Animal wastes-derived capped and bactericidal silver nanoparticles induce immuno-physiological responses in fish for multiple stress resilience in the One-Health Approach

Animal wastes-derived capped and bactericidal silver nanoparticles induce immuno-physiological responses in fish for multiple stress resilience in the One-Health Approach | 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 Animal wastes-derived capped and bactericidal silver nanoparticles induce immuno-physiological responses in fish for multiple stress resilience in the One-Health Approach Sowa o Lamare, K K Krishnani, Neeraj Kumar, Madhuri Pathak, Ajay Upadhyay, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4215264/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 present study elucidates the extracellular synthesis of capped silver nanoparticles using processing waste of sheep and swine by dispensing with addition of any capping agent and advantage of avoiding agglomeration and loss of nanosized characteristics of AgNPs. The synthesis of Ag-NPs was ascertained by UV-VIS spectrophotometry of yellowish-brown suspension at 400–410 nm. The Ag-NPs were further characterized using a HR-TEM, which confirmed that the Ag-NPs were primarily spherical and had a size range of 5-100 nm with a maximum frequency fall between 5–20 nm, 21–30 nm, 31–50 nm and a few falls within 51–100 nm. Ag-NPs synthesized using sheep and pig wastes are characterized by DLS, which confirmed the high stability of Ag-NPs with a zeta potential of -27 and − 32 mV respectively. Biomolecules and biological extracts of animal wastes act as biogenic reducing and capping agents. Based on the zone of inhibition, Ag-NPs biosynthesized using sheep waste showed high bactericidal properties against Aeromonas hydrophila, Edwardsiella tarda , and Micrococcus luteus as compared to swine waste-derived AgNPs. The chronic toxicity analysis of biosynthesized Ag-NPs on Pangasianodon hypophthalmus was carried out using stress biomarkers such as an antioxidant enzyme, AChE, and metabolic enzyme activity. Chronic toxicity of synthesized Ag-NPs was found to increase with increased sub-lethal ammonia concentration and temperature. The findings of this study revealed that biosynthesis of capped and non-agglomerated Ag-NPs can be undertaken by using animal wastes for their potential application in aquaculture based on the properties observed in characterization, bactericidal activity, and physiological responses of the fish. Sheep and Swine intestines Biosynthesis Colloidal silver nanoparticles Toxicity test on Pangasianodon hypophthalmus Bactericidal activity and physiological responses Multiple stress resilience Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Novel therapeutic approaches replacing inefficient antibiotics are in high demand to overcome increasing multidrug-resistant (MDR), extensively drug-resistant (XDR), and totally drug-resistant (TDR). According to the US Environmental Protection Agency (USEPA), nanomaterials are as particles of matter that is between 1 and 100 nanometres (nm) in diameter, where a unique phenomenon enables novel applications including various physicochemical properties (optical, electrical, magnetic, etc.), such as large surface area, high surface energy, and quantum confinement (Kumar and Seth 2021 ). Nanoparticles have unique properties due to their high specific surface area and a fraction of surface atoms, roughly 40–50%, and have an extraordinary potential for reactivity (Pournori et al. 2017 ). Therefore, nanomaterials have attracted much attention for their distinct properties unavailable in conventional macroscopic materials (Annamalai & Nallamuthu 2016 ). Due to their unique properties, nanomaterials have diverse applications in areas such as electronics, medical devices, cosmetics, food packaging, water treatment, fuel cells, biosensors, and environmental remediation (Chakraborty and Krishnani 2022 ). This has led to the production of nanomaterials on a large scale. Aquaculture nanotechnology can revolutionize the aquaculture field with its application to rapid disease detection, DNA vaccines, nutrient delivery, water filtration, water purification, water quality monitoring, construction materials, nanomedicine, and nano-sensors can be used to monitor aquaculture practices and improve fish health. Silver nanoparticles are one of the most exploited nanomaterials owing to their germicidal and anti-inflammatory properties; they are used in burn treatment, socks, detergents, soaps, water, and air filters, bedding, and other medical and industrial textiles (Bar-Ilan et al. 2009 ). About 30% of nanoproducts are known to contain silver nanoparticles (Khan et al. 2015 ). The stabilization of AgNPs becomes a challenging research area because of their highly active surface atoms, the aggregation, and deactivation by secondary nucleation and recrystallization and hence restricting their wide industrial applications. The majority of nanoparticle synthesis methods depend on the use of chemical capping agents like surfactants, polymers, and thiols (Niu and Li 2014 ), which have a strong interaction with the surface of AgNPs and hence act as good stabilizing agents. However, these chemical capping agents are nonbiodegradable, toxic, and difficult to detach from the surface of the nanoparticles. Furthermore, concerns about the materials produced by these expensive chemical and physical methods usually carry traces of toxic chemicals which could present catastrophic effects on the environment leading to biological hazards (Sidhu et al. 2022 ; Saeb et al. 2014 ). There is always a pressing need to explore green capping agents in order to secure the biological system and the environment (Sharma et al., 2019 ). Biogenic synthesis of nanoparticles is superior in comparison to the physical and chemical synthesis of nanoparticles as it is cost-effective, eco-friendly, and involvement of biomolecule-based capping agents like proteins, amino acids, lipids, and carbohydrates, which provide specific functional groups on the surface of nanoparticles (Das et al. 2023 ; Chakraborty et al. 2023 ). Various plant-derived products have been employed to synthesize nanomaterials (Sidhu et al. 2022 ). However, animal-based materials are rarely used for the biosynthesis of nanoparticles, despite the fact that biomolecules of animal wastes can serve as reducing and capping agents (Krishnani et al. 2022 ). There is a need to improve nanoparticle characteristics like small size, long-term stability, and biocompatibility and enhance antimicrobial activity by preventing agglomeration and controlling surface energy, dispersion, and electrostatic and steric hindrance. In the present scenario of climate change, the temperature of water bodies increases, which leads to increases in the toxicity of metallic and ammonical pollutants (Kumar et al. 2017 ; Abisha et al. 2022 ). In aquaculture, ammonia nitrogen is the final product of protein catabolism and metabolism, which is the most common environmental limiting factor (Arunkumar et al. 2023 ). Heavy metals such as cadmium are known to increase in toxicity with an increase in temperature (Guinot et al. 2012 ), which in turn affects the physiological function of the fish, such as thermal tolerance, growth, metabolism, food consumption, reproductive success, and the ability to maintain internal homeostasis are adversely affected. The contaminants of environmental concern, including heavy metals and metalloids, together with contaminants of emerging concern, including organic micropollutants, have been newly coined for the first time as contaminants of environmental and emerging concern (Krishnani et al. 2023 ). Excessive and non-judicial use of antibiotic agents has significantly increased the number of drug-resistant pathogens. When applied within the permissible limits of tissue retention, silver nanoparticles provide protection and benefit and do not pose any threat to environmental hazards (Chakraborty et al. 2013 ). Silver nanoparticles provide protection against Aeromonas veronii biovar sobria , high temperature, and Pb toxicity when used at 0.5 mg/kg feed (Kumar et al. 2018 ). The production of meat from farm to table produces waste at various stages. The processing procedure of animals such as cows, sheep, goats, pigs, chickens, and turkeys leaves litter such as bones, hides, and blood. The meat/edible portion of a cow accounts for 50–54%, sheep/goat 52%, pig 60–62%, chicken 68–72%, turkey 78%, and the other portion is turned into waste (Jha and Prasad 2016 ). The waste produced from the processing house or abattoir is left or thrown into the environment; if left unutilized, the waste creates unappealing and unhygienic surroundings. The use of certain animals and their byproducts for the synthesis of nanoparticles has been done, i.e., cockroaches (Jha and Prasad, 2013 ), fish scales of Labeo rohita (Sinha et al. 2014 ), cobweb (Lateef et al. 2016 ), goat fur (Akintayo et al. 2020 ). With only 52% of sheep and 60–62% of pig meat consumed for human consumption, effective utilization of the waste material to produce nano products via the biological method and an understanding of the product are required prior to its application. Several studies on the ecotoxicity of nanoparticles were undertaken on different species, like Labeo rohita, Lates calcarifer, Channa striatus, Pangasianodon hypophthalmus, Zebra fish , etc (Sarkar et al. 2014 ). Ecotoxicity analysis in the present study was carried out on Pangasianodon hypophthalmus fingerling as it is one of the most cultured species globally owing to its high growth rate, increased disease resistance, good taste, high resistance to poor water quality, and ability to survive in high stocking densities. A present study was carried out on sheep and swine waste (intestine) thrown out as a waste product for the synthesis of silver nanoparticles, and their characterization, bactericidal activity, and evaluation of the toxicity on the physiological stress response activity of Pangasianodon hypophthalmus under stressed environment. 2. Materials and methods 2.1 Green synthesis of silver nanoparticles (Ag-NPs) Pig rearing is integral to the way of life of a high tribal population in the North-Eastern States of India, where pig meat is considered an important food item (Roy et al. 2017 ). Animal waste, primarily intestine, was obtained from a Meghalaya slaughterhouse shortly after the animals were dissected. To remove undesired elements such as blood, undigested food, or any metallic contamination, the intestines were cleansed numerous times with water. 2.1.1 Preparation of extract from sheep and swine intestines To obtain an extract, a known quantity (2 gm) of sheep and pig processing waste was gently crushed in a porcelain mortar and pestle in the presence of 25 ml of distilled water, phosphate buffer (PB) and phosphate buffer saline (PBS) each to get a suspension as and when required. Filter paper and subsequently a syringe filter (0.45 µm) were used to filter the suspension/extract. The extract's pH was maintained at 7.2 by employing a NaOH solution, which was optimum for reducing agents and a stabilizer in the synthesis. The pH of the extract needs to be maintained because the process is affected by pH, which determines the size and shape of silver nanoparticles. 2.1.2 Biosynthesis of silver nanoparticles Sheep and pig intestine extracts were mixed with a 3 mM silver nitrate solution at 1:1, 1:2, 1:3, and 1:4 ratios and then kept in a rotating shaker for 3 hours at room temperature and then incubated overnight for complete reduction of the AgNO 3 . Once the suspensions turned yellowish-brown, the mixture was centrifuged at 9000 rpm for 15 minutes and rinsed three times with distilled water to eliminate any unconverted silver ions. The silver nanoparticles were gathered in a pellet and preserved for later investigation. 2.2 Characterization of silver nanoparticles The biosynthesized Ag-NPs were assessed using a High-resolution Transmission Electron Microscope (HR-TEM-JEOL-JEM 2100 F 120/200 kV) operating at an acceleration voltage of 200 kV, a Dynamic Light Scattering (DLS) particles size analyzer (Horiba Scientific Nanoparticles Analyzer nano Partica SZ-100 series, Kyoto, Japan), and UV–Visible spectrophotometer equipped with a 1-cm quartz cell. The plausible mechanisms between Ag-NPs and the functional groups present in the sheep and pig intestine extracts were predicted using the Fourier Transform Infrared (FTIR) spectrometer (ThermoFisher) using the potassium bromide (KBr) pellet technique. The sample was scanned from the 4,000 to 400 cm − 1 wave number. 2.3 Bactericidal activity An agar well diffusion method was used to test the bactericidal activity of Ag-NPs synthesized using extracts of sheep and pig wastes. The Ag-NPs were tested against Edwardsiella tarda (ATCC 15947, Ref 0845P, LOT 845-38-30, HiMedia, India) and Aeromonas hydrophilla (ATCC 49140, Lot No. 637-55-6, Ref No. 0637P, HiMedia, India) both Gram-negative bacteria and Micrococcus luteus , (ATCC 10240, lot 689-80-3, Ref 0689P, HiMedia, India) a gram-positive bacterium. Bacteria from overnight cultures were poured onto a sterile agar plate and left for solidification. Four wells were prepared using a sterile micropipette tip, of which two of the wells were filled with 50 µl (250 µg) and 100 µl (500 µg) of 5 mg of Ag-NPs/ml of sterile distilled water. As a control, 100 µl of streptomycin and 100 µl of sterile distilled water were added to the other two wells. After 24 hours of incubation at 37° C, the antibacterial capabilities of the produced Ag-NPs were assessed using the zone of inhibition around the well. 2.4 Experimental animal, design, and conditions for toxicity analysis of biosynthesized Ag-NPs Pangasianodon hypophthalmus fingerlings were procured from West Bengal. The fish were treated with a prophylactic dip in salt solution (2%) and then acclimatized for a period of two weeks prior to the start of the experiment. The experiment was carried out in a rectangular glass aquarium measuring 60’’× 30" × 30". The acclimatized Pangasianodon hypophthalmus fingerlings with an average weight of 8 grams were divided into five groups in triplicate, following a completely randomized design, with each group containing 10 fingerlings at ICAR-NIASM-Baramati. Throughout the course of the experiment, the fish were fed a practical diet (35% Protein) and non-stop aeration was provided to all the tanks by a compressed air pump. The five toxicity groups were designed as control (no exposure to Ag-NPs and temperature), T1 (Exposure to Ag-NPs), T2 (Concurrent exposure to Ag-NPs and NH3), T3 (Concurrent exposure to Ag-NPs and high temperature), and T4 (Concurrent exposure to Ag-NPs, NH 3 , and High temperature). The duration of the exposure was 21 days with 7 doses each containing 1/10th of the LC 50 , 1.8 ppm (NH 4 ) 2 SO 4, and a temperature at 34° C was maintained with a thermostatic heater. The water was exchanged manually by siphoning out two-thirds of the water every 72 hours, and then the doses were added. 