Progress Series , 171, 199-208. https://www.int-res.com/abstracts/meps/v171/p199-208/. Dayras, P., Bialais, C., Sadovskaya, I., Lee, M. C., Lee, J. S., & Souissi, S. (2021). Microalgal Diet Influences the Nutritive Quality and Reproductive Investment of the Cyclopoid Copepod (Original Research). Frontiers in Marine Science , 8. doi:ARTN 6975610.3389/fmars.2021.697561. Arteaga Quico, C., Mariano Astocondor, M., & Aquino Ortega, R. (2021). Dietary supplementation with Chlorella peruviana improve the growth and innate immune response of rainbow trout Oncorhynchus mykiss fingerlings. Aquaculture , 533, 736117. doi:10.1016/j.aquaculture.2020.736117. Raji, A. A., Alaba, P. A., Yusuf, H., Abu Bakar, N. H., Mohd Taufek, N., Muin, H., et al. (2018). Fishmeal replacement with Spirulina Platensis and Chlorella vulgaris in African catfish (Clarias gariepinus) diet: Effect on antioxidant enzyme activities and haematological parameters. Res Vet Sci , 119, 67-75. doi:10.1016/j.rvsc.2018.05.013. Kang'ombe, J., Brown, J. A., & Halfyard, L. C. (2006). Effect of using different types of organic animal manure on plankton abundance, and on growth and survival of (Boulenger) in ponds. Aquaculture Research , 37(13), 1360-1371. doi:10.1111/j.1365-2109.2006.01569.x. Munro, P. D., Barbour, A., & Birkbeck, T. H. (1994). Comparison of the Gut Bacterial-Flora of Start-Feeding Larval Turbot Reared under Different Conditions. Journal of Applied Bacteriology , 77(5), 560-566. doi:DOI 10.1111/j.1365-2672.1994.tb04402.x. Samat, N. A., Yusoff, F. M., Rasdi, N. W., & Karim, M. (2020). Enhancement of Live Food Nutritional Status with Essential Nutrients for Improving Aquatic Animal Health: A Review. Ashraf, M. (2009). Biotechnological approach of improving plant salt tolerance using antioxidants as markers. Biotechnol Adv , 27(1), 84-93. doi:10.1016/j.biotechadv.2008.09.003. Sun, X. H., Liang, Z. L., Zou, J. X., & Wang, L. X. (2013). Seasonal variation in community structure and body length of dominant copepods around artificial reefs in Xiaoshi Island, China. Chinese Journal of Oceanology and Limnology , 31(2), 282-289. doi:10.1007/s00343-013-2088-0. Talpur, M. A. D., & Ikhwanuddin, M. (2012). Effects of stress tests on larvae of blue swimming crab, Portunus pelagicus (Linnaeus, 1758). Advances in Environmental Biology , 6, 1909-1915. Tables Table I. Nutrition profile of zooplanktonic feeds used in aquaculture. Protein ( % ) Carbohydrate ( % ) Fat ( % ) DHA ( % ) EPA ( % ) Ash ( % ) Energy value (KJ/g) Reference Artemia 55 15 40 2.68 0.81 9.4 18.97 John et al., 2004, Martinez et al., 2023 Rotifer 32.1 14.62 19.85 13.7 9.3 5.2 17.61 Jeeja et al., 2011, Hamre, 2016 Copepods 82 68 24 34.4 17.4 9.5 31 Støttrup., 2003, Meeren et al., 2008 Table 2. Geolocation for zooplankton sample collection. Area Latitude Longitude Cauvery river 10°50'15.4"N 78°41'51.2"E Mathur seasonal lake 10°41'40.8"N 78°44'14.0"E Gundur lake 10°43'32.5"N 78°43'36.6"E Kumbakudi pond 10°42'14.5"N 78°45'00.1"E Balancing reservoir 10°42'22.8"N 78°49'00.1"E Thuvakudi village pond 10°45'04.5"N 78°50'13.0"E Table 3. Zooplanktons from Trichy's Cauvery River during monsoon 2018-19. Zooplankton Months November 2018 December 2018 January 2019 Protozoa 224 ± 5.2 401 ± 4.1 221 ± 4.5 Rotifer 533 ± 5.2 494 ± 5.1 408 ± 5.1 Copepoda 1952 ± 4.5 911 ± 2.9 1983 ± 5.5 Cladocera 275 ± 5.5 195 ± 4.2 312 ± 2.6 Ostracoda 101 ± 3.6 86 ± 2.7 123 ± 5.5 Table 4. Evaluation of the biosafety of copepods before use as live food organisms Bacterial isolates Gram positive Gram negative Hemolysis cocci rods cocci rods α β γ 36 12 23 - 1 - - 36 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4674332","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":333136510,"identity":"8750e903-247b-4f3a-ac86-504450a4b30e","order_by":0,"name":"Fazal Husain","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCUlEQVRIie2PsUrEQBCGJ02uCVy7cJg8gTBHQKzOt7CeJeA2J1heGRD2ynuAFHmFHAfWC1vY+AABLRKE1FtapHA3ClqY3NkJ7lf8Oyzz8TMAHs8fZJbbaEDZDNwIsQvVTCiRW6YvBdNBodOUAeRD16Qye2wNwQsvt1o20PeivNatbVnF5/mIElHGCDpePfEtBhJv9883aJUsvVA/K1dAyiqaV8AlC3KrFOQUxR9GlGje3r85pdy1kkGPYlkIM62wLBxa8tq2QIiULNZHWlgXXhLqtKptC5fpslqs7xTh+C3RXLzWZqPPyp3omOnjJCnEwZjNKh5TPsDPl9ysvv+cRJL/Ztvj8Xj+A+9kGWVQ9p5YmQAAAABJRU5ErkJggg==","orcid":"","institution":"National Botanical Research Institute","correspondingAuthor":true,"prefix":"","firstName":"Fazal","middleName":"","lastName":"Husain","suffix":""}],"badges":[],"createdAt":"2024-07-02 12:48:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4674332/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4674332/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":61388169,"identity":"c1089463-1452-4ec7-af42-5f4dd2e44638","added_by":"auto","created_at":"2024-07-30 07:30:16","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":109951,"visible":true,"origin":"","legend":"\u003cp\u003eDiagrammatic representation of the male and female \u003cem\u003eAcanthocyclops \u003c/em\u003esp.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4674332/v1/1a46d114366df9da3cd46f34.png"},{"id":61387541,"identity":"a7c93289-fa84-4799-afca-c256e23035bc","added_by":"auto","created_at":"2024-07-30 07:22:16","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":44318,"visible":true,"origin":"","legend":"\u003cp\u003eLog\u003csub\u003e10 \u003c/sub\u003evalues (mean ± SD) of viable counts for a. KAF061; b. KAF124; c. KAF135; and d. commercial probiotics at 4°C, 27°C, and 37°C storage temperatures, incubated with fish feed for 0, 5, 10, 15, 20, 30, and 45 days.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4674332/v1/90cba0a131bb02a44b19d345.png"},{"id":61387542,"identity":"73cf6c37-7289-4860-99a4-5e5e8d4a2f92","added_by":"auto","created_at":"2024-07-30 07:22:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":60656,"visible":true,"origin":"","legend":"\u003cp\u003eOptimization of copepod culture in Group 1: water (W), Group 2: water + fish fecal matter (W+F), Group 3: water + \u003cem\u003eSpirulina\u003c/em\u003esp.+ \u003cem\u003eChlorella\u003c/em\u003e sp. (W+S+C), Group 4: water + fish fecal matter + \u003cem\u003eSpirulina\u003c/em\u003esp. (W+F+S), Group 5: water + fish fecal matter + \u003cem\u003eChlorella \u003c/em\u003esp. (W+F+C) and Group 6: water + fish fecal matter + \u003cem\u003eSpirulina\u003c/em\u003e +\u003cem\u003e Chlorella\u003c/em\u003e (W+F+S+C). *P\u0026lt;0.05,\u003c/p\u003e\n\u003cp\u003e**P\u0026lt;0.01, ***P\u0026lt;0.001, #: nonsignificant\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4674332/v1/53ef3a335bf039924c52bb07.png"},{"id":61387544,"identity":"a9a65eeb-5c7f-448d-aecc-278f4bdf22b8","added_by":"auto","created_at":"2024-07-30 07:22:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":737752,"visible":true,"origin":"","legend":"\u003cp\u003eImages showing copepods \u003cem\u003eAcanthocyclops \u003c/em\u003esp\u003cem\u003e.\u003c/em\u003e view under phase-contrast microscopy after incubating with probiotics KAF061 at different time points a) 0 min, b) 30 min, c) 60 min, d) 90 min, e) 120 min and f) 180 min (representative images).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4674332/v1/8d42e66ca1cc7dd26322748b.png"},{"id":66822059,"identity":"dff1b450-4382-48cd-b221-1819b6c666c5","added_by":"auto","created_at":"2024-10-16 22:16:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1854714,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4674332/v1/bb49991e-cb9f-404d-9142-9512e2971f50.