2.5 Sample preparation for different biochemical parameters Gill, liver, kidney, and brain tissues of fish were collected from all experimental groups under aseptic conditions and weight. The tissues were homogenized (5% w/v) separately in chilled sucrose solution (0.25 M) in a glass tube using a Teflon-coated mechanical tissue homogenizer (Omni tissues master homogenize, Kennesaw, GA). The tubes were kept on ice to avoid denaturation of the enzymes during the homogenization. The homogenates were centrifuged at 5000 rpm for 20 min at 4°C in a cooling centrifuge (Eppendorf AG, 5430 R, Hamburg, Germany). Protein contents in the supernatants were quantified following the method of Lowry et al. ( 1951 ), using bovine serum albumin as a standard. The supernatants were collected and stored at -20 degrees Celsius until further analysis. 2.6 Antioxidant enzyme activities Superoxide dismutase (SOD) (EC 1.15.1.1) activity was measured by the method of Misra and Fridovich ( 1972 ). The assay was based on the oxidation of the epinephrine-adrenochrome transition by the enzyme. Solution of 1.5 ml carbonate bicarbonate buffer (0.1M; pH-10.2), 50 µl tissue homogenate and 0.5 ml of epinephrine substrate solutions (freshly prepared) were mixed well and the increase in absorbance was read at 480 nm for 3 min. Catalase (CAT) (EC 1.11.1.6) activity was determined by the method of Takahara et al. ( 1960 ). A solution of 2.45 ml of phosphate buffer (50 mM; pH-7), 50 µl of tissue homogenate, and 1 ml of hydrogen peroxide substrate solutions (Freshly prepared) was mixed well and the decrease in absorbance was read at 240 nm for 3 min. 2.7 Metabolic enzymes Aspartate aminotransaminase (EC.2.6.1.1) and alanine amino transaminase (EC.2.6.1.2) activities were measured using the Wootton method of 1964, where the concentration is estimated by the release of oxaloacetate and pyruvate, respectively. Lactate dehydrogenase (Lactate NAD1 oxidoreductase; EC.1.1.1.27) was assayed using (Wroblewski and LaDue 1955), in which 0.2 mM sodium pyruvate was used as a substrate and the reading was taken at an absorbance of 340 nm. Mate dehydrogenase (NAD + oxidoreductase: EC.1.1.1.37) was estimated employing the Ochoa method of 1955, where 1 mg of oxaloacetate/ml of chilled triple distilled water was used as a substrate. 2.8 Neurotransmitter enzyme activities Acetylcholinesterase (EC 3.1.1.7) was measured by the method of Hestrin ( 1949 ). The activity was spectrophotometrically measured as the increase in absorbance of the sample at 540 nm. 2.9 Statistical analysis The data were statistically analyzed by the statistical package for the social sciences (SPSS) version 16.0 (SPSS, Chicago, IL), in which the data were subjected to one-way ANOVA followed by Duncan's multiple range tests to determine the significant differences between the means. Comparisons were made at a 5% probability level. 3. Results 3.1. Synthesis of Ag NPs using animal wastes One Health approach integrates various components of the livestock-human-plant-environment interface to prevent health issues with the objective of achieving food and nutritional security, besides the surrounding environment, and social, economic, and political well-being (Krishnani et al. 2023 ). In this context, the use of by-products of one component for the well-being of another component can be an effective strategy in achieving the One-Health concept. In the present study, livestock wastes have been used for the synthesis of AgNPs for application in aquaculture. Silver nanoparticles (Ag-NPs) were synthesized using the collected intestine extracts of the Pig and Sheep in distilled water and buffers (phosphate buffer and phosphate buffer saline) and 3 mM of silver nitrate as the precursor in the ratios of 1:1 to 1:4. Change in colour of the suspension (intestine extract and silver nitrate) was observed after 24 hours of incubation, the colour varies from yellowish brown to dark brown which designates the formation of Ag-NPs. Synthesis of Ag NPs using sheep and pig waste was optimum at the ratios of 1:1 and 1:4 respectively, which clearly indicates that pig waste extract was a more effective reducing agent as compared to sheep waste extract. Hence further scaling-ups were done at these two ratios only for their large-scale use in experimental work. 3.2 Characterization of the synthesized particles The study elucidates the biosynthesis of Ag-NPs from sheep and pig processing waste, mainly the intestine, and their bactericidal activity and influence on the physiology of fish in the presence of other stressors. The mixture of animal intestine extract and silver nitrate turns yellowish-brown after overnight incubation, indicating the formation of Ag-NPs. The particle's zeta potential obtained via DLS shows that the particles have a zeta potential of -27 mV and − 32 mV as shown in Fig. 1 A. The UV spectrum of the Ag-NPs synthesized via a biological method using sheep and pig intestines as a reducing and capping agent was observed at a wavelength of 400–410 nm (Figure-2). The HR-TEM analysis is useful in nanoparticle characterization studies for determining the shape, size, and morphology of Ag-NPs. The size of the particles obtained using HRTEM ranges from 5-100 nm, with a maximum frequency of between 5–20 nm, and 20–30, and a few falling within 50–100 nm and more than 100 nm (Figs. 3 and 4 ). The magnified image of HR-TEM confirmed that the Ag-NPs were capped with biomolecules and uniform and primarily spherical in shape with the average particle size ranging from 10 ± 5 to 80 ± 20 nm, without significant agglomeration. The lowest-sized range of silver nanoparticles achieved was synthesized from distilled water and phosphate buffer from the intestine of sheep followed by the pig. The Ag NPs synthesized from the pig intestine had more nanoparticles in the range of 30–50 nm, whereas Ag NPs synthesized from the sheep intestine ranged from 5–20 nm followed by 21–30 nm. Proteins present in synthesized AgNPs may be responsible for the efficient capping and stabilization of nanoparticles and this was further confirmed by the FTIR spectrum. In the present study, the FTIR spectrum shows absorption bands at 3271, 2918–2922, 2849, 1579–1632, 1514–1537, 1454, 1397–1403, 1329, 1236, and 1029–1052 cm − 1 indicating the presence of a capping agent with the nanoparticles (Figure-5). The broad band at 3271 cm − 1 in the spectra corresponds to NH amide stretching vibration indicating the presence of amino acids/protein. Bands at 2918–2922 and 2849 cm − 1 region arose from C–H stretching. The band at 1579–1632 cm − 1 in the spectra corresponds to C–N and C–C stretching indicating the presence of proteins (Prakash et al., 2013 ). The weaker band at 1579–1632 cm − 1 corresponds to the amide I (NH) C = O group arising due to carbonyl stretch in proteins. The band at 1454 cm − 1 was assigned for N–H stretch vibration present in the amide linkages of the proteins. These functional groups have a role in the stability/capping of AgNP as reported previously (Niraimathi et al., 2013 , Prakash et al., 2013 ). The bands at 1236 and 1029–1052 cm − 1 were assigned for the C–N (amines) stretch vibration of the proteins. The FTIR spectrum indicated the presence of protein in samples of silver nanoparticles, which further confirms that the secondary structure of proteins is not affected because of their interaction with Ag + ions or nanoparticles. It has been reported by Nicholas et al. (2010) that proteins can bind to nanoparticles either through their free amine groups or cysteine residues. Thus, AgNPs are stabilized and capped by proteins. 3.2 Antioxidant status In Figure-6, the antioxidant status (SOD and catalase) of the gills and liver of Pangasianodon hypophthalmus fingerlings exposed to Ag-NPs individually or concurrently with NH 3 and high-temperature are illustrated. The antioxidant enzyme increases significantly ( p < 0.05) after successful exposure to different toxicity groups as compared to the control. The highest antioxidant enzyme activity was observed in the toxicity group exposed to the combined effect of Ag-NPs, NH 3 , and high temperature, followed by the group exposed to the concurrent effect of Ag-NPs and high temperature, and then Ag-NPs individually in the case of catalase enzyme. The SOD activity showed no significant difference ( p < 0.05) between the toxicity groups exposed to Ag-NPs individually and combined with NH 3 or high temperature, except for the group exposed concurrently to Ag-NPs, NH 3, and high temperature. 3.3 Metabolic enzymes The metabolic stress enzyme activity in terms of alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and malate dehydrogenase (MDH) of Pangasianodon hypophthalmus fingerling is illustrated in Figs. 7 and 8 . The activities of ALT (Figure-7A) and AST (Figure-7B) in the liver and kidneys of the treatment group increased significantly ( p < 0.05) as compared to the control. The highest AST and ALT activities of the liver and kidney were observed in the group exposed to the combined effect of Ag-NPs, ammonia, and high temperature. The groups exposed to the other treatments do not show significant differences ( p < 0.05) between each other in terms of the ALT activity of the liver. In the case of the kidney, the most elevated treatment group was followed by the group exposed to Ag-NPs and high temperature, the group exposed to Ag-NPs and NH 3 , and then Ag-NPs individually. The LDH and MDH (Figures- 8 A) activities of the liver increased significantly ( p < 0.05) in the toxicity group as compared to the control. The group that was exposed to the combined effect of Ag-NPs, NH 3 , and high temperatures had the most elevated activity. The group exposed to Ag-NPs individually or combined with NH 3 or at high temperature showed no significant differences ( p < 0.05) between each other in terms of LDH and MDH activity in the liver. 3.4 Neurotransmitter enzyme The neurotransmitter activity in the form of acetylcholinesterase (AChE) in the brain of Pangasianodon hypophthalmus is illustrated in Figure-8B. Brain AChE activities were noticeably inhibited ( p < 0.05) in the group exposed to Ag-NPs individually or concurrently with ammonia and temperature, or both, as compared to control. The highest inhibition was observed in the group exposed to the concurrent effects of Ag-NPs, NH 3, and high temperatures. The inhibition effect between the groups exposed to Ag-NPs and NH 3 and the group exposed to Ag-NPs and high temperature did not show a significant difference ( p < 0.05). The lowest inhibition was observed in the group treated with Ag-NPs individually. 3.5 Bactericidal activity As shown in Fig. 9 , Ag-NPs synthesized using sheep waste in DW, PB and PBS have high bactericidal properties against the tested gram-negative ( A. hydrophila , and E. tarda) and gram-positive bacteria ( M. luteus) , as compared pig waste derived AgNPs. The zone of inhibition (in mm) at 50 µl (250 µg) and 100 µl (500 µg) of 5 mg of pig and sheep waste mediated Ag-NPs in DW, PB and PBS per ml of sterile distilled water are given in Table-1. Gram-negative Aeromonas hydrophila and Edwardsiella tarda have been shown to have the highest sensitivity against the biosynthesized Ag-NPs as compared to the Gram-positive bacteria Micrococcus luteus . The bactericidal activity of AgNPs is influenced by their size and shape and oxidative stress induction and the release of silver ions, which thus induces a viable but non-culturable state (VBNC) in silver-exposed bacteria or bacterial death (de Silva et al. 2021 ; Konigs et al. 2015 ). Table 1. Bactericidal properties of Ag-NPs synthesized from swine/pig and sheep intestines Zone of inhibition (in mm) Swine Intestine mediated AgNPs SI. No Content M. luteus A. hydrophila E. tarda 1 100 µl Ag-NPs PBS 11.6 10 12 PB 10 11 13 DW 12 15 14 2 50 µl Ag-NPs PBS 9 6 8 PB 6 7.6 8.6 DW 10 13 13 Sheep Intestine mediated Ag NPs 1 100 µl Ag-NPs PBS 14 15 19 PB 14 19 16 DW 16 20 22 2 50 µl Ag-NPs PBS 9 10 16 PB 10 15 11 DW 10 15 18 (PBS-phosphate buffer saline, PB-Phosphate buffer, and DW-Distilled water) 4. Discussion Pig species such as yolk shire, ham shire, and Landrace are among the most cultured species for human consumption globally. Pigs are euryphages in nature as they consume a large variety of foods that may contain various contaminants such as heavy metals, pesticides, etc., so they are equipped with multiple mechanisms to overcome different stressors. The biosynthesis of Ag-NPs from the pig intestine may be accomplished by the activity of a protein known as metallothionein. Metallothionein is a cysteine-rich metal-binding protein present in all eukaryotes that play an essential role in intracellular metal distribution, and accumulation, and as a reducing functional group for reducing metal ions, chelation, and accumulation of metal particles in the cells (Yuan et al. 2019 ). Jha and Prasad ( 2016 ) investigated the formation of ZnO nanoparticles using goat waste as a reducing and capping agent, leading to the conclusion that "from insects to mammals, metallothionein genes are induced in response to heavy metal load, and we can emphatically perceive that even in the case of dead animal tissues, their crucial molecules are thermodynamically flexible to liberate or absorb energy. In this scenario, the liberated energy is most likely responsible for the microscale to nanoscale phase transformation. In the present study, the colour of the suspension after 24 hours of incubation was yellow-brown, which could be due to the excitation of surface plasmon resonance of the synthesized Ag-NPs. The variations in colour, if compared with other findings for the biosynthesized Ag-NPs, are due to the composition of biomolecules responsible for reducing silver nitrate to silver nanoparticles (Akintayo et al. 2020 ). The zeta potential of the synthesized Ag-NPs from sheep waste and pig waste was − 27 mV and − 32 mV respectively, which showed that the synthesized Ag-NPs were highly stable. A Zeta potential of greater than 30 mV or less than − 30 mV is indicative of a stable system (Abdelmoteleb et al. 2017 ). The size of the Ag-NPs obtained via DLS is larger than the size obtained via HRTEM due to the method employed as the DLS measured the hydrodynamic radius (Saha et al. 2017 ). Biomolecules like proteins, amino acids, lipids, and carbohydrates provide specific functional groups on the surface of nanoparticles (Marisca et al. 2019). These biomolecules act as capping agents that prevent agglomeration and steric hindrance, alter the biological activity and surface chemistry and stabilize the interaction of nanoparticles within the preparation medium. The various biogenic capping agents, including biomolecules and biological extracts of plants and microorganisms, have been highlighted (Sidhu et al. 2022 ). Several reports are in line with the findings of the present study. Kakakhel et al. (2020) reported a cheaper and eco-friendly protocol for synthesizing silver nanoparticles from animal blood where the UV spectrum was 422 nm and the achieved size of the Ag-NPs was 20–50 nm. Kumar et al. ( 2018 ) synthesized silver nanoparticles from the gills of Channa Striata and reported that the average size obtained via DLS was 297 nm and the mean zeta potential was − 34 mV. Another study in line with the present finding is the synthesis of silver nanoparticles from E. coli transformed with the Candida albicans metallothionein gene. The production of silver nanoparticles is more from the transformed bacteria than from the non-transformed bacteria. The biosynthesis of other metallic nanoparticles, such as zinc nanoparticles, has been elucidated in which the biomolecules responsible for the reduction of silver nitrate to silver nanoparticles were metallothionein (Jha and Prasad 2016 ). The toxicity of AgNPs on Anabas testudineus was evaluated, determining a 96-h LC 50 value of 25.46 mg l − 1 (Chakraborty et al. 2023 ) and analysis of physiological data and integrated biomarker responses reveal that concentrations of 1/10th, 1/25th, and 1/50th of the LC 50 can induce stress in the fish, while exposure to 1/100th of the LC 50 shows minimal to no stress response. The toxicity of silver nanoparticles in the presence of sub-lethal ammonia and high temperatures was concluded, meaning that silver nanoparticle toxicity in the presence of other abiotic stressors increases. The stress tolerance of aquatic organisms is greatly influenced by contaminants such as heavy metals, ammonia species, pesticides, etc. (Kumar et al. 2018 ). The increased concentration of pollutants and the temperature rise induce stress on the aquatic organisms, which is reflected in terms of elevated antioxidant enzymes, protein, carbohydrate metabolic enzymes, etc. (Kumar et al. 2018 ). In our present study, the antioxidant enzyme (Catalase and SOD) activities were elevated, succeeding the exposure to different toxicity groups. The increase in activity may be because the Ag-NPs, individually or concurrently with either NH 3 or temperature, or both, induce stress on the fingerling, which leads to the production of antioxidants to control the excessive production of reactive oxygen species. The stress on the fingerling contributes to the metallic nature of the nanoparticles, and the presence of transition metals increases the production of reactive oxygen species (ROS), resulting in oxidative stress (Rajkumar et al. 2015 ). The transaminase enzymes (ALT & AST) in the present study were greatly influenced by the Ag-NPs. ALT and AST are used as biomarkers for stress induced by contaminants by several authors (Kumar et al. 2018 , 2017 ; Reddy et al. 2012). In an aquatic organism, transaminase enzyme activity increases during stress conditions. The increase in the activities of AST and ALT might be due to the mobilization of aspartate and alanine through gluconeogenesis for glucose production to cope with the induced stress. Increased transaminase activity levels may also be attributed to cellular damage, increased plasma membrane permeability, or altered metabolism of enzymes. Increased transaminase activities indicate an adaptive physiological response to combat energy demand (Reddy et al. 2012). LDH and MDH catalyze the oxidation of malate and lactate to pyruvate at a strategic point between glycolysis and the citric acid cycle, serving in the terminal step of glycolysis (Reddy et al. 2012). In our study, the carbohydrate enzymes are elevated following exposure to different toxicity groups. The increase in activity might be accounted for because lactate and malate serve as the primary substrate for gluconeogenesis during anaerobic metabolism for glucose production to meet the energy demand during stress, which led to the higher activity of the LDH and MDH. The increased activity of LDH and MDH during stress has been reported by Kumar et al. 2018 ; Reddy et al. 2012; Defo et al. 2019 ). Acetylcholine esterase is an essential enzyme in the modulation of neuromuscular impulse transmission; the central role of AChE is to separate both acetylcholine and the cholinergic signal molecule from its receptors in the plasma membrane (Myrzakhanova et al. 2013 ). AChE has been employed as a stress biomarker for pollution or contaminants in the aquatic environment. The activity of AChE is known to be inhibited in the presence of pollutants such as heavy metals (Kumar et al. 2017 , 2018 ; Ahmad et al. 2016 ; Hayat et al. 2017 ; Muthappa et al. 2014 ; Gupta et al. 2014 ). The present work's neurotransmitter enzyme acetylcholine esterase (AChE) was drastically inhibited upon subsequent exposure to Ag-NPs, individually or concurrently with ammonia and high treatment. The inhibition of AChE may be due to the metal ions binding to the terminal-OH and-SH functional groups as they bind with the allosteric sites that cause the conformational changes, making the substrate fail to bind at the specific site of the enzymes (Kumar et al. 2016; Hayat et al. 2017 ; Ahmad et al. 2016 ). The synthesized silver nanoparticles at a concentration of 50 µl (250 µg) and 100 µl (500 µg) of 5 mg of Ag-NPs/ml of sterile distilled water exhibited good antibacterial activity against gram-negative and gram-positive bacteria. The bactericidal properties of the biosynthesized Ag-NPs have been linked to the interaction of Ag-NPs with sulfur and phosphorous-containing constituents of the bacterial cell, which initiates cell killing by attacking the respiratory chain and cell division (Mahendra et al. 2009). The difference in the inhibition zone formation of Ag-NPs might be due to the cell wall component of bacteria (Chaloupka et al. 2010 ) and the particle size of the synthesized Ag-NPs (Awwad et al. 2020 ). Gram-negative bacteria lack the thick peptidoglycan layer, causing bacterial cell wall destruction easily as compared to Gram-positive bacteria which have thick protective peptidoglycan layer (Slavin et al. 2017 ). Bactericidal activity of recombinant Elastin-like biopolymer composed of a polyhistidine domain with Ag + against Gram-negative bacteria prevalent in coastal shrimp aquaculture has successfully been demonstrated by Krishnani et al. ( 2014 ). In the present study, the zone of inhibition of AgNPs was found to be bigger in the cases of Gram-negative bacteria prevalent in freshwater aquaculture as compared to Gram-positive bacteria. The known antibacterial mechanisms of Ag NPs are the physical direct interaction of extremely sharp edges of AgNPs with cell wall membrane; ROS generation; Trapping the bacteria within the aggregated AgNPs; Oxidative stress; Interruption in the glycolysis process of the bacteria; DNA damaging; Ion release; and inducing the viable but non-culturable state of pathogens (VBNC) (Krishnani et al. 2022 ). 5. Conclusion The present study concluded that silver nanoparticle synthesis can be achieved by using processed waste from abattoirs, mainly the intestine of sheep and pigs, with the conviction that waste from animals can effectively be used and that waste from one point can be a resource for another point in the circular bioresource utilization. The synthesized silver nanoparticles show good bactericidal properties against different Gram-positive and Gram-negative bacteria. Further, the toxicity analysis has also provided insight into the effect of co-stressors on the toxicity of silver nanoparticles. The chronic toxicity analysis of the biosynthesized Ag-NPs on fish carried out using stress biomarkers was found to increase with increased sub-lethal ammonia concentration and temperature. Biomolecules present in animal wastes are an excellent source of distinctive functional groups that have been utilized in modulating the surface of the nanoparticles due to the availability of natural binding sites. Overall, it can be concluded from the study that the waste generated after the slaughter of a sheep and pig, primarily the intestine, can be utilized to produce silver nanoparticles. With the increasing resistance of bacteria to antibiotics, biosynthesized Ag-NPs if used within the optimum concentration, have the potential for application in the health management of aquaculture species as they have exhibited antibacterial properties without any adverse effect on the fish. Ag NPs are one of the promising candidates as alternatives to synthetic antibiotics, for preventing antimicrobial resistance in the aquatic environment. Scaling-up of silver nanoparticle synthesis using animal waste extracts is worthy to be researched further to mitigate climate change-induced stresses for developing climate resilience in fish and to prevent antimicrobial resistance in One-Health Fisheries and other sectors of agriculture. Declarations Acknowledgments The authors are grateful to the Directors of the ICAR-Central Institute of Fisheries Education and the ICAR-National Institute of Abiotic Stress Management for providing their encouragement to carry out the research work and providing an Institute project to the corresponding author. Ethics statement The use of animals conforms to the existing laws in India. The care and treatment of animals used in this study were in accordance with the guidelines of the CPCSEA [(Committee for the Purpose of Control and Supervision of Experiments on Animals, (Ministry of Environment & Forests (Animal Welfare Division), Government of India] on the care and use of animals in scientific research. The study protocol and experimental endpoints were approved by the institutional research committee (Board of Studies) and the authority of the ICAR Institute (MA-09-12, M.F.Sc. synopsis approved by Board of Studies, and degree awarded by the University in September 2021). Funding ICAR is gratefully acknowledged for awarding a fellowship to the student and providing an Institute project to the corresponding author. Competing Interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Author Contributions Mr. Sowa o Lamare and Dr. Krishnani contributed to the study’s conception and design. Material Preparation, data collection, and analysis were performed by Sowa o Lamare and Dr. Krishnani. The first draft of the manuscript was written by Sowa and Krishnani and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Name of authors Contribution of authors Mr. Sowa o Lamare Data curation; Execution of Experimental work; Validation; Data interpretation, Compilation, and preparation. Dr. K K Krishnani Conceptualization; Synthesis and experimental protocols, Outline & design, Overall guidance, Review & Editing Dr. Neeraj Kumar Methodology, Inputs related to enzyme activity; Physiological responses of fish, Review & Editing Dr. Madhuri Pathak Inputs related to pathogenic bacteria, Review & Editing Dr Ajay Upadhyay Inputs related to methodology, Review Dr. Biplab Sarkar Inputs related to Ag NPs, FTIR, Review & Editing Dr A K Verma Inputs related to abiotic stresses, Review & Editing Ms Puja Chakraborty Inputs related to biotic stresses, Review & Editing Dr NK Chadha Inputs related to application in aquaculture, Review & Editing Availability of data and materials All data generated or analyzed during this study are included in this article. The data that support the findings of this study are available from the corresponding author. 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Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine, 90(1), 210–213. https://doi.org/10.3181/00379727-90-21985. Yuan, Q., Bomma, M., & Xiao, Z. (2019). Enhanced silver nanoparticle synthesis by Escherichia coli transformed with Candida albicans Metallothioneins Gene. Materials, 12(24), 4180. https://doi.org/10.3390/ma12244180. 