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Probiotic enrichment of copepod (Acanthocyclops sp.) towards improving fish survival, nutritional content, optimal growth and sustainability","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAquaculture hatcheries and nurseries need live feed culture to support fry and fingerling survival, growth, and supply year-round. This is crucial because early hatched fish larvae need prepared nutrition. Finfish and shellfish hatcheries rely heavily on live feed culture because they have an immature digestive system and lack enzyme synthesis. Live feeds provide a healthy diet for fries and fingerlings. Previous research has demonstrated that live feed is essential for the growth and development of newborn fish fingerlings. Aquaculture hatcheries' newly hatched fish fry and fingerlings solely depend on live feeds of microalgae, copepods, rotifers, and Artemia [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Among its copepods, there is a superior zooplankton that has a minimal size and provides highly suitable nutrition for fish fry.\u003c/p\u003e \u003cp\u003eHatcheries use microalgae such as \u003cem\u003eNannochloropsis\u003c/em\u003e, \u003cem\u003eChlorella\u003c/em\u003e sp., \u003cem\u003eChaetoceros, Tetraselmis, Scenedesmus, Pavlova, Phaeodactylum, Skeletonema\u003c/em\u003e, and \u003cem\u003eThalassiosira\u003c/em\u003e to grow larval fish, mollusks, and crustaceans. Artemia to their well-established production and rearing methods in aquaculture, artemia and rotifers are the preferred zooplankton. Copepods are better for you than Artemia and rotifers (\u003cb\u003eTable\u0026nbsp;1\u003c/b\u003e) because they have more good things for you, like polyunsaturated fatty acids (PUFA) and especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. However, these lipids are scarce; therefore, enrichment is generally needed. Copepods are the favourite diet for fish fry and fingerlings, especially during early hunting, because their zig-zag movement and brief gliding phase provide visual stimulation and also clean rearing tanks by nibbling on algae and detritus [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Additionally, copepods reduce ecosystem acidification and global warming [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRotifers are fish larvae's live feed because of their wide size range (100 to 300 mm), high fecundity, ability to grow in high population density, resistance to variable temperatures and salinities, ability to be supplemented with minimal nutritional supplements, and slow swimming movements that make them easy to hunt [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Aquaculture has used rotifers, Brachionus sp., as live feed since the 1970s [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The larvae of most marine fish, crabs, and mollusks filter water to feed. They need minimal prey size in their early stages [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Even though rotifers are resistant to abiotic factors [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], Birnavirus or fungal infections can stop them from producing a lot of fish. This means that they must be cultured all year, which takes more time and costs more money than seasonal outdoor rearing and often leads to complete culture losses [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eArtemia is a popular aquaculture live feed because of its availability and high salinity tolerance. We grow \u003cem\u003eArtemia salina, A. sinica, A. persimilis, A. urmiana\u003c/em\u003e, and \u003cem\u003eA. tibetiana\u003c/em\u003e for their easy storage and hatching. Artemia's nutritional content needs enrichment, but technology has solved this problem. Live feeding with arthritis causes issues such as unpredictable supply, poor nutrition, shifting pricing, and cyst shortages. Growers may need to adapt pricing or fish larvae diets. Despite these drawbacks, artemia remains popular due to its many advantages. In order to tackle these issues, scholars are now investigating copepods as prospective substitutes for live-feed aquaculture. Artemia, like rotifers, necessitates vital elements such as fatty acids, minerals, and vitamins; nonetheless, contemporary enrichment technologies have facilitated its utilization. Despite Artemia's widespread use, its limitations have prompted research into copepods. Copepods exhibit a range of life phases, including eggs, nauplius, copepodite, and maturity, which enables them to meet diverse size needs within aquatic settings. Copepods have the ability to provide viable solutions to the challenges associated with the practice of live-feeding Artemia.\u003c/p\u003e \u003cp\u003eProbiotics are beneficial bacteria that protect host animals from dangerous microbial infections, stimulate quick development, and boost activity. Microbial community expansion in hatcheries is a major cause of large mortalities, as well as uneven survival and growth. In hatchery cultures, probiotics have replaced antibiotics and their microbial control hazards. Note that probiotic strains have varied health advantages. Aquaculture uses live foods such as Artemia and rotifers, pellet food, direct water addition, and injection to provide probiotics. Effective probiotic distribution reduces illness onset, treatment, and prevention costs. By enriching copepods with probiotic bacteria and directly inoculating them into the target animals' digestive tracts, probiotics and copepods can enhance aquatic fish rearing by providing better nutritional content. This study cultivates copepods inside and enriches them with probiotic bacteria.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e2.1.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eSample collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe collected freshwater zooplankton samples from multiple ponds and stagnant water bodies in and around the Cauvery River in Tamil Nadu, India (\u003cstrong\u003eTable 2\u003c/strong\u003e). Prior to transportation to the laboratory for further processing, the samples were filtered using a 70 µm mesh. The collection took place during the morning hours, around 6 a.m., before the surface water temperature began to rise.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eProbiotic\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eBacillus subtilis\u003c/em\u003e KAF061, \u003cem\u003eBacillus cereus\u003c/em\u003e KAF124, \u003cem\u003eBacillus thuringiensis\u003c/em\u003e KAF135, and a commercial probiotic called \u003cem\u003eBacillus subtilis\u003c/em\u003e were used in this study to find possible probiotic strains that would help increase the number of copepods. Husain et al. [9] revealed the powerful probiotics KAF124 and KAF135. \u003cem\u003eB. subtilis\u003c/em\u003e, a commercially available probiotic, served as a positive control in this investigation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eMorphology \u0026amp; Anatomy of \u003cem\u003eAcanthocyclops\u0026nbsp;\u003c/em\u003esp\u003cem\u003e.\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWitty [10] described the morphological analysis using stereomicroscopy. Copepods were the most numerous groups, which were isolated zooplanktons, followed by rotifers, Cladocera, protozoa, and Ostracoda (\u003cstrong\u003eTable 3\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eCopepod analysis in water samples: isolation, identification, and hemolysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNylon mesh with a 300 µm hole size was used to isolate the copepods from surface water samples. An inverted microscope at 40x magnification evaluated the samples after 24 h of stabilization. Copepods were the most prevalent zooplankton and had sophisticated eyes, sexual dimorphism, colouring, and viable female egg sacs [11]. For research purposes, copepods were mass-produced in the lab.