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-4215264","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":291063087,"identity":"f3e87409-01c2-46f1-b1c3-ece296dd8ea9","order_by":0,"name":"Sowa o Lamare","email":"","orcid":"","institution":"ICAR-CIFE-Central Institute of Fisheries Education (Deemed University","correspondingAuthor":false,"prefix":"","firstName":"Sowa","middleName":"o","lastName":"Lamare","suffix":""},{"id":291063089,"identity":"e6a79432-ca24-4e7e-a413-ad7271fbe8ef","order_by":1,"name":"K K Krishnani","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABC0lEQVRIie2OsWrDMBCGzxzIyxWvEg5+BgWBTCHQZ8kbdMyQBoPB3pLVfZcOAkG8FDoXOqQENLcUCl1K5eJ0KCJpt1L0Lfo53cf9AJHIH4QjsOGVMAbK8OszqX6miPqUAgdlDCDNiWKiTd3u+QZU6cP+cvkwUT0+vizgqpAGm11AyZHKaedATyyVqts60pYpcQu9kiZpZUApkFhOBmb8MzDrFQJRwXZ+XSUNDyqpG5UhvFtSNeHbMSVH0IOi+RDOGkvSn/NXlvMNhBVRkxad4coX88raErdMn1fSqAzDCr/rHX8ys2mX9b7Yq73INnZ/Xy1WBUtbF1IO5veBtAAY2jzC6pf7kUgk8o/5AEg+TfpMRTb9AAAAAElFTkSuQmCC","orcid":"","institution":"ICAR-Indian Institute of Agricultural Biotechnology","correspondingAuthor":true,"prefix":"","firstName":"K","middleName":"K","lastName":"Krishnani","suffix":""},{"id":291063090,"identity":"af4d45e4-7a78-49ce-bc74-9b830c6ac36c","order_by":2,"name":"Neeraj Kumar","email":"","orcid":"","institution":"ICAR-National Institute of Abiotic Stress Management","correspondingAuthor":false,"prefix":"","firstName":"Neeraj","middleName":"","lastName":"Kumar","suffix":""},{"id":291063091,"identity":"4b835fc8-2e47-4786-a074-7597bf9397be","order_by":3,"name":"Madhuri Pathak","email":"","orcid":"","institution":"ICAR-CIFE-Central Institute of Fisheries Education (Deemed University","correspondingAuthor":false,"prefix":"","firstName":"Madhuri","middleName":"","lastName":"Pathak","suffix":""},{"id":291063093,"identity":"33adfa8a-5873-483a-8a8d-ff2e4336e76c","order_by":4,"name":"Ajay Upadhyay","email":"","orcid":"","institution":"ICAR-National Research Centre for Grapes, Manjiri Farm","correspondingAuthor":false,"prefix":"","firstName":"Ajay","middleName":"","lastName":"Upadhyay","suffix":""},{"id":291063096,"identity":"fc1c72e4-0cd9-475b-a033-c5fb8f798050","order_by":5,"name":"Biplab Sarkar","email":"","orcid":"","institution":"ICAR-Indian Institute of Agricultural Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Biplab","middleName":"","lastName":"Sarkar","suffix":""},{"id":291063097,"identity":"7f301436-b2bc-40cb-8c0d-1a685fc167c7","order_by":6,"name":"AK Verma","email":"","orcid":"","institution":"ICAR-CIFE-Central Institute of Fisheries Education (Deemed University","correspondingAuthor":false,"prefix":"","firstName":"AK","middleName":"","lastName":"Verma","suffix":""},{"id":291063098,"identity":"204edd35-4a0d-4e93-9d09-5209f15f35d7","order_by":7,"name":"Puja Chakraborty","email":"","orcid":"","institution":"ICAR-CIFE-Central Institute of Fisheries Education (Deemed University","correspondingAuthor":false,"prefix":"","firstName":"Puja","middleName":"","lastName":"Chakraborty","suffix":""},{"id":291063099,"identity":"900ac959-0f05-4064-b6f2-5ff4b079a2b0","order_by":8,"name":"NK Chadha","email":"","orcid":"","institution":"ICAR-CIFE-Central Institute of Fisheries Education (Deemed University","correspondingAuthor":false,"prefix":"","firstName":"NK","middleName":"","lastName":"Chadha","suffix":""}],"badges":[],"createdAt":"2024-04-04 01:59:45","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4215264/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4215264/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":54737761,"identity":"f888048b-8bbb-41ef-a446-c5aa60ca4c42","added_by":"auto","created_at":"2024-04-16 04:48:23","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":190615,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of the synthesized Ag-NPs using DLS - Zeta potential\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4215264/v1/224d06535773d7b8b6b457a9.png"},{"id":54737760,"identity":"13350142-3017-459a-b22c-3fb818257e29","added_by":"auto","created_at":"2024-04-16 04:48:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":70612,"visible":true,"origin":"","legend":"\u003cp\u003eUV-Visible spectra of Ag-NPs synthesized using sheep and pig wastes\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4215264/v1/c07c1f567eea9d326aedbb24.png"},{"id":54738060,"identity":"0c4860aa-a200-4e74-be52-479d4b609181","added_by":"auto","created_at":"2024-04-16 04:56:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":534953,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of the synthesized sheep-waste mediated Ag-NPs using HR-TEM. (A). DW, (B). PB, (C). PBS\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4215264/v1/ee83c16eaec90996395172a5.png"},{"id":54738061,"identity":"2b14498f-a6ed-4b0e-a5d0-219888d50628","added_by":"auto","created_at":"2024-04-16 04:56:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":503646,"visible":true,"origin":"","legend":"\u003cp\u003eFigure-3: Characterization of pig-waste mediated Ag-NPs using HR-TEM. (A). DW, (B). PB, (C). PBS\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4215264/v1/076bbc4e01bad58264be62c2.png"},{"id":54737767,"identity":"b12f593a-1089-4aa1-b9ef-b7844f2adb5f","added_by":"auto","created_at":"2024-04-16 04:48:23","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":172429,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR of biosynthesized silver nanoparticles stabilized by animal waste proteins\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4215264/v1/e1ee2bf73b27628da555f47a.png"},{"id":54737763,"identity":"dc04ca53-4201-47ad-b5c1-730e5e3cc953","added_by":"auto","created_at":"2024-04-16 04:48:23","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":53839,"visible":true,"origin":"","legend":"\u003cp\u003e6(A). Catalase activity and 6(B). SOD activity in the liver and gills of \u003cem\u003ePangasianodon hypophthalmus \u003c/em\u003eexposed to different toxicity groups of Ag-NPs. Different letters above the bar indicate significant differences.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4215264/v1/0836573b970784a174adc0fb.png"},{"id":54737765,"identity":"507d83ad-cd6d-402a-baa9-aca501d02972","added_by":"auto","created_at":"2024-04-16 04:48:23","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":67988,"visible":true,"origin":"","legend":"\u003cp\u003e7(A). ALT, and 7(B). AST activity in liver and kidney of \u003cem\u003ePangasianodon hypophthalmus \u003c/em\u003eexposed to different toxicity groups of Ag-NPs. Different letters above the bar indicate significant difference.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4215264/v1/69c44097f3c7f1ea57e6f071.png"},{"id":54737766,"identity":"1e032b91-c64a-4a81-a9b7-6a11fbbbc86c","added_by":"auto","created_at":"2024-04-16 04:48:23","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":58330,"visible":true,"origin":"","legend":"\u003cp\u003e8(A). LDH and MDH activity in the liver and 8(B). AChE activity in the brain of \u003cem\u003ePangasianodon hypophthalmus \u003c/em\u003eexposed to different toxicity groups of Ag-NPs. Different letters above the bar indicate significant differences.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-4215264/v1/03725a5230ceb467aa82f2b3.png"},{"id":54737768,"identity":"9ac3d678-9234-4fac-b0e8-6f43bc8169bb","added_by":"auto","created_at":"2024-04-16 04:48:23","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1094355,"visible":true,"origin":"","legend":"\u003cp\u003eBactericidal activity of synthesized Ag-NPs against \u0026nbsp;(9A) \u003cem\u003eM. luteus, \u003c/em\u003e(9B)\u003cem\u003e A. hydrophila,\u003c/em\u003e and (9C) \u003cem\u003eE. tarda \u003c/em\u003e(S-Streptomycin, DW-sterile Distilled water, 50 µl (250 µg) and 100 µl (500 µg) of 5 mg AgNPs/ml sterile distilled water).\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-4215264/v1/c49a1abb248b1e0e6b18ac34.png"},{"id":55265263,"identity":"253a129f-9da1-4bca-8a75-9f7b84e96442","added_by":"auto","created_at":"2024-04-25 02:00:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3487655,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4215264/v1/09e77864-4cc8-4aff-9cc2-49d5255646ee.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Animal wastes-derived capped and bactericidal silver nanoparticles induce immuno-physiological responses in fish for multiple stress resilience in the One-Health Approach","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eNovel therapeutic approaches replacing inefficient antibiotics are in high demand to overcome increasing multidrug-resistant (MDR), extensively drug-resistant (XDR), and totally drug-resistant (TDR). According to the US Environmental Protection Agency (USEPA), nanomaterials are as particles of matter that is between 1 and 100 nanometres (nm) in diameter, where a unique phenomenon enables novel applications including various physicochemical properties (optical, electrical, magnetic, etc.), such as large surface area, high surface energy, and quantum confinement (Kumar and Seth \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNanoparticles have unique properties due to their high specific surface area and a fraction of surface atoms, roughly 40\u0026ndash;50%, and have an extraordinary potential for reactivity (Pournori et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Therefore, nanomaterials have attracted much attention for their distinct properties unavailable in conventional macroscopic materials (Annamalai \u0026amp; Nallamuthu \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Due to their unique properties, nanomaterials have diverse applications in areas such as electronics, medical devices, cosmetics, food packaging, water treatment, fuel cells, biosensors, and environmental remediation (Chakraborty and Krishnani \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This has led to the production of nanomaterials on a large scale. Aquaculture nanotechnology can revolutionize the aquaculture field with its application to rapid disease detection, DNA vaccines, nutrient delivery, water filtration, water purification, water quality monitoring, construction materials, nanomedicine, and nano-sensors can be used to monitor aquaculture practices and improve fish health.\u003c/p\u003e \u003cp\u003eSilver nanoparticles are one of the most exploited nanomaterials owing to their germicidal and anti-inflammatory properties; they are used in burn treatment, socks, detergents, soaps, water, and air filters, bedding, and other medical and industrial textiles (Bar-Ilan et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). About 30% of nanoproducts are known to contain silver nanoparticles (Khan et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The stabilization of AgNPs becomes a challenging research area because of their highly active surface atoms, the aggregation, and deactivation by secondary nucleation and recrystallization and hence restricting their wide industrial applications. The majority of nanoparticle synthesis methods depend on the use of chemical capping agents like surfactants, polymers, and thiols (Niu and Li \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), which have a strong interaction with the surface of AgNPs and hence act as good stabilizing agents. However, these chemical capping agents are nonbiodegradable, toxic, and difficult to detach from the surface of the nanoparticles. Furthermore, concerns about the materials produced by these expensive chemical and physical methods usually carry traces of toxic chemicals which could present catastrophic effects on the environment leading to biological hazards (Sidhu et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Saeb et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). There is always a pressing need to explore green capping agents in order to secure the biological system and the environment (Sharma et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Biogenic synthesis of nanoparticles is superior in comparison to the physical and chemical synthesis of nanoparticles as it is cost-effective, eco-friendly, and involvement of biomolecule-based capping agents like proteins, amino acids, lipids, and carbohydrates, which provide specific functional groups on the surface of nanoparticles (Das et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Chakraborty et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Various plant-derived products have been employed to synthesize nanomaterials (Sidhu et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, animal-based materials are rarely used for the biosynthesis of nanoparticles, despite the fact that biomolecules of animal wastes can serve as reducing and capping agents (Krishnani et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). There is a need to improve nanoparticle characteristics like small size, long-term stability, and biocompatibility and enhance antimicrobial activity by preventing agglomeration and controlling surface energy, dispersion, and electrostatic and steric hindrance.