\u003c/p\u003e\n\u003cp\u003eHusain et al. (2022) provided instructions for the hemolysis test, a method for identifying potentially harmful microorganisms on the outside and inside of copepods. We homogenized the copepods and plated them on nutrient agar at 37 °C for 24 h. We formed separate colonies on blood agar plates and examined blood cell lysis in cultures after 72 h [12].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eProbiotic survival at varied temperatures with commercial fish feed\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the shelf life, we blended autoclaved commercial fish feed (0.5 g) with KAF061, KAF124, and KAF135 (6.5 x 107 CFU/mL) probiotic isolates and incubated them at 4, 27, and 37 °C. After 5, 10, 15, 20, 30, and 45 days, we tested the bacterial isolate's survival using plate counts at different storage temperatures. We quantified the logarithmic colony-forming unit (CFU) values [13].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eMass Culture of Copepods\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eusing \u003cem\u003eSpirulina\u003c/em\u003esp\u003cem\u003e.\u003c/em\u003e and \u003cem\u003eChlorella\u003c/em\u003esp\u003cem\u003e.\u003c/em\u003e as\u0026nbsp;feed: Cultivation and\u0026nbsp;feeding\u0026nbsp;strategies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe used \u003cem\u003eSpirulina\u003c/em\u003e sp. for copepod mass culture, and we cultivated pure \u003cem\u003eSpirulina\u003c/em\u003e sp. culture in Zarrouk's medium [14] with the following contents: (g/L) NaHCO3, 16.8; K2HPO4 0.5; NaNO3, 2.5; K2SO4, 1.0; NaCl, 1.0; MgSO4-7H20, 0.2; CaCl2, 0.04; FeSO4-7H2O, 0.01; EDTA, 0.08; Solution A5, (g/L) H3BO3, 2.86; MnCl2-4H2O, 1.81; ZnSO4-7H2O, 0.222; CuSO4 5—1 ml/L; Solution B6, (mg/L, NH4VO3, 22.96; KCr (SO4)2 - 12H2O, 192.0; NiSO4-6H2O, 44.8; Na2PO4-2H2O, 17.94; TiOSO4H2SO4 - 8H2O, 61.1; and Co(NO3)2 - 6H2O, 43.98) optimized for copepod feed. The culture broth was incubated at 27 °C with a 10 mg/L inoculWe measured the optical density (OD) of the culture broth at 450 nm every 24 h for 10 days. every 24 h for 10 days. We produced the growth curve using the OD values of the broth culture.\u003c/p\u003e\n\u003cp\u003eSimilarly, copepod mass culture used \u003cem\u003eChlorella\u003c/em\u003e sp. as food. A BG11 medium [15] was used to grow \u003cem\u003eChlorella\u003c/em\u003e sp. It had 1.5 g of NaNO3, 0.04 g of K2HPO4∙2H2O, 0.075 g of MgSO4∙7H2O, 0.036 g of CaCl2∙2H2O, 0.006 g of citric acid, 0.02 g of Na2CO3, 0.006 g of ferric ammonium citrate, 0.001 g of Na-EDTA, and 1 mL of trace metal A5.\u0026nbsp;The trace metal A5 solution had 2.86 H3BO3, 1.81 MnCl2 ∙ 4H2O, 0.22 ZnSO4∙7H2O, 0.39 NaMoO4∙2H2O, 0.079 CuSO4∙5H2O, and 0.05 CoCl2∙6H2O (g/L).\u0026nbsp;The solution was incubated at 27 °C with a 10 mg/L inoculum in BG11 media. We measured the optical density (OD) of the \u003cem\u003eChlorella\u003c/em\u003e sp. culture broth at 450 nm every 24 h for 10 days. We plotted the growth curve using the OD values of the broth culture.\u003c/p\u003e\n\u003cp\u003eWe used Whatman No. 1 paper to isolate the cultures of \u003cem\u003eSpirulina\u003c/em\u003e sp. and \u003cem\u003eChlorella\u003c/em\u003e sp. To make it more suitable for copepods, we diluted it in sterile water.\u003c/p\u003e\n\u003cp\u003eWe mixed \u003cem\u003eSpirulina\u003c/em\u003e sp. and \u003cem\u003eChlorella\u003c/em\u003e sp. to feed copepods. Once we collected the pure cultures of \u003cem\u003eSpirulina\u003c/em\u003e sp. and \u003cem\u003eChlorella\u003c/em\u003e sp., we diluted each culture to 10 mg/L and blended them at a 1:1 ratio.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.7.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eCopepod culture, media composition and optimisation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe optimized the culture media composition of copepods in 20 L culture tanks using various diets. Group 1: water alone (10 L) (W), Group 2: water + fish feces matter (0.5 g/L) (W+F), Group 3: water (10 L) + \u003cem\u003eChlorella\u0026nbsp;\u003c/em\u003esp. (10 mg/L) (W+S+C) + \u003cem\u003eSpirulina\u003c/em\u003e sp. (10 mg/L), Group 4: water + \u003cem\u003eSpirulina\u003c/em\u003e sp. + fish feces, Group 5: water + \u003cem\u003eChlorella\u003c/em\u003e sp.+ fish feces, and Group 6: water + \u003cem\u003eSpirulina\u003c/em\u003e sp. + \u003cem\u003eChlorella\u003c/em\u003e sp. + fish feces (W+S+C+F). I inoculated each tank with 25 copepods. We aerated the tank for 1 hour at 8 a.m. and 7 p.m. during the experiment. The light and dark conditions lasted 12 h. Room temperature (27 ± 1 °C). We manually counted copepods under a stereomicroscope from the culture broth every 5 days [16].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.8.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eOptimization and visualization of copepod\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ebio enrichment\u0026nbsp;with probiotic bacteria\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCopepods were washed in sterilized water (1.5 mL) in groups of 6 to optimize bio-enrichment. Before adding the probiotic culture, they were kept at room temperature for 12 h. We cultured probiotic bacteria KAF061, 124, 135 and commercial probiotics in nutritional broth for 12 h, and then cleaned it twice with sterilized water. Approximately 3 µL of bacterial strains (≈2.4 × 107 CFU/mL) were introduced and maintained at room temperature. We visualized the bioenriched copepods using a phase-contrast microscope (Nikon, Inverted Microscope, Japan) after 0, 30, 60, 90, and 180 min and took images.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.9.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe investigations in this study were performed in triplicate and analysed using GraphPad Prism 8 software's one-way analysis of variance (ANOVA) and student's t-test. The dataset was presented as mean and standard deviation (SD) values for triplicate measurements. We evaluated the statistical significance at three different levels: P = 0.05, P = 0.01, and P = 0.001.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eCopepod isolation, identification, and safety evaluation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMultiple features were detected, including sexual dimorphism, with smaller males 0.8–1.0 mm shorter than females 1.0–1.4 mm. The medial setae were the longest, and the lateral seta was located along the caudal ramus, one-third of the posterior end.\u0026nbsp;All copepods lack complex eyes. Males have two geniculate initial antennae, whereas females have straight ones that seldom reach the genital segment. The initial endopod segment of the fourth leg was long and broad, with a smooth inner border. Finally, the caudal rami base displayed no development (\u003cstrong\u003eFig.1\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eCopepod microorganisms were analysed to determine their biosafety. Copepods yielded 36 bacterial strains. These strains included 12 Gram-positive cocci, 23 Gram-positive rods, and 1 Gram-negative rod. All isolates showed gamma (γ) hemolysis, (\u003cstrong\u003eTable 4\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eProbiotic bacterial culture in fish feed at different storage temperatures and time intervals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt varied storage temperatures (4, 27, and 37 °C), probiotic bacterial culture (KAF061, 124, 135 \u0026amp; commercial probiotic) with sterile fish feed was tested for its viability at 0, 5, 10, 15, 20, 30, and 45 days. KAF061 had the highest CFU (5.85 log\u003csub\u003e10\u003c/sub\u003e) after 45 days at 4 °C (\u003cstrong\u003eFig. 2a\u003c/strong\u003e), comparable to commercial probiotics (5.9 log\u003csub\u003e10\u003c/sub\u003e). After 45 days at 4 °C, KAF124 and KAF135 had log\u003csub\u003e10\u003c/sub\u003e 5.42 and 5.14, respectively (\u003cstrong\u003eFig. 2b \u0026amp; c\u003c/strong\u003e). All isolates had the greatest log10 values at 4 °C, not 27 or 37 °C (\u003cstrong\u003eFig. 2a, b, c, d\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eAnalysing different cultural media for copepod growth with microalgae and fish feces\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe six culture media sets were created utilising two microalgae (\u003cem\u003eSpirulina\u003c/em\u003esp\u003cem\u003e.\u0026nbsp;\u003c/em\u003e\u0026amp; \u003cem\u003eChlorella\u003c/em\u003esp\u003cem\u003e.\u003c/em\u003e) and fish faeces to determine the best copepod growth medium. The copepods spent 30 days at room temperature. The growth rate was much greater (P\u0026lt;0.01) in group 5 (W + F + C), with 190 ± 5.5 individuals on day 30. Group 3 (W + S + C) included 182.3 ± 4.6 people, followed by group 6 (W + F +S + C) with 171.3 ± 5.2 individuals (\u003cstrong\u003eFig 3\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eImpact of probiotic microorganisms on copepod gastrointestinal system\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo improve the bioenrichment study, 200 copepods were separated into four groups of 50. These groups were incubated with probiotic microorganisms. Group I was cultured with KAF061, Group II with KAF124, Group III with KAF135, and Group IV with a commercial probiotic. The copepods were collected at 0, 30, 60, 90, 120, and 180 minutes and inspected using a phase-contrast microscope. The copepod's stomach tract was empty at 0 minutes. The copepod's intestines were partially filled with probiotics for 30 and 60 minutes. Probiotics filled the gastrointestinal system to capacity after 90 minutes. As the copepod digested, the gastrointestinal tract shrank (\u003cstrong\u003eFig 4\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eAquaculture has a multitude of benefits that have substantial global significance. The aforementioned factors include enhanced nutritional composition, heightened market demand, rapid growth rates, diverse flavor profiles, an effective feed conversion ratio, local employment prospects, the possibility of substantial financial gains, foreign currency generation, and simplified upkeep requirements. During the first phases of fish fingerling and crustacean larval development, the presence of phytoplankton and zooplankton is of utmost importance. According to Samat et al. [17], these organisms play a critical role in serving as the fundamental foundation of the diet and a valuable reservoir of vital nutrients. Consequently, it is essential for hatcheries to engage in the cultivation and preservation of a sufficient quantity of phytoplankton and zooplankton. This is critical to safeguarding the well-being and optimal growth of juvenile fish, shielding them from possible outbreaks of infectious diseases and malnutrition disorders.\u003c/p\u003e\n\u003cp\u003eThe Food and Agriculture Organization [18] has highlighted the impracticality and potential introduction of environmental illnesses associated with relying on wild monospecies procurement. To tackle this issue, it is usual practice to cultivate microorganisms such as \u003cem\u003eChlorella\u003c/em\u003e sp. and \u003cem\u003eSpirulina\u003c/em\u003e sp., which possess a substantial protein content for zooplankton growth and are good feed for fish fry and fingerlings. People frequently use these microorganisms to produce fish fry and fingerlings, as well as for human and veterinary applications. Due to their high protein and pigment content, they are considered special cyanobacterial species and are used as space food supplements. Moreover, studies [19, 20] have proven that \u003cem\u003eSpirulina\u003c/em\u003e sp.'s pigment, C-Phycocyanin, has a high antioxidant value and prolongs cataract progression in animal models. Moreover, copepods, which are diminutive crustaceans, demonstrate abundant proliferation in many aquatic habitats, presenting considerable diversity in terms of size, species composition, and nutritional content. It is advantageous to ensure a consistent and ample provision of copepods during the initial developmental phases of fish larvae due to their essential role as a nutrient source, encompassing vital fatty acids, vitamins, and minerals. This distinguishes copepods from alternative live feeds, such as rotifer and artemia [17]. Scholars have focused their attention on improving copepod nauplii's cultural conditions and nutritional makeup in order to use them as live feed and/or vectors [21].\u003c/p\u003e\n\u003cp\u003eLive feeding helps newborn finfish and shellfish survive, grow quickly, establish strong innate immunity, and avoid nutritional abnormalities [22]. Copepods' high nutritional content, digestibility, size, coloration, and zig-zag movements make them easier for fry and fingerlings to hunt [23]. In many hatcheries and estuaries, fish, prawns, and mollusc fingerlings rely solely on copepods. Small and large hatcheries, nursery management systems, and ornamental fish farms supply copepods for their fry and fingerlings. The present study concludes that copepod enrichment is very essential in fish hatcheries to enhance fry and fingerling survival, rapid growth, and immunity. Copepod-supplied halibut fish fingerlings had greater survival, metamorphosis, colour, and eye development than Artemia and rotifers [24], proving copepods are healthier diets for fish larvae [25].\u003c/p\u003e\n\u003cp\u003eCopepods, collected from the Cauvery River in Tamil Nadu, India, were the dominant zooplankton in the specific natural environment, indicating their adaptability, optimal environmental condition, nutrition availability, etc. Interestingly, Mitsuka and Henry [26] reported similar findings in the Paranapanema River in Brazil. The composition and abundance of zooplankton populations are complex due to various biotic and abiotic interactions in lacustrine and lotic ecosystems. The teardrop body shape and large egg sacks identify the female copepods. Acanthocyclops sp. is considered the most prevalent freshwater species based on morphological traits [27].\u003c/p\u003e\n\u003cp\u003eUnlike other proteinaceous marine primary producers, microalgae, notably \u003cem\u003eChlorella\u003c/em\u003e sp. and \u003cem\u003eSpirulina\u003c/em\u003e sp., have rich amino acid profiles, pigments, vitamins, and minerals [28]. They provide 60% digestible proteins, amino acids, beta-carotene, vitamins, and minerals [29]. \u003cem\u003eChlorella\u003c/em\u003e sp. inhibits superoxide dismutase and lipid peroxidation better [30]. This study utilized fish poop as a crucial additional food source for \u003cem\u003eChlorella\u003c/em\u003e sp. and \u003cem\u003eSpirulina\u003c/em\u003e sp., revealing significant growth, consistent with previous findings [31].