\u003c/p\u003e \u003cp\u003eIn the present scenario of climate change, the temperature of water bodies increases, which leads to increases in the toxicity of metallic and ammonical pollutants (Kumar et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Abisha et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In aquaculture, ammonia nitrogen is the final product of protein catabolism and metabolism, which is the most common environmental limiting factor (Arunkumar et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Heavy metals such as cadmium are known to increase in toxicity with an increase in temperature (Guinot et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), which in turn affects the physiological function of the fish, such as thermal tolerance, growth, metabolism, food consumption, reproductive success, and the ability to maintain internal homeostasis are adversely affected. The contaminants of environmental concern, including heavy metals and metalloids, together with contaminants of emerging concern, including organic micropollutants, have been newly coined for the first time as contaminants of environmental and emerging concern (Krishnani et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Excessive and non-judicial use of antibiotic agents has significantly increased the number of drug-resistant pathogens. When applied within the permissible limits of tissue retention, silver nanoparticles provide protection and benefit and do not pose any threat to environmental hazards (Chakraborty et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Silver nanoparticles provide protection against \u003cem\u003eAeromonas veronii biovar sobria\u003c/em\u003e, high temperature, and Pb toxicity when used at 0.5 mg/kg feed (Kumar et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe production of meat from farm to table produces waste at various stages. The processing procedure of animals such as cows, sheep, goats, pigs, chickens, and turkeys leaves litter such as bones, hides, and blood. The meat/edible portion of a cow accounts for 50\u0026ndash;54%, sheep/goat 52%, pig 60\u0026ndash;62%, chicken 68\u0026ndash;72%, turkey 78%, and the other portion is turned into waste (Jha and Prasad \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The waste produced from the processing house or abattoir is left or thrown into the environment; if left unutilized, the waste creates unappealing and unhygienic surroundings. The use of certain animals and their byproducts for the synthesis of nanoparticles has been done, i.e., cockroaches (Jha and Prasad, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), fish scales of \u003cem\u003eLabeo rohita\u003c/em\u003e (Sinha et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), cobweb (Lateef et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), goat fur (Akintayo et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWith only 52% of sheep and 60\u0026ndash;62% of pig meat consumed for human consumption, effective utilization of the waste material to produce nano products via the biological method and an understanding of the product are required prior to its application. Several studies on the ecotoxicity of nanoparticles were undertaken on different species, like \u003cem\u003eLabeo rohita, Lates calcarifer, Channa striatus, Pangasianodon hypophthalmus, Zebra fish\u003c/em\u003e, etc (Sarkar et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Ecotoxicity analysis in the present study was carried out on \u003cem\u003ePangasianodon hypophthalmus\u003c/em\u003e fingerling as it is one of the most cultured species globally owing to its high growth rate, increased disease resistance, good taste, high resistance to poor water quality, and ability to survive in high stocking densities. A present study was carried out on sheep and swine waste (intestine) thrown out as a waste product for the synthesis of silver nanoparticles, and their characterization, bactericidal activity, and evaluation of the toxicity on the physiological stress response activity of \u003cem\u003ePangasianodon hypophthalmus\u003c/em\u003e under stressed environment.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Green synthesis of silver nanoparticles (Ag-NPs)\u003c/h2\u003e \u003cp\u003ePig rearing is integral to the way of life of a high tribal population in the North-Eastern States of India, where pig meat is considered an important food item (Roy et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Animal waste, primarily intestine, was obtained from a Meghalaya slaughterhouse shortly after the animals were dissected. To remove undesired elements such as blood, undigested food, or any metallic contamination, the intestines were cleansed numerous times with water.\u003c/p\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003e2.1.1 Preparation of extract from sheep and swine intestines\u003c/h2\u003e \u003cp\u003eTo obtain an extract, a known quantity (2 gm) of sheep and pig processing waste was gently crushed in a porcelain mortar and pestle in the presence of 25 ml of distilled water, phosphate buffer (PB) and phosphate buffer saline (PBS) each to get a suspension as and when required. Filter paper and subsequently a syringe filter (0.45 \u0026micro;m) were used to filter the suspension/extract. The extract's pH was maintained at 7.2 by employing a NaOH solution, which was optimum for reducing agents and a stabilizer in the synthesis. The pH of the extract needs to be maintained because the process is affected by pH, which determines the size and shape of silver nanoparticles.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.1.2 Biosynthesis of silver nanoparticles\u003c/h2\u003e \u003cp\u003eSheep and pig intestine extracts were mixed with a 3 mM silver nitrate solution at 1:1, 1:2, 1:3, and 1:4 ratios and then kept in a rotating shaker for 3 hours at room temperature and then incubated overnight for complete reduction of the AgNO\u003csub\u003e3\u003c/sub\u003e. Once the suspensions turned yellowish-brown, the mixture was centrifuged at 9000 rpm for 15 minutes and rinsed three times with distilled water to eliminate any unconverted silver ions. The silver nanoparticles were gathered in a pellet and preserved for later investigation.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Characterization of silver nanoparticles\u003c/h2\u003e \u003cp\u003eThe biosynthesized Ag-NPs were assessed using a High-resolution Transmission Electron Microscope (HR-TEM-JEOL-JEM 2100 F 120/200 kV) operating at an acceleration voltage of 200 kV, a Dynamic Light Scattering (DLS) particles size analyzer (Horiba Scientific Nanoparticles Analyzer nano Partica SZ-100 series, Kyoto, Japan), and UV\u0026ndash;Visible spectrophotometer equipped with a 1-cm quartz cell. The plausible mechanisms between Ag-NPs and the functional groups present in the sheep and pig intestine extracts were predicted using the Fourier Transform Infrared (FTIR) spectrometer (ThermoFisher) using the potassium bromide (KBr) pellet technique. The sample was scanned from the 4,000 to 400 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e wave number.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Bactericidal activity\u003c/h2\u003e \u003cp\u003eAn agar well diffusion method was used to test the bactericidal activity of Ag-NPs synthesized using extracts of sheep and pig wastes. The Ag-NPs were tested against \u003cem\u003eEdwardsiella tarda\u003c/em\u003e (ATCC 15947, Ref 0845P, LOT 845-38-30, HiMedia, India) and \u003cem\u003eAeromonas hydrophilla\u003c/em\u003e (ATCC 49140, Lot No. 637-55-6, Ref No. 0637P, HiMedia, India) both Gram-negative bacteria and \u003cem\u003eMicrococcus luteus\u003c/em\u003e, (ATCC 10240, lot 689-80-3, Ref 0689P, HiMedia, India) a gram-positive bacterium. Bacteria from overnight cultures were poured onto a sterile agar plate and left for solidification. Four wells were prepared using a sterile micropipette tip, of which two of the wells were filled with 50 \u0026micro;l (250 \u0026micro;g) and 100 \u0026micro;l (500 \u0026micro;g) of 5 mg of Ag-NPs/ml of sterile distilled water. As a control, 100 \u0026micro;l of streptomycin and 100 \u0026micro;l of sterile distilled water were added to the other two wells. After 24 hours of incubation at 37\u0026deg; C, the antibacterial capabilities of the produced Ag-NPs were assessed using the zone of inhibition around the well.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Experimental animal, design, and conditions for toxicity analysis of biosynthesized Ag-NPs\u003c/h2\u003e \u003cp\u003e \u003cem\u003ePangasianodon hypophthalmus\u003c/em\u003e fingerlings were procured from West Bengal. The fish were treated with a prophylactic dip in salt solution (2%) and then acclimatized for a period of two weeks prior to the start of the experiment. The experiment was carried out in a rectangular glass aquarium measuring 60\u0026rsquo;\u0026rsquo;\u0026times; 30\" \u0026times; 30\". The acclimatized \u003cem\u003ePangasianodon hypophthalmus\u003c/em\u003e fingerlings with an average weight of 8 grams were divided into five groups in triplicate, following a completely randomized design, with each group containing 10 fingerlings at ICAR-NIASM-Baramati. Throughout the course of the experiment, the fish were fed a practical diet (35% Protein) and non-stop aeration was provided to all the tanks by a compressed air pump. The five toxicity groups were designed as control (no exposure to Ag-NPs and temperature), T1 (Exposure to Ag-NPs), T2 (Concurrent exposure to Ag-NPs and NH3), T3 (Concurrent exposure to Ag-NPs and high temperature), and T4 (Concurrent exposure to Ag-NPs, NH\u003csub\u003e3\u003c/sub\u003e, and High temperature). The duration of the exposure was 21 days with 7 doses each containing 1/10th of the LC\u003csub\u003e50\u003c/sub\u003e, 1.8 ppm (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4,\u003c/sub\u003e and a temperature at 34\u0026deg; C was maintained with a thermostatic heater. The water was exchanged manually by siphoning out two-thirds of the water every 72 hours, and then the doses were added.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Sample preparation for different biochemical parameters\u003c/h2\u003e \u003cp\u003eGill, liver, kidney, and brain tissues of fish were collected from all experimental groups under aseptic conditions and weight. The tissues were homogenized (5% w/v) separately in chilled sucrose solution (0.25 M) in a glass tube using a Teflon-coated mechanical tissue homogenizer (Omni tissues master homogenize, Kennesaw, GA). The tubes were kept on ice to avoid denaturation of the enzymes during the homogenization. The homogenates were centrifuged at 5000 rpm for 20 min at 4\u0026deg;C in a cooling centrifuge (Eppendorf AG, 5430 R, Hamburg, Germany). Protein contents in the supernatants were quantified following the method of Lowry et al. (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1951\u003c/span\u003e), using bovine serum albumin as a standard. The supernatants were collected and stored at -20 degrees Celsius until further analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Antioxidant enzyme activities\u003c/h2\u003e \u003cp\u003eSuperoxide dismutase (SOD) (EC 1.15.1.1) activity was measured by the method of Misra and Fridovich (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1972\u003c/span\u003e). The assay was based on the oxidation of the epinephrine-adrenochrome transition by the enzyme. Solution of 1.5 ml carbonate bicarbonate buffer (0.1M; pH-10.2), 50 \u0026micro;l tissue homogenate and 0.5 ml of epinephrine substrate solutions (freshly prepared) were mixed well and the increase in absorbance was read at 480 nm for 3 min. Catalase (CAT) (EC 1.11.1.6) activity was determined by the method of Takahara et al. (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e1960\u003c/span\u003e). A solution of 2.45 ml of phosphate buffer (50 mM; pH-7), 50 \u0026micro;l of tissue homogenate, and 1 ml of hydrogen peroxide substrate solutions (Freshly prepared) was mixed well and the decrease in absorbance was read at 240 nm for 3 min.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Metabolic enzymes\u003c/h2\u003e \u003cp\u003eAspartate aminotransaminase (EC.2.6.1.1) and alanine amino transaminase (EC.2.6.1.2) activities were measured using the Wootton method of 1964, where the concentration is estimated by the release of oxaloacetate and pyruvate, respectively. Lactate dehydrogenase (Lactate NAD1 oxidoreductase; EC.1.1.1.27) was assayed using (Wroblewski and LaDue 1955), in which 0.2 mM sodium pyruvate was used as a substrate and the reading was taken at an absorbance of 340 nm. Mate dehydrogenase (NAD\u0026thinsp;+\u0026thinsp;oxidoreductase: EC.1.1.1.37) was estimated employing the Ochoa method of 1955, where 1 mg of oxaloacetate/ml of chilled triple distilled water was used as a substrate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Neurotransmitter enzyme activities\u003c/h2\u003e \u003cp\u003eAcetylcholinesterase (EC 3.1.1.7) was measured by the method of Hestrin (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1949\u003c/span\u003e). The activity was spectrophotometrically measured as the increase in absorbance of the sample at 540 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Statistical analysis\u003c/h2\u003e \u003cp\u003eThe data were statistically analyzed by the statistical package for the social sciences (SPSS) version 16.0 (SPSS, Chicago, IL), in which the data were subjected to one-way ANOVA followed by Duncan's multiple range tests to determine the significant differences between the means. Comparisons were made at a 5% probability level.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1. Synthesis of Ag NPs using animal wastes\u003c/h2\u003e\n \u003cp\u003eOne Health approach integrates various components of the livestock-human-plant-environment interface to prevent health issues with the objective of achieving food and nutritional security, besides the surrounding environment, and social, economic, and political well-being (Krishnani et al. \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e). In this context, the use of by-products of one component for the well-being of another component can be an effective strategy in achieving the One-Health concept. In the present study, livestock wastes have been used for the synthesis of AgNPs for application in aquaculture. Silver nanoparticles (Ag-NPs) were synthesized using the collected intestine extracts of the Pig and Sheep in distilled water and buffers (phosphate buffer and phosphate buffer saline) and 3 mM of silver nitrate as the precursor in the ratios of 1:1 to 1:4. Change in colour of the suspension (intestine extract and silver nitrate) was observed after 24 hours of incubation, the colour varies from yellowish brown to dark brown which designates the formation of Ag-NPs. Synthesis of Ag NPs using sheep and pig waste was optimum at the ratios of 1:1 and 1:4 respectively, which clearly indicates that pig waste extract was a more effective reducing agent as compared to sheep waste extract. Hence further scaling-ups were done at these two ratios only for their large-scale use in experimental work.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 Characterization of the synthesized particles\u003c/h2\u003e\n \u003cp\u003eThe study elucidates the biosynthesis of Ag-NPs from sheep and pig processing waste, mainly the intestine, and their bactericidal activity and influence on the physiology of fish in the presence of other stressors. The mixture of animal intestine extract and silver nitrate turns yellowish-brown after overnight incubation, indicating the formation of Ag-NPs. The particle\u0026apos;s zeta potential obtained via DLS shows that the particles have a zeta potential of -27 mV and \u0026minus;\u0026thinsp;32 mV as shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA. The UV spectrum of the Ag-NPs synthesized via a biological method using sheep and pig intestines as a reducing and capping agent was observed at a wavelength of 400\u0026ndash;410 nm (Figure-2).\u003c/p\u003e\n \u003cp\u003eThe HR-TEM analysis is useful in nanoparticle characterization studies for determining the shape, size, and morphology of Ag-NPs. The size of the particles obtained using HRTEM ranges from 5-100 nm, with a maximum frequency of between 5\u0026ndash;20 nm, and 20\u0026ndash;30, and a few falling within 50\u0026ndash;100 nm and more than 100 nm (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). The magnified image of HR-TEM confirmed that the Ag-NPs were capped with biomolecules and uniform and primarily spherical in shape with the average particle size ranging from 10\u0026thinsp;\u0026plusmn;\u0026thinsp;5 to 80\u0026thinsp;\u0026plusmn;\u0026thinsp;20 nm, without significant agglomeration. The lowest-sized range of silver nanoparticles achieved was synthesized from distilled water and phosphate buffer from the intestine of sheep followed by the pig. The Ag NPs synthesized from the pig intestine had more nanoparticles in the range of 30\u0026ndash;50 nm, whereas Ag NPs synthesized from the sheep intestine ranged from 5\u0026ndash;20 nm followed by 21\u0026ndash;30 nm.\u003c/p\u003e\n \u003cp\u003eProteins present in synthesized AgNPs may be responsible for the efficient capping and stabilization of nanoparticles and this was further confirmed by the FTIR spectrum. In the present study, the FTIR spectrum shows absorption bands at 3271, 2918\u0026ndash;2922, 2849, 1579\u0026ndash;1632, 1514\u0026ndash;1537, 1454, 1397\u0026ndash;1403, 1329, 1236, and 1029\u0026ndash;1052 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e indicating the presence of a capping agent with the nanoparticles (Figure-5). The broad band at 3271 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in the spectra corresponds to NH amide stretching vibration indicating the presence of amino acids/protein. Bands at 2918\u0026ndash;2922 and 2849 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e region arose from C\u0026ndash;H stretching. The band at 1579\u0026ndash;1632 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in the spectra corresponds to C\u0026ndash;N and C\u0026ndash;C stretching indicating the presence of proteins (Prakash et al., \u003cspan class=\"CitationRef\"\u003e2013\u003c/span\u003e). The weaker band at 1579\u0026ndash;1632 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e corresponds to the amide I (NH) C\u0026thinsp;=\u0026thinsp;O group arising due to carbonyl stretch in proteins. The band at 1454 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was assigned for N\u0026ndash;H stretch vibration present in the amide linkages of the proteins. These functional groups have a role in the stability/capping of AgNP as reported previously (Niraimathi et al., \u003cspan class=\"CitationRef\"\u003e2013\u003c/span\u003e, Prakash et al., \u003cspan class=\"CitationRef\"\u003e2013\u003c/span\u003e). The bands at 1236 and 1029\u0026ndash;1052 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e were assigned for the C\u0026ndash;N (amines) stretch vibration of the proteins. The FTIR spectrum indicated the presence of protein in samples of silver nanoparticles, which further confirms that the secondary structure of proteins is not affected because of their interaction with Ag\u003csup\u003e+\u003c/sup\u003e ions or nanoparticles. It has been reported by Nicholas et al. (2010) that proteins can bind to nanoparticles either through their free amine groups or cysteine residues. Thus, AgNPs are stabilized and capped by proteins.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 Antioxidant status\u003c/h2\u003e\n \u003cp\u003eIn Figure-6, the antioxidant status (SOD and catalase) of the gills and liver of \u003cem\u003ePangasianodon hypophthalmus\u003c/em\u003e fingerlings exposed to Ag-NPs individually or concurrently with NH\u003csub\u003e3\u003c/sub\u003e and high-temperature are illustrated. The antioxidant enzyme increases significantly (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) after successful exposure to different toxicity groups as compared to the control. The highest antioxidant enzyme activity was observed in the toxicity group exposed to the combined effect of Ag-NPs, NH\u003csub\u003e3\u003c/sub\u003e, and high temperature, followed by the group exposed to the concurrent effect of Ag-NPs and high temperature, and then Ag-NPs individually in the case of catalase enzyme. The SOD activity showed no significant difference (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) between the toxicity groups exposed to Ag-NPs individually and combined with NH\u003csub\u003e3\u003c/sub\u003e or high temperature, except for the group exposed concurrently to Ag-NPs, NH\u003csub\u003e3,\u003c/sub\u003e and high temperature.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3 Metabolic enzymes\u003c/h2\u003e\n \u003cp\u003eThe metabolic stress enzyme activity in terms of alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and malate dehydrogenase (MDH) of \u003cem\u003ePangasianodon hypophthalmus\u003c/em\u003e fingerling is illustrated in Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e. The activities of ALT (Figure-7A) and AST (Figure-7B) in the liver and kidneys of the treatment group increased significantly (\u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05) as compared to the control. The highest AST and ALT activities of the liver and kidney were observed in the group exposed to the combined effect of Ag-NPs, ammonia, and high temperature. The groups exposed to the other treatments do not show significant differences (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) between each other in terms of the ALT activity of the liver. In the case of the kidney, the most elevated treatment group was followed by the group exposed to Ag-NPs and high temperature, the group exposed to Ag-NPs and NH\u003csub\u003e3\u003c/sub\u003e, and then Ag-NPs individually.\u003c/p\u003e\n \u003cp\u003eThe LDH and MDH (Figures-\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eA) activities of the liver increased significantly (\u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05) in the toxicity group as compared to the control. The group that was exposed to the combined effect of Ag-NPs, NH\u003csub\u003e3\u003c/sub\u003e, and high temperatures had the most elevated activity. The group exposed to Ag-NPs individually or combined with NH\u003csub\u003e3\u003c/sub\u003e or at high temperature showed no significant differences (\u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05) between each other in terms of LDH and MDH activity in the liver.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4 Neurotransmitter enzyme\u003c/h2\u003e\n \u003cp\u003eThe neurotransmitter activity in the form of acetylcholinesterase (AChE) in the brain of \u003cem\u003ePangasianodon hypophthalmus\u003c/em\u003e is illustrated in Figure-8B. Brain AChE activities were noticeably inhibited (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the group exposed to Ag-NPs individually or concurrently with ammonia and temperature, or both, as compared to control. The highest inhibition was observed in the group exposed to the concurrent effects of Ag-NPs, NH\u003csub\u003e3,\u003c/sub\u003e and high temperatures. The inhibition effect between the groups exposed to Ag-NPs and NH\u003csub\u003e3\u003c/sub\u003e and the group exposed to Ag-NPs and high temperature did not show a significant difference (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The lowest inhibition was observed in the group treated with Ag-NPs individually.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003e3.5 Bactericidal activity\u003c/h2\u003e\n \u003cp\u003eAs shown in Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e, Ag-NPs synthesized using sheep waste in DW, PB and PBS have high bactericidal properties against the tested gram-negative (\u003cem\u003eA. hydrophila\u003c/em\u003e, and \u003cem\u003eE. tarda)\u003c/em\u003e and gram-positive bacteria (\u003cem\u003eM. luteus)\u003c/em\u003e, as compared pig waste derived AgNPs. The zone of inhibition (in mm) at 50 \u0026micro;l (250 \u0026micro;g) and 100 \u0026micro;l (500 \u0026micro;g) of 5 mg of pig and sheep waste mediated Ag-NPs in DW, PB and PBS per ml of sterile distilled water are given in Table-1. Gram-negative \u003cem\u003eAeromonas hydrophila\u003c/em\u003e and \u003cem\u003eEdwardsiella tarda\u003c/em\u003e have been shown to have the highest sensitivity against the biosynthesized Ag-NPs as compared to the Gram-positive bacteria \u003cem\u003eMicrococcus luteus\u003c/em\u003e. The bactericidal activity of AgNPs is influenced by their size and shape and oxidative stress induction and the release of silver ions, which thus induces a viable but non-culturable state (VBNC) in silver-exposed bacteria or bacterial death (de Silva et al. \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e; Konigs et al. \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eTable 1. Bactericidal properties of Ag-NPs synthesized from swine/pig and sheep intestines\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"564\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"99.46808510638297%\" colspan=\"11\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Zone of inhibition (in mm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0.5319148936170213%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"99.46808510638297%\" colspan=\"11\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Swine Intestine mediated AgNPs\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0.5319148936170213%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.479573712255773%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSI. No\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"32.85968028419183%\" colspan=\"4\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eContent\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.00532859680284%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eM. luteus\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.00532859680284%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eA. hydrophila\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.117229129662523%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eE. tarda\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0.5328596802841918%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.479573712255773%\" rowspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.136767317939608%\" colspan=\"2\" rowspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e100 \u0026micro;l Ag-NPs\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.72291296625222%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePBS\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.00532859680284%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e11.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.00532859680284%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.117229129662523%\" valign=\"top\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0.5328596802841918%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.142857142857142%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePB\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.792207792207794%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.792207792207794%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.493506493506494%\" valign=\"top\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0.7792207792207793%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.142857142857142%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eDW\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.792207792207794%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.792207792207794%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.493506493506494%\" valign=\"top\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0.7792207792207793%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.479573712255773%\" rowspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.136767317939608%\" colspan=\"2\" rowspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e50 \u0026micro;l Ag-NPs\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.72291296625222%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePBS\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.00532859680284%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.00532859680284%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.117229129662523%\" valign=\"top\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0.5328596802841918%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.142857142857142%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePB\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.792207792207794%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.792207792207794%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e7.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.493506493506494%\" valign=\"top\"\u003e\n \u003cp\u003e8.