\u003c/p\u003e\n\u003cp\u003eCopepods prefer microalgae and fish feces owing to their nutritional needs and the variety of carbon and nitrogen sources [32]. Copepods containing microalgae improve fish survival, growth, fecundity, and nutritional storage [32]. Techniques such as mono- or poly-microalgal culture boost output, but species-specific needs vary. This led to the creation of a copepod growth medium utilizing \u003cem\u003eChlorella\u003c/em\u003e sp., \u003cem\u003eSpirulina\u003c/em\u003e sp., and fish feces. In tanks containing \u003cem\u003eChlorella\u003c/em\u003e sp. and fish feces, growth was faster than in other media. \u003cem\u003eChlorella\u003c/em\u003e sp. has always been a better supplement for fish fingerlings in terms of survival, growth, innate immunity, and hematological markers [33]. The findings show that \u003cem\u003eChlorella\u003c/em\u003e sp. outperforms \u003cem\u003eSpirulina\u003c/em\u003e sp. and the copepod Acanthocyclops sp. Both \u003cem\u003eChlorella\u003c/em\u003e sp. and \u003cem\u003eSpirulina\u003c/em\u003e sp. contain single-cell protein, antioxidant enzymes, minerals, and vitamins; however, \u003cem\u003eChlorella\u003c/em\u003e sp. is the superior choice due to its smaller size, round shape, greenish color, higher fat content, and calorie count [34]. Previous research [35] examined the impacts of chicken, cow, and pig dung on phytoplankton, zooplankton, and Tilapia rendalli juveniles, observing that chicken manure enhanced phytoplankton. In fish-manure-treated tanks, copepod development was faster than in the control tank. Traditional outdoor copepod culture uses animal dung, soybean meal, and NPK to nourish the phytoplankton, copepods' main food supply [35].\u003c/p\u003e\n\u003cp\u003eStudies have shown that recirculation systems and probiotics can minimize mass mortalities in intense batch cultures [36]. Copepods do not need nutrient enrichment for growth and reproduction, unlike rotifers and artemia [32]. In this work, copepods were bioenriched with probiotics to boost aquaculture output for rapid growth, fry and fingerling survival, and disease resistance. This bioenrichment method delivers the indigenous beneficial bacteria directly to young fish's digestive systems through copepods, which are both a vector and a preferred food source [9]. Probiotic treatment increases a fish's ultimate body weight, body length, and specific growth rate (SGR), especially while eating [37]. Research on feeding native probiotics to young copepods to help them stay healthy and live longer is lacking [17], but more research is necessary to make aquaculture sustainable.\u003c/p\u003e\n\u003cp\u003ePrevious research by Husain et al. [9] examined the probiotic properties of KAF061, 124, and 135. This research examined probiotic viability at 4, 27, and 37 °C for optimum storage up to 45 days. Probiotics survived best at 4 °C, compared to 27 and 37 °C. We observed the least decline in survival at 27 and 37 °C, indicating their potential for growth at 27 °C over extended storage. Ashraf et al. [38] found comparable probiotic preservation results. Most commercial probiotics are stored and shipped at 27 °C. These findings demonstrate that commercial feed formulas can store the discovered probiotic at all temperatures analysed in this investigation.\u003c/p\u003e\n\u003cp\u003eCopepods' bacterial composition before probiotic supplementation lacked 𝛼 and β hemolytic pathogens. Copepod probiotic enrichment lasted 180 min with 30-min intervals. Microscopy and bacterial cell count revealed that probiotic absorption peaked after 90 minutes. These findings align with the earlier research by Sun et al. [39], which confirmed that the gut size of copepods decreased after 90 minutes of probiotic enrichment, possibly due to digestion or forceful release. Due to their nutritional richness and simplicity of handling, copepods are the most popular live food [40]. The research found that probiotic-enriched copepods helped fish fingerlings survive and develop, making them effective carriers for antibiotics, vaccinations, growth supplements, and probiotics.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe study concluded that copepods are the best life feed for fry and fingerling growth and survival. The bioenrichment of indigenous probiotics with copepods further enhance their growth, survival, and disease resistance. Storage of probiotics with feed at 4 °C shows maximum survival in feed.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author thanks Bharathidasan University for financial support, especially the URF fellowship (FH) and University Instrumentation Centre for facility support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author FH.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eM\u0026aelig;hre, H. K., Hamre, K., \u0026amp; Elvevoll, E. O. (2013). 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Live feeds for early stages of fish rearing. \u003cem\u003eAquaculture Research\u003c/em\u003e, 41(5), 613-640. doi:10.1111/j.1365-2109.2009.02242.x.\u003c/li\u003e\n \u003cli\u003eComps, M., \u0026amp; Menu, B. (1997). Infectious diseases affecting mass production of the marine rotifer Brachionus plicatilis. \u003cem\u003eHydrobiologia\u003c/em\u003e, 358(1), 179-183. doi:Doi 10.1023/A:1003101201928.\u003c/li\u003e\n \u003cli\u003eHusain, F., Duraisamy, S., Balakrishnan, S., Ranjith, S., Chidambaram, P., \u0026amp; Kumarasamy, A. (2022). Phenotypic assessment of safety and probiotic potential of native isolates from marine fish Moolgarda seheli towards sustainable aquaculture. \u003cem\u003eBiologia (Bratisl)\u003c/em\u003e, 77(3), 775-790. doi:10.1007/s11756-021-00957-w.\u003c/li\u003e\n \u003cli\u003eWitty, L. (2004). Practical Guide to Identifying Freshwater Crustacean Zooplankton, 2nd edition, Cooperative Freshwater Ecology Unit, 2004.\u003c/li\u003e\n \u003cli\u003eDodson, S. (1994). Morphological Analysis of Wisconsin (U.S.A.) Species of the Acanthocyclops Vernalis Group (Copepoda: Cyclopoida). \u003cem\u003eJournal of Crustacean Biology\u003c/em\u003e, 14(1), 113-131. doi:10.1163/193724094x00515.\u003c/li\u003e\n \u003cli\u003ePieniz, S., Andreazza, R., Anghinoni, T., Camargo, F., \u0026amp; Brandelli, A. (2014). Probiotic potential, antimicrobial and antioxidant activities of strain LAB18s. \u003cem\u003eFood Control\u003c/em\u003e, 37, 251-256. doi:10.1016/j.foodcont.2013.09.055.\u003c/li\u003e\n \u003cli\u003eAshraf, M. (2009). Biotechnological approach of improving plant salt tolerance using antioxidants as markers. \u003cem\u003eBiotechnol Adv\u003c/em\u003e, 27(1), 84-93. doi:10.1016/j.biotechadv.2008.09.003.\u003c/li\u003e\n \u003cli\u003eZarrouk, C. (1966). Contribution \u0026agrave; l\u0026apos;\u0026eacute;tude d\u0026apos;une cyanophyc\u0026eacute;e.\u003c/li\u003e\n \u003cli\u003eAl-Rikabey, M. N., \u0026amp; Al-Mayah, A. M. (2018). Cultivation of Chlorella vulgaris in BG-11 media using Taguchi method. \u003cem\u003eJ Adv Res Dynamic\u003c/em\u003e, 10(7), 19-30.\u003c/li\u003e\n \u003cli\u003eLi, S. W., Huang, Y. X., \u0026amp; Liu, M. Y. (2020). Transcriptome profiling reveals the molecular processes for survival of Lysinibacillus fusiformis strain 15-4 in petroleum environments. \u003cem\u003eEcotoxicol Environ Saf\u003c/em\u003e, 192, 110250. doi:10.1016/j.ecoenv.2020.110250.\u003c/li\u003e\n \u003cli\u003eSamat, N. A., Yusoff, F. M., Rasdi, N. W., \u0026amp; Karim, M. (2020). Enhancement of Live Food Nutritional Status with Essential Nutrients for Improving Aquatic Animal Health: A Review.\u003c/li\u003e\n \u003cli\u003eFood and Agriculture Organization of the United Nations (2016), FAOSTAT Database, FAO. (1 December 2016; www.fao.org/faostat)\u003c/li\u003e\n \u003cli\u003eMarzorati, S., Schievano, A., Id\u0026agrave;, A., \u0026amp; Verotta, L. (2020). Carotenoids, chlorophylls and phycocyanin from Spirulina: supercritical CO2 and water extraction methods for added value products cascade (10.1039/C9GC03292D). \u003cem\u003eGreen Chemistry\u003c/em\u003e, 22(1), 187-196. doi:10.1039/c9gc03292d.\u003c/li\u003e\n \u003cli\u003eCiti, V., Torre, S., Flori, L., Usai, L., Aktay, N., Dunford, N. T., et al. (2024). Nutraceutical Features of the Phycobiliprotein C-Phycocyanin: Evidence from Arthrospira platensis (Spirulina).\u003c/li\u003e\n \u003cli\u003ePatra, S. K., \u0026amp; Mohamed, K. S. (2003). Enrichment of nauplii with the probiotic yeast and its resistance against a pathogenic. \u003cem\u003eAquaculture International\u003c/em\u003e, 11(5), 505-514. doi:Doi 10.1023/B:Aqui.0000004193.40039.54.\u003c/li\u003e\n \u003cli\u003eBurr, G., Gatlin, D., \u0026amp; Ricke, S. (2005). Microbial ecology of the gastrointestinal tract of fish and the potential application of prebiotics and probiotics in finfish aquaculture. \u003cem\u003eJournal of the World Aquaculture Society\u003c/em\u003e, 36(4), 425-436. doi:DOI 10.1111/j.1749-7345.2005.tb00390.x.\u003c/li\u003e\n \u003cli\u003eRayner, T. A., Hwang, J. S., \u0026amp; Hansen, B. W. (2017). Minimizing the use of fish oil enrichment in live feed by use of a self-enriching calanoid copepod. \u003cem\u003eJournal of Plankton Research\u003c/em\u003e, 39(6), 1004-1011. doi:10.1093/plankt/fbx021.\u003c/li\u003e\n \u003cli\u003eShields, R. J., Bell, J. G., Luizi, F. S., Gara, B., Bromage, N. R., \u0026amp; Sargent, J. R. (1999). Natural copepods are superior to enriched artemia nauplii as feed for halibut larvae (Hippoglossus hippoglossus) in terms of survival, pigmentation and retinal morphology: relation to dietary essential fatty acids. \u003cem\u003eJ Nutr\u003c/em\u003e, 129(6), 1186-94. doi:10.1093/jn/129.6.1186.\u003c/li\u003e\n \u003cli\u003eSanthanam, P., Muralisankar, T., Devi, K. N., Krishnaveni, N., Divya, M., Gowthami, A., et al. (2023). Algae materials. In K. Arunkumar, A. Arun, R. Raja, \u0026amp; R. Palaniappan (Eds.), \u003cem\u003eAlgae Materials\u003c/em\u003e (pp. 435-449). Academic Press. doi:10.1016/b978-0-443-18816-9.00001-0.\u003c/li\u003e\n \u003cli\u003eMitsuka, P. M., \u0026amp; Henry, R. (2002). The fate of copepod populations in the Paranapanema River (Sao Paulo, Brazil), downstream from the Jurumirim Dam. \u003cem\u003eBrazilian Archives of Biology and Technology\u003c/em\u003e, 45(4), 479-490. doi:Doi 10.1590/S1516-89132002000600012.\u003c/li\u003e\n \u003cli\u003eAlekseev, V. R., Miracle, M. R., \u0026amp; Vicente, E. (2020). Redescription of Acanthocyclops vernalis (Fischer, 1853) and Acanthocyclops robustus (Sars, 1863) from neotypes, with special reference to their distinction from Acanthocyclops americanus (Marsh, 1892) and its invasion of Eurasia. \u003cem\u003eLimnetica\u003c/em\u003e, 40(1), 57-78. doi:10.23818/limn.40.05.\u003c/li\u003e\n \u003cli\u003eMacias-Sancho, J., Poersch, L. H., Bauer, W., Romano, L. A., Wasielesky, W., \u0026amp; Tesser, M. B. (2014). Fishmeal substitution with Arthrospira (in a practical diet for: Effects on growth and immunological parameters. \u003cem\u003eAquaculture\u003c/em\u003e, 426, 120-125. doi:10.1016/j.aquaculture.2014.01.028.\u003c/li\u003e\n \u003cli\u003eCapelli, B., \u0026amp; Cysewski, G. R. (2010). Potential health benefits of spirulina microalgae*. \u003cem\u003eNutrafoods\u003c/em\u003e, 9(2), 19-26. doi:10.1007/bf03223332.\u003c/li\u003e\n \u003cli\u003eBengwayan, P. T., Laygo, J. C., Pacio, A. E., Poyaoan, J. L. Z., Rebugio, J. F., \u0026amp; Yuson, A. L. L. (2010). A comparative study on the antioxidant property of Chlorella (Chlorella sp.) tablet and glutathione tablet. \u003cem\u003eE-International Scientific Research Journal\u003c/em\u003e, 2(1), 25-35.\u003c/li\u003e\n \u003cli\u003eJuanita, U.-R., Dennis, A. H., \u0026amp; Michael, R. R. (1998). Analysis of copepod fecal pellet carbon using a high temperature combustion method. \u003cem\u003eMarine Ecology Progress Series\u003c/em\u003e, 171, 199-208. https://www.int-res.com/abstracts/meps/v171/p199-208/.\u003c/li\u003e\n \u003cli\u003eDayras, P., Bialais, C., Sadovskaya, I., Lee, M. C., Lee, J. S., \u0026amp; Souissi, S. (2021). Microalgal Diet Influences the Nutritive Quality and Reproductive Investment of the Cyclopoid Copepod (Original Research). \u003cem\u003eFrontiers in Marine Science\u003c/em\u003e, 8. doi:ARTN 6975610.3389/fmars.2021.697561.\u003c/li\u003e\n \u003cli\u003eArteaga Quico, C., Mariano Astocondor, M., \u0026amp; Aquino Ortega, R. (2021). Dietary supplementation with Chlorella peruviana improve the growth and innate immune response of rainbow trout Oncorhynchus mykiss fingerlings. \u003cem\u003eAquaculture\u003c/em\u003e, 533, 736117. doi:10.1016/j.aquaculture.2020.736117.\u003c/li\u003e\n \u003cli\u003eRaji, A. A., Alaba, P. A., Yusuf, H., Abu Bakar, N. H., Mohd Taufek, N., Muin, H., et al. (2018). Fishmeal replacement with Spirulina Platensis and Chlorella vulgaris in African catfish (Clarias gariepinus) diet: Effect on antioxidant enzyme activities and haematological parameters. \u003cem\u003eRes Vet Sci\u003c/em\u003e, 119, 67-75. doi:10.1016/j.rvsc.2018.05.013.\u003c/li\u003e\n \u003cli\u003eKang\u0026apos;ombe, J., Brown, J. A., \u0026amp; Halfyard, L. C. (2006). Effect of using different types of organic animal manure on plankton abundance, and on growth and survival of (Boulenger) in ponds. \u003cem\u003eAquaculture Research\u003c/em\u003e, 37(13), 1360-1371. doi:10.1111/j.1365-2109.2006.01569.x.\u003c/li\u003e\n \u003cli\u003eMunro, P. D., Barbour, A., \u0026amp; Birkbeck, T. H. (1994). Comparison of the Gut Bacterial-Flora of Start-Feeding Larval Turbot Reared under Different Conditions. \u003cem\u003eJournal of Applied Bacteriology\u003c/em\u003e, 77(5), 560-566. doi:DOI 10.1111/j.1365-2672.1994.tb04402.x.\u003c/li\u003e\n \u003cli\u003eSamat, N. A., Yusoff, F. M., Rasdi, N. W., \u0026amp; Karim, M. (2020). Enhancement of Live Food Nutritional Status with Essential Nutrients for Improving Aquatic Animal Health: A Review.\u003c/li\u003e\n \u003cli\u003eAshraf, M. (2009). Biotechnological approach of improving plant salt tolerance using antioxidants as markers. \u003cem\u003eBiotechnol Adv\u003c/em\u003e, 27(1), 84-93. doi:10.1016/j.biotechadv.2008.09.003.\u003c/li\u003e\n \u003cli\u003eSun, X. H., Liang, Z. L., Zou, J. X., \u0026amp; Wang, L. X. (2013). Seasonal variation in community structure and body length of dominant copepods around artificial reefs in Xiaoshi Island, China. \u003cem\u003eChinese Journal of Oceanology and Limnology\u003c/em\u003e, 31(2), 282-289. doi:10.1007/s00343-013-2088-0.\u003c/li\u003e\n \u003cli\u003eTalpur, M. A. D., \u0026amp; Ikhwanuddin, M. (2012). Effects of stress tests on larvae of blue swimming crab, Portunus pelagicus (Linnaeus, 1758). \u003cem\u003eAdvances in Environmental Biology\u003c/em\u003e, 6, 1909-1915.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable I.\u0026nbsp;\u003c/strong\u003eNutrition profile of zooplanktonic feeds used in aquaculture.