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0.7792207792207793%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.142857142857142%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eDW\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.792207792207794%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.792207792207794%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.493506493506494%\" valign=\"top\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0.7792207792207793%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"12\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSheep Intestine mediated Ag NPs\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.504424778761061%\" colspan=\"2\" rowspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.353982300884955%\" colspan=\"2\" rowspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e100 \u0026micro;l Ag-NPs\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.68141592920354%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePBS\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.4070796460177%\" valign=\"top\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.4070796460177%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.646017699115045%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.142857142857142%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePB\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.01298701298701%\" valign=\"top\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.01298701298701%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.83116883116883%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.142857142857142%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eDW\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.01298701298701%\" valign=\"top\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.01298701298701%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.83116883116883%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.504424778761061%\" colspan=\"2\" rowspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.353982300884955%\" colspan=\"2\" rowspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e50 \u0026micro;l Ag-NPs\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.68141592920354%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePBS\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.4070796460177%\" valign=\"top\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.4070796460177%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.646017699115045%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.142857142857142%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePB\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.01298701298701%\" valign=\"top\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.01298701298701%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.83116883116883%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.142857142857142%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eDW\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.01298701298701%\" valign=\"top\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.01298701298701%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.83116883116883%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e(PBS-phosphate buffer saline, PB-Phosphate buffer, and DW-Distilled water)\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003ePig species such as yolk shire, ham shire, and Landrace are among the most cultured species for human consumption globally. Pigs are euryphages in nature as they consume a large variety of foods that may contain various contaminants such as heavy metals, pesticides, etc., so they are equipped with multiple mechanisms to overcome different stressors. The biosynthesis of Ag-NPs from the pig intestine may be accomplished by the activity of a protein known as metallothionein. Metallothionein is a cysteine-rich metal-binding protein present in all eukaryotes that play an essential role in intracellular metal distribution, and accumulation, and as a reducing functional group for reducing metal ions, chelation, and accumulation of metal particles in the cells (Yuan et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Jha and Prasad (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) investigated the formation of ZnO nanoparticles using goat waste as a reducing and capping agent, leading to the conclusion that \"from insects to mammals, metallothionein genes are induced in response to heavy metal load, and we can emphatically perceive that even in the case of dead animal tissues, their crucial molecules are thermodynamically flexible to liberate or absorb energy. In this scenario, the liberated energy is most likely responsible for the microscale to nanoscale phase transformation.\u003c/p\u003e \u003cp\u003eIn the present study, the colour of the suspension after 24 hours of incubation was yellow-brown, which could be due to the excitation of surface plasmon resonance of the synthesized Ag-NPs. The variations in colour, if compared with other findings for the biosynthesized Ag-NPs, are due to the composition of biomolecules responsible for reducing silver nitrate to silver nanoparticles (Akintayo et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The zeta potential of the synthesized Ag-NPs from sheep waste and pig waste was \u0026minus;\u0026thinsp;27 mV and \u0026minus;\u0026thinsp;32 mV respectively, which showed that the synthesized Ag-NPs were highly stable. A Zeta potential of greater than 30 mV or less than \u0026minus;\u0026thinsp;30 mV is indicative of a stable system (Abdelmoteleb et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The size of the Ag-NPs obtained via DLS is larger than the size obtained via HRTEM due to the method employed as the DLS measured the hydrodynamic radius (Saha et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Biomolecules like proteins, amino acids, lipids, and carbohydrates provide specific functional groups on the surface of nanoparticles (Marisca et al. 2019). These biomolecules act as capping agents that prevent agglomeration and steric hindrance, alter the biological activity and surface chemistry and stabilize the interaction of nanoparticles within the preparation medium. The various biogenic capping agents, including biomolecules and biological extracts of plants and microorganisms, have been highlighted (Sidhu et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Several reports are in line with the findings of the present study. Kakakhel et al. (2020) reported a cheaper and eco-friendly protocol for synthesizing silver nanoparticles from animal blood where the UV spectrum was 422 nm and the achieved size of the Ag-NPs was 20\u0026ndash;50 nm. Kumar et al. (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) synthesized silver nanoparticles from the gills of \u003cem\u003eChanna Striata\u003c/em\u003e and reported that the average size obtained via DLS was 297 nm and the mean zeta potential was \u0026minus;\u0026thinsp;34 mV. Another study in line with the present finding is the synthesis of silver nanoparticles from \u003cem\u003eE. coli\u003c/em\u003e transformed with the \u003cem\u003eCandida albicans\u003c/em\u003e metallothionein gene. The production of silver nanoparticles is more from the transformed bacteria than from the non-transformed bacteria. The biosynthesis of other metallic nanoparticles, such as zinc nanoparticles, has been elucidated in which the biomolecules responsible for the reduction of silver nitrate to silver nanoparticles were metallothionein (Jha and Prasad \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe toxicity of AgNPs on \u003cem\u003eAnabas testudineus\u003c/em\u003e was evaluated, determining a 96-h LC\u003csub\u003e50\u003c/sub\u003e value of 25.46 mg l\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Chakraborty et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and analysis of physiological data and integrated biomarker responses reveal that concentrations of 1/10th, 1/25th, and 1/50th of the LC\u003csub\u003e50\u003c/sub\u003e can induce stress in the fish, while exposure to 1/100th of the LC\u003csub\u003e50\u003c/sub\u003e shows minimal to no stress response. The toxicity of silver nanoparticles in the presence of sub-lethal ammonia and high temperatures was concluded, meaning that silver nanoparticle toxicity in the presence of other abiotic stressors increases. The stress tolerance of aquatic organisms is greatly influenced by contaminants such as heavy metals, ammonia species, pesticides, etc. (Kumar et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The increased concentration of pollutants and the temperature rise induce stress on the aquatic organisms, which is reflected in terms of elevated antioxidant enzymes, protein, carbohydrate metabolic enzymes, etc. (Kumar et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In our present study, the antioxidant enzyme (Catalase and SOD) activities were elevated, succeeding the exposure to different toxicity groups. The increase in activity may be because the Ag-NPs, individually or concurrently with either NH\u003csub\u003e3\u003c/sub\u003e or temperature, or both, induce stress on the fingerling, which leads to the production of antioxidants to control the excessive production of reactive oxygen species. The stress on the fingerling contributes to the metallic nature of the nanoparticles, and the presence of transition metals increases the production of reactive oxygen species (ROS), resulting in oxidative stress (Rajkumar et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe transaminase enzymes (ALT \u0026amp; AST) in the present study were greatly influenced by the Ag-NPs. ALT and AST are used as biomarkers for stress induced by contaminants by several authors (Kumar et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Reddy et al. 2012). In an aquatic organism, transaminase enzyme activity increases during stress conditions. The increase in the activities of AST and ALT might be due to the mobilization of aspartate and alanine through gluconeogenesis for glucose production to cope with the induced stress. Increased transaminase activity levels may also be attributed to cellular damage, increased plasma membrane permeability, or altered metabolism of enzymes. Increased transaminase activities indicate an adaptive physiological response to combat energy demand (Reddy et al. 2012).\u003c/p\u003e \u003cp\u003eLDH and MDH catalyze the oxidation of malate and lactate to pyruvate at a strategic point between glycolysis and the citric acid cycle, serving in the terminal step of glycolysis (Reddy et al. 2012). In our study, the carbohydrate enzymes are elevated following exposure to different toxicity groups. The increase in activity might be accounted for because lactate and malate serve as the primary substrate for gluconeogenesis during anaerobic metabolism for glucose production to meet the energy demand during stress, which led to the higher activity of the LDH and MDH. The increased activity of LDH and MDH during stress has been reported by Kumar et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Reddy et al. 2012; Defo et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAcetylcholine esterase is an essential enzyme in the modulation of neuromuscular impulse transmission; the central role of AChE is to separate both acetylcholine and the cholinergic signal molecule from its receptors in the plasma membrane (Myrzakhanova et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). AChE has been employed as a stress biomarker for pollution or contaminants in the aquatic environment. The activity of AChE is known to be inhibited in the presence of pollutants such as heavy metals (Kumar et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Ahmad et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Hayat et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Muthappa et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Gupta et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The present work's neurotransmitter enzyme acetylcholine esterase (AChE) was drastically inhibited upon subsequent exposure to Ag-NPs, individually or concurrently with ammonia and high treatment. The inhibition of AChE may be due to the metal ions binding to the terminal-OH and-SH functional groups as they bind with the allosteric sites that cause the conformational changes, making the substrate fail to bind at the specific site of the enzymes (Kumar et al. 2016; Hayat et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Ahmad et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe synthesized silver nanoparticles at a concentration of 50 \u0026micro;l (250 \u0026micro;g) and 100 \u0026micro;l (500 \u0026micro;g) of 5 mg of Ag-NPs/ml of sterile distilled water exhibited good antibacterial activity against gram-negative and gram-positive bacteria. The bactericidal properties of the biosynthesized Ag-NPs have been linked to the interaction of Ag-NPs with sulfur and phosphorous-containing constituents of the bacterial cell, which initiates cell killing by attacking the respiratory chain and cell division (Mahendra et al. 2009). The difference in the inhibition zone formation of Ag-NPs might be due to the cell wall component of bacteria (Chaloupka et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) and the particle size of the synthesized Ag-NPs (Awwad et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGram-negative bacteria lack the thick peptidoglycan layer, causing bacterial cell wall destruction easily as compared to Gram-positive bacteria which have thick protective peptidoglycan layer (Slavin et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Bactericidal activity of recombinant Elastin-like biopolymer composed of a polyhistidine domain with Ag\u0026thinsp;+\u0026thinsp;against Gram-negative bacteria prevalent in coastal shrimp aquaculture has successfully been demonstrated by Krishnani et al. (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In the present study, the zone of inhibition of AgNPs was found to be bigger in the cases of Gram-negative bacteria prevalent in freshwater aquaculture as compared to Gram-positive bacteria. The known antibacterial mechanisms of Ag NPs are the physical direct interaction of extremely sharp edges of AgNPs with cell wall membrane; ROS generation; Trapping the bacteria within the aggregated AgNPs; Oxidative stress; Interruption in the glycolysis process of the bacteria; DNA damaging; Ion release; and inducing the viable but non-culturable state of pathogens \u003cem\u003e(VBNC)\u003c/em\u003e (Krishnani et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThe present study concluded that silver nanoparticle synthesis can be achieved by using processed waste from abattoirs, mainly the intestine of sheep and pigs, with the conviction that waste from animals can effectively be used and that waste from one point can be a resource for another point in the circular bioresource utilization. The synthesized silver nanoparticles show good bactericidal properties against different Gram-positive and Gram-negative bacteria. Further, the toxicity analysis has also provided insight into the effect of co-stressors on the toxicity of silver nanoparticles. The chronic toxicity analysis of the biosynthesized Ag-NPs on fish carried out using stress biomarkers was found to increase with increased sub-lethal ammonia concentration and temperature. Biomolecules present in animal wastes are an excellent source of distinctive functional groups that have been utilized in modulating the surface of the nanoparticles due to the availability of natural binding sites. Overall, it can be concluded from the study that the waste generated after the slaughter of a sheep and pig, primarily the intestine, can be utilized to produce silver nanoparticles. With the increasing resistance of bacteria to antibiotics, biosynthesized Ag-NPs if used within the optimum concentration, have the potential for application in the health management of aquaculture species as they have exhibited antibacterial properties without any adverse effect on the fish. Ag NPs are one of the promising candidates as alternatives to synthetic antibiotics, for preventing antimicrobial resistance in the aquatic environment. Scaling-up of silver nanoparticle synthesis using animal waste extracts is worthy to be researched further to mitigate climate change-induced stresses for developing climate resilience in fish and to prevent antimicrobial resistance in One-Health Fisheries and other sectors of agriculture.