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"764\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.219895287958115%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.99476439790576%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eProtein (\u003c/strong\u003e%\u003cstrong\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.31413612565445%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCarbohydrate (\u003c/strong\u003e%\u003cstrong\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.591623036649215%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eFat (\u003c/strong\u003e%\u003cstrong\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.031413612565444%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eDHA (\u003c/strong\u003e%\u003cstrong\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.37696335078534%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eEPA (\u003c/strong\u003e%\u003cstrong\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.722513089005235%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAsh (\u003c/strong\u003e%\u003cstrong\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.780104712041885%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eEnergy value (KJ/g)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.968586387434556%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eReference\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.219895287958115%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eArtemia\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.99476439790576%\" valign=\"top\"\u003e\n \u003cp\u003e55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.31413612565445%\" valign=\"top\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.591623036649215%\" valign=\"top\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.031413612565444%\" valign=\"top\"\u003e\n \u003cp\u003e2.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.37696335078534%\" valign=\"top\"\u003e\n \u003cp\u003e0.81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.722513089005235%\" valign=\"top\"\u003e\n \u003cp\u003e9.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.780104712041885%\" valign=\"top\"\u003e\n \u003cp\u003e18.97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.968586387434556%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eJohn et al., 2004,\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eMartinez et al., 2023\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.219895287958115%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eRotifer\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.99476439790576%\" valign=\"top\"\u003e\n \u003cp\u003e32.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.31413612565445%\" valign=\"top\"\u003e\n \u003cp\u003e14.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.591623036649215%\" valign=\"top\"\u003e\n \u003cp\u003e19.85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.031413612565444%\" valign=\"top\"\u003e\n \u003cp\u003e13.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.37696335078534%\" valign=\"top\"\u003e\n \u003cp\u003e9.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.722513089005235%\" valign=\"top\"\u003e\n \u003cp\u003e5.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.780104712041885%\" valign=\"top\"\u003e\n \u003cp\u003e17.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.968586387434556%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eJeeja et al., 2011,\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eHamre, 2016\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.219895287958115%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCopepods\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.99476439790576%\" valign=\"top\"\u003e\n \u003cp\u003e82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.31413612565445%\" valign=\"top\"\u003e\n \u003cp\u003e68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.591623036649215%\" valign=\"top\"\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.031413612565444%\" valign=\"top\"\u003e\n \u003cp\u003e34.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.37696335078534%\" valign=\"top\"\u003e\n \u003cp\u003e17.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.722513089005235%\" valign=\"top\"\u003e\n \u003cp\u003e9.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.780104712041885%\" valign=\"top\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.968586387434556%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSt\u0026oslash;ttrup., 2003, \u0026nbsp;Meeren et al., 2008\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u003c/strong\u003e Geolocation for zooplankton sample collection.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"419\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"47.01670644391408%\" valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eArea\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.821002386634845%\" valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eLatitude\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.162291169451073%\" valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eLongitude\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"47.01670644391408%\" valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eCauvery river\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.821002386634845%\"\u003e\n \u003cp\u003e10\u0026deg;50\u0026apos;15.4\u0026quot;N\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.162291169451073%\"\u003e\n \u003cp\u003e78\u0026deg;41\u0026apos;51.2\u0026quot;E\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"47.01670644391408%\" valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eMathur seasonal\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003elake\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.821002386634845%\"\u003e\n \u003cp\u003e10\u0026deg;41\u0026apos;40.8\u0026quot;N\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.162291169451073%\"\u003e\n \u003cp\u003e78\u0026deg;44\u0026apos;14.0\u0026quot;E\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"47.01670644391408%\" valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eGundur lake\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.821002386634845%\"\u003e\n \u003cp\u003e10\u0026deg;43\u0026apos;32.5\u0026quot;N\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.162291169451073%\"\u003e\n \u003cp\u003e78\u0026deg;43\u0026apos;36.6\u0026quot;E\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"47.01670644391408%\" valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eKumbakudi\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003epond\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.821002386634845%\"\u003e\n \u003cp\u003e10\u0026deg;42\u0026apos;14.5\u0026quot;N\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.162291169451073%\"\u003e\n \u003cp\u003e78\u0026deg;45\u0026apos;00.1\u0026quot;E\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"47.01670644391408%\" valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eBalancing\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ereservoir\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.821002386634845%\"\u003e\n \u003cp\u003e10\u0026deg;42\u0026apos;22.8\u0026quot;N\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.162291169451073%\"\u003e\n \u003cp\u003e78\u0026deg;49\u0026apos;00.1\u0026quot;E\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"47.01670644391408%\" valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eThuvakudi\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003evillage\u0026nbsp;pond\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.821002386634845%\"\u003e\n \u003cp\u003e10\u0026deg;45\u0026apos;04.5\u0026quot;N\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.162291169451073%\"\u003e\n \u003cp\u003e78\u0026deg;50\u0026apos;13.0\u0026quot;E\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3.\u0026nbsp;\u003c/strong\u003eZooplanktons from Trichy\u0026apos;s Cauvery River during monsoon 2018-19.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.61640798226164%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eZooplankton\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"77.38359201773837%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eMonths\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"34.95702005730659%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eNovember 2018\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"34.383954154727796%\" valign=\"top\" style=\"width: 19.3566%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDecember 2018\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.659025787965614%\" valign=\"top\" style=\"width: 23.0366%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eJanuary 2019\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.61640798226164%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eProtozoa\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.0509977827051%\" valign=\"top\"\u003e\n \u003cp\u003e224 \u0026plusmn; 5.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.607538802660756%\" valign=\"top\" style=\"width: 19.3566%;\"\u003e\n \u003cp\u003e401 \u0026plusmn; 4.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.725055432372507%\" valign=\"top\" style=\"width: 23.0366%;\"\u003e\n \u003cp\u003e221 \u0026plusmn; 4.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.61640798226164%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eRotifer\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.0509977827051%\" valign=\"top\"\u003e\n \u003cp\u003e533 \u0026plusmn; 5.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.607538802660756%\" valign=\"top\" style=\"width: 19.3566%;\"\u003e\n \u003cp\u003e494 \u0026plusmn; 5.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.725055432372507%\" valign=\"top\" style=\"width: 23.0366%;\"\u003e\n \u003cp\u003e408 \u0026plusmn; 5.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.61640798226164%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCopepoda\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.0509977827051%\" valign=\"top\"\u003e\n \u003cp\u003e1952 \u0026plusmn; 4.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.607538802660756%\" valign=\"top\" style=\"width: 19.3566%;\"\u003e\n \u003cp\u003e911 \u0026plusmn; 2.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.725055432372507%\" valign=\"top\" style=\"width: 23.0366%;\"\u003e\n \u003cp\u003e1983 \u0026plusmn; 5.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.61640798226164%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCladocera\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.0509977827051%\" valign=\"top\"\u003e\n \u003cp\u003e275 \u0026plusmn; 5.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.607538802660756%\" valign=\"top\" style=\"width: 19.3566%;\"\u003e\n \u003cp\u003e195 \u0026plusmn; 4.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.725055432372507%\" valign=\"top\" style=\"width: 23.0366%;\"\u003e\n \u003cp\u003e312 \u0026plusmn; 2.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.61640798226164%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eOstracoda\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.0509977827051%\" valign=\"top\"\u003e\n \u003cp\u003e101 \u0026plusmn; 3.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.607538802660756%\" valign=\"top\" style=\"width: 19.3566%;\"\u003e\n \u003cp\u003e86 \u0026plusmn; 2.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.725055432372507%\" valign=\"top\" style=\"width: 23.0366%;\"\u003e\n \u003cp\u003e123 \u0026plusmn; 5.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4.\u003c/strong\u003e Evaluation of the biosafety of copepods before use as live food organisms\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"617\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.883116883116884%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eBacterial isolates\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.707792207792206%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eGram positive\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.7012987012987%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eGram negative\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.707792207792206%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eHemolysis\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.933723196881093%\"\u003e\n \u003cp\u003e\u003cstrong\u003ecocci\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.933723196881093%\"\u003e\n \u003cp\u003e\u003cstrong\u003erods\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.230019493177387%\"\u003e\n \u003cp\u003e\u003cstrong\u003ecocci\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.230019493177387%\"\u003e\n \u003cp\u003e\u003cstrong\u003erods\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.89083820662768%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026alpha;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.89083820662768%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026beta;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.89083820662768%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026gamma;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.85575364667747%\" valign=\"top\"\u003e\n \u003cp\u003e36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.910858995137763%\" valign=\"top\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.910858995137763%\" valign=\"top\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.831442463533225%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.831442463533225%\" valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.886547811993516%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.886547811993516%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.886547811993516%\" valign=\"top\"\u003e\n \u003cp\u003e36\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Bioenrichment, Live feed culture, Acanthocyclops sp., Copepods, Bacillus subtilis","lastPublishedDoi":"10.21203/rs.3.rs-4674332/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4674332/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe article illustrates a thorough examination of the importance of live feed culture in aquaculture, especially in nurseries. Live feed is essential for aquaculture's sustainable development and ensuring a steady supply of fry and fingerlings. After being washed in sterilized water, copepods were bio-enriched with probiotic bacterial isolates (KAF061, 124, \u0026amp; 135) and commercial probiotics. A phase-contrast microscopic analysis confirmed the bioenrichment of copepods. We assessed the nutritional composition of the live feed culture using proximate analysis, revealing a greater protein content in microalgae, copepods, rotifers, and artemia compared to commercial fish feed. Based on these findings, the probiotic-rich live feed culture has a lot of potential for improving the nutritional content of fish, mollusks, and crustaceans that are still larvae. This could lead to better growth and survival rates for fry and fingerlings. These findings have significant implications for long-term aquaculture practices in developing low-cost and ecologically acceptable live feed alternatives for growth and survival.\u003c/p\u003e","manuscriptTitle":"Probiotic enrichment of copepod (Acanthocyclops sp.) towards improving fish survival, nutritional content, optimal growth and sustainability","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-30 07:22:12","doi":"10.21203/rs.3.rs-4674332/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"93c06861-43ed-4d98-8f4b-7bc52c80be15","owner":[],"postedDate":"July 30th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-10-16T22:08:23+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-30 07:22:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4674332","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4674332","identity":"rs-4674332","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}