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe authors\u003c/strong\u003e are grateful to the Directors of the ICAR-Central Institute of Fisheries Education and the ICAR-National Institute of Abiotic Stress Management for providing their encouragement to carry out the research work and providing an Institute project to the corresponding author. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEthics statement\u003c/em\u003e\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe use of animals conforms to the existing laws in India. The care and treatment of animals used in this study were in accordance with the guidelines of the CPCSEA [(Committee for the Purpose of Control and Supervision of Experiments on Animals, (Ministry of Environment \u0026amp; Forests (Animal Welfare Division), Government of India] on the care and use of animals in scientific research. The study protocol and experimental endpoints were approved by the institutional research committee (Board of Studies) and the authority of the ICAR Institute (MA-09-12, M.F.Sc. synopsis approved by Board of Studies, and degree awarded by the University in September 2021).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eICAR is gratefully acknowledged for awarding a fellowship to the student and providing an Institute project to the corresponding author. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMr. Sowa o Lamare and Dr. Krishnani contributed to the study\u0026rsquo;s conception and design. Material Preparation, data collection, and analysis were performed by Sowa o Lamare and Dr. Krishnani. The first draft of the manuscript was written by Sowa and Krishnani and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u0026nbsp;\u003c/em\u003e\u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"603\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.245847176079735%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eName of authors\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"73.75415282392026%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eContribution of authors\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.245847176079735%\" valign=\"top\"\u003e\n \u003cp\u003eMr. Sowa o Lamare\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"73.75415282392026%\" valign=\"top\"\u003e\n \u003cp\u003eData curation; Execution of Experimental work; Validation; Data interpretation, Compilation, and preparation.\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.245847176079735%\" valign=\"top\"\u003e\n \u003cp\u003eDr. K K Krishnani\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"73.75415282392026%\" valign=\"top\"\u003e\n \u003cp\u003eConceptualization; Synthesis and experimental protocols, Outline \u0026amp; design, Overall guidance, Review \u0026amp; Editing\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.245847176079735%\" valign=\"top\"\u003e\n \u003cp\u003eDr. Neeraj Kumar\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"73.75415282392026%\" valign=\"top\"\u003e\n \u003cp\u003eMethodology, Inputs related to enzyme activity; Physiological responses of fish, Review \u0026amp; Editing \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.245847176079735%\" valign=\"top\"\u003e\n \u003cp\u003eDr. Madhuri Pathak\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"73.75415282392026%\" valign=\"top\"\u003e\n \u003cp\u003eInputs related to pathogenic bacteria, Review \u0026amp; Editing\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.245847176079735%\" valign=\"top\"\u003e\n \u003cp\u003eDr Ajay Upadhyay\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"73.75415282392026%\" valign=\"top\"\u003e\n \u003cp\u003eInputs related to methodology, Review\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.245847176079735%\" valign=\"top\"\u003e\n \u003cp\u003eDr. Biplab Sarkar\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"73.75415282392026%\" valign=\"top\"\u003e\n \u003cp\u003eInputs related to Ag NPs, FTIR, Review \u0026amp; Editing\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.245847176079735%\" valign=\"top\"\u003e\n \u003cp\u003eDr A K Verma\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"73.75415282392026%\" valign=\"top\"\u003e\n \u003cp\u003eInputs related to abiotic stresses, Review \u0026amp; Editing\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.245847176079735%\" valign=\"top\"\u003e\n \u003cp\u003eMs Puja Chakraborty\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"73.75415282392026%\" valign=\"top\"\u003e\n \u003cp\u003eInputs related to biotic stresses, Review \u0026amp; Editing\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.245847176079735%\" valign=\"top\"\u003e\n \u003cp\u003eDr NK Chadha\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"73.75415282392026%\" valign=\"top\"\u003e\n \u003cp\u003eInputs related to application in aquaculture, Review \u0026amp; Editing\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this article. The data that support the findings of this study are available from the corresponding author. \u003cem\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eCode availability\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe consent of all the authors of this article has been obtained for submitting the article to this Journal.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAbdelmoteleb, A., Valdez-Salas, B., Cece\u0026ntilde;a-Duran, C., Tzintzun-Camacho, O., Guti\u0026eacute;rrez-Miceli, F., Grimaldo-Juarez, O., \u0026amp; Gonz\u0026aacute;lez-Mendoza, D. (2017). 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(2014). Lipotropes promote immunobiochemical plasticity and protect fish against low-dose pesticide-induced oxidative stress. Cell Stress and Chaperones, 19(1), 61\u0026ndash;81. https://doi.org/10.1007/s12192-013-0434-y.\u003c/li\u003e\n \u003cli\u003eMyrzakhanova, M., Gambardella, C., Falugi, C., Gatti, A. M., Tagliafierro, G., Ramoino, P., Bianchini, P., \u0026amp; Diaspro, A. (2013). Effects of nanosilver exposure on cholinesterase activities, CD41, and CDF/LIF-like expression in ZebraFish (\u003cem\u003eDanio rerio\u003c/em\u003e) larvae. BioMed Research International, 2013, 205183. https://doi.org/10.1155/2013/205183.\u003c/li\u003e\n \u003cli\u003eNiu, Z., \u0026amp; Li, Y. (2014). Removal and Utilization of Capping Agents in Nanocatalysis. \u003cem\u003eChem. Mater.\u003c/em\u003e 26 (1), 72\u0026ndash;83. doi:10.1021/cm4022479.\u003c/li\u003e\n \u003cli\u003eNiraimathi, K.L., Sudha, V., Lavanya, R. \u0026amp; Brindha, P. (2013). Biosynthesis of silver nanoparticles using \u003cem\u003eAlternanthera sessilis\u003c/em\u003e (Linn.) extract and their antimicrobial, antioxidant activities, Colloids and Surfaces B: Biointerfaces, 102: 288-291.\u003c/li\u003e\n \u003cli\u003eOchoa, S. (1955). Malic dehydrogenase and \u0026ldquo;Malic\u0026rdquo; enzyme. In S. P. Coloric \u0026amp; N. Kaplan (Eds.), Methods of enzymology, Vol I (pp. 735\u0026ndash;745). Academic Press.\u003c/li\u003e\n \u003cli\u003ePournori, B., Paykan Heyrati, F., \u0026amp; Dorafshan, S. (2017). Histopathological changes in various tissues of striped catfish, \u003cem\u003ePangasianodon hypophthalmus\u003c/em\u003e, fed on dietary nucleotides and exposed to water-borne silver nanoparticles or silver nitrate. Iranian Journal of Aquatic Animal Health, 3(2), 36\u0026ndash;52. https://doi.org/10.29252/ijaah.3.2.36.\u003c/li\u003e\n \u003cli\u003ePrakash, P., Gnanaprakasam P., Emmanuel, R., Arokiyaraj, S., Saravanan, M. (2013). Green synthesis of silver nanoparticles from leaf extract of \u003cem\u003eMimusops elengi\u003c/em\u003e, Linn. for enhanced antibacterial activity against multi-drug resistant clinical isolates, Colloids and Surfaces B: Biointerfaces, 108, 255-259.\u003c/li\u003e\n \u003cli\u003eRajkumar, K S, Kanipandian, N. \u0026amp; Ramasamy, \u0026amp; Thirumurugan. (2015). Toxicity assessment on haemotology, biochemical and histopathological alterations of silver nanoparticles-exposed freshwater fish \u003cem\u003eLabeo rohita\u003c/em\u003e. Applied Nanoscience, 6(1), 19\u0026ndash;29. https://doi.org/10.1007/s13204-015-0417-7.\u003c/li\u003e\n \u003cli\u003eReddy, S. J. (2012). Impact of heavy metals on changes in metabolic biomarkers of carp fish. International Journal of Bioassays, 1(12), 227\u0026ndash;232.\u003c/li\u003e\n \u003cli\u003eRoy, D.C., Gogoi, R., \u0026amp; Laskar, S.K. (2017). Enrofloxacin Residue Detection in Marketed Pork of North East India. 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Part A, Molecular and Biomolecular Spectroscopy, 131, 413\u0026ndash;423. https://doi.org/10.1016/j.saa.2014.04.065.\u003c/li\u003e\n \u003cli\u003eSlavin, Y.N., Asnis, J., H\u0026auml;feli, U.O., Bach, H. (2017). Metal nanoparticles: understanding the mechanisms behind antibacterial activity. J Nanobiotechnology. 15(1): 65. doi: 10.1186/s12951-017-0308-z.\u003c/li\u003e\n \u003cli\u003eTakahara, S., Hamilton, H. B., Neel, J. V., Kobara, T. Y., Ogura, Y., \u0026amp; Nishimura, E. T. (1960). Hypocatalasemia: A new genetic carrier state. Journal of Clinical Investigation, 39(4), 610\u0026ndash;619. https://doi.org/10.1172/JCI104075.\u003c/li\u003e\n \u003cli\u003eWootton, I. D. P. (1964). Microanalysis in medical biochemistry. J and A Churchill Ltd. London. Proceedings of the Society for Experimental Biology and Medicine, 90, 101\u0026ndash;103.\u003c/li\u003e\n \u003cli\u003eWr\u0026oacute;blewski, F., \u0026amp; Ladue, J. S. (1955). Lactic dehydrogenase activity in blood. Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine, 90(1), 210\u0026ndash;213. https://doi.org/10.3181/00379727-90-21985.\u003c/li\u003e\n \u003cli\u003eYuan, Q., Bomma, M., \u0026amp; Xiao, Z. (2019). Enhanced silver nanoparticle synthesis by Escherichia coli transformed with Candida \u003cem\u003ealbicans Metallothioneins\u003c/em\u003e Gene. Materials, 12(24), 4180. https://doi.org/10.3390/ma12244180.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"Sheep and Swine intestines, Biosynthesis, Colloidal silver nanoparticles, Toxicity test on Pangasianodon hypophthalmus, Bactericidal activity and physiological responses, Multiple stress resilience","lastPublishedDoi":"10.21203/rs.3.rs-4215264/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4215264/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe present study elucidates the extracellular synthesis of capped silver nanoparticles using processing waste of sheep and swine by dispensing with addition of any capping agent and advantage of avoiding agglomeration and loss of nanosized characteristics of AgNPs. The synthesis of Ag-NPs was ascertained by UV-VIS spectrophotometry of yellowish-brown suspension at 400\u0026ndash;410 nm. The Ag-NPs were further characterized using a HR-TEM, which confirmed that the Ag-NPs were primarily spherical and had a size range of 5-100 nm with a maximum frequency fall between 5\u0026ndash;20 nm, 21\u0026ndash;30 nm, 31\u0026ndash;50 nm and a few falls within 51\u0026ndash;100 nm. Ag-NPs synthesized using sheep and pig wastes are characterized by DLS, which confirmed the high stability of Ag-NPs with a zeta potential of -27 and \u0026minus;\u0026thinsp;32 mV respectively. Biomolecules and biological extracts of animal wastes act as biogenic reducing and capping agents. Based on the zone of inhibition, Ag-NPs biosynthesized using sheep waste showed high bactericidal properties against \u003cem\u003eAeromonas hydrophila, Edwardsiella tarda\u003c/em\u003e, and \u003cem\u003eMicrococcus luteus\u003c/em\u003e as compared to swine waste-derived AgNPs. The chronic toxicity analysis of biosynthesized Ag-NPs on \u003cem\u003ePangasianodon hypophthalmus\u003c/em\u003e was carried out using stress biomarkers such as an antioxidant enzyme, AChE, and metabolic enzyme activity. Chronic toxicity of synthesized Ag-NPs was found to increase with increased sub-lethal ammonia concentration and temperature. The findings of this study revealed that biosynthesis of capped and non-agglomerated Ag-NPs can be undertaken by using animal wastes for their potential application in aquaculture based on the properties observed in characterization, bactericidal activity, and physiological responses of the fish.\u003c/p\u003e","manuscriptTitle":"Animal wastes-derived capped and bactericidal silver nanoparticles induce immuno-physiological responses in fish for multiple stress resilience in the One-Health Approach","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-16 04:48:18","doi":"10.21203/rs.3.rs-4215264/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":"bcdf47bb-87a6-4455-8a6e-645a18e6eab4","owner":[],"postedDate":"April 16th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-04-20T18:42:47+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-16 04:48:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4215264","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4215264","identity":"rs-4215264","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}