Ginger nanoparticles enhancing the immune responses in fish exposed to glyphosate -

Ginger nanoparticles enhancing the immune responses in fish exposed to glyphosate

In this study investigated the toxicity of commercial glyphosate-induced oxidative stress, immune suppression, liver and kidney dysfunction, and the protective role of ginger or ginger nanoparticles in Nile tilapia. Fish were allocated into four groups: the first group presented the control without glyphosate toxicity and ginger feeding, the second group intoxicated with glyphosate at 0.6 mg/L and fed ginger free diet, the third group intoxicated with glyphosate and fed ginger at 2.5 g/kg, and the fourth group intoxicated with glyphosate and fed ginger nanoparticles at 2.5 g/kg. Fish were kept under the experimental conditions for four weeks, and the samples of blood and tissues were collected after 2 and 4 weeks. Ginger nanoparticles are superior to the standard ginger form in enhancing the antioxidative and immune responses of Nile tilapia exposed to glyphosate

Material and methods

Ginger nanoparticles and test diets preparation

Ginger was obtained from the local market as a fine powder and stored in glass containers in the refrigerator until further use. Ginger nanoparticles were prepared using a planetary ball mill at 550 rpm for 4 h till they reached size 100 nm. Commercial basal diets were crushed to a fine powder and split into three parts, with the first diet serving as the control. At a 2.5 g/kg diet dosage, the second and third diets were properly incorporated with ginger and ginger nanoparticles, respectively. Ginger and ginger nanoparticles were diluted in distilled water and mixed with a crushed diet to produce dough. The soft dough was re-pelleted using a meat mincer. The prepared pellets (2–3 mm) were kept at room temperature for 48 h to dry out, sealed in clean, dry plastic bags, and stored at 4 °C until needed.

Fish and experimental procedure

A total of 250 healthy all males Nile tilapia (25.71±0.5 g) have been obtained from a private fish farm. Then, they were allocated in two fiberglass tanks (750 L capacity) provided with continuous aeration for two weeks acclimation period. During this period, fish were hand-fed daily with the control diet at a 3% body weight twice daily (8:00 and 16:00). Fish were stocked in twelve glass aquaria (89 × 30 × 29 cm) at 15 fish per aquarium. The aquaria present four groups in triplicates, and each aquarium is supplied with continuous aeration. The first group was fed a basal diet and kept in glyphosate-free water (control). The second group was intoxicated with glyphosate at 0.6 mg/L and fed the basal diet. The third group was intoxicated with glyphosate and fed ginger at 2.5 g/kg. The fourth group was intoxicated with glyphosate and fed ginger nanoparticles at 2.5 g/kg. The water from groups two to four was partially exchanged three times weekly, and the dose of glyphosate (Roundup 48%) was maintained within the required concentration (0.6 mg/L) in each aquarium. Meanwhile, the water was renewed with dechlorinated tap water in the control group. Glyphosate lethal concentration (LC50; 12 mg/L) was calculated and fish was exposed to 1/20 of LC50 (0.6 mg/L). All fish have received the experimental diets twice daily at a feeding rate of 3% of the total body weight for four weeks under 12 h day:12 h night photoperiod regime. Water quality was maintained at 25±1 °C, 5.1±0.2 mg/L, 0.23±0.07 mg/L, and 7.2±0.2 for the temperature, dissolved oxygen, ammonia concentration, and pH, respectively. The water exchange was done daily to eliminate fecal matter and uneaten food to maintain water quality parameters. The particle size distribution and zeta potential of ginger nanoparticles were determined using Zetasizer. The ginger nanoparticle morphology was observed under transmission electron microscopy at an accelerating voltage of 200 K.V.

Blood and tissue collection

Blood and tissue samples were collected from all treated groups after 2 weeks of exposure and at the end of the experiment (4 weeks). Fish were anesthetized with MS-222 (100 μg/mL), then blood samples from nine fish per treatment (3 fish/ replicate) were collected without anticoagulant. To separate serum, these samples were left in a slant position for 30 min and centrifuged at 3000 r.p.m for 15 min. The serum was collected and kept at -20 ˚C for immunological and biochemical assays. For evaluation of antioxidant enzyme activity, liver and gills specimens were taken from nine fish per treatment (3 fish/ replicate), homogenized in 9 volumes of ice-cold 0.05 Mm potassium phosphate bufer saline pH 7.4 using a glass homogenizer. The homogenates were centrifuged at 6000 r.p.m for 15 min at 4˚C. The resultant supernatant was used to determine malondialdehyde (MDA) and reduced glutathione (GSH). Serum biochemical and immune assays, Liver and gills antioxidant assay was determined based on the standard protocol

Results

Characterization of ginger nanoparticles

The size distribution of ginger nanoparticles analyzed by Zetasizer for size determination showed an average diameter of 906.9 d.nm. TEM image showed that ginger nanoparticles were circular in shape.

Growth performance

The obtained results indicated that Nile tilapia-fed ginger or ginger nanoparticles after 4-week exposure to sublethal concentration of glyphosate had no signifcant diferences in the case of final weight and specifc growth rate.

Liver and kidney‑related metabolites

Fish exposed to glyphosate showed the highest ALT and AST activities, while fish in the control group had the lowest ALT and AST activities after 2 and 4 weeks. Interestingly, fish-fed ginger nanoparticles showed lower ALT and AST activities than the glyphosate intoxicated group. Further, urea and creatinine levels showed the lowest values meaningfully in fish fed the control diet without glyphosate toxicity, and fish fed ginger nanoparticles with glyphosate toxicity after 2 and 4 weeks

Blood proteins

The blood total protein, albumin, and globulin levels were meaningfully increased in the control group and severely reduced in fish exposed to glyphosate toxicity after 2 and 4 weeks. No signifcant diferences were seen between the control and fish intoxicated with glyphosate and fed ginger nanoparticles. Moreover, fish fed dietary ginger and exposed to glyphosate had higher total protein, albumin, and globulin levels. Fish fed dietary ginger had higher glucose and cortisol levels than the control and lower glucose and cortisol levels than the glyphosate intoxicated group after 2 and 4 weeks.

Stress‑related markers

Fish exposed to glyphosate showed the highest glucose and cortisol levels, while fish in the control group had the lowest glucose and cortisol levels after 2 and 4 weeks. Fish fed dietary ginger had higher glucose and cortisol levels than the control and lower glucose and cortisol levels than the glyphosate intoxicated group after 2 and 4 weeks.

Liver and gills antioxidant status

In the gills and liver tissues, fish exposed to glyphosate showed the lowest glutathione (GSH) levels, while fish in the control group and fish intoxicated with glyphosate and fed ginger nanoparticles had the highest GSH after 2 and 4 weeks. In the gills and liver tissues, fish exposed to glyphosate showed the highest malondialdehyde (MDA) levels, while fish in the control group and fish intoxicated with glyphosate and fed ginger nanoparticles had the lowest MDA levels after 2 and 4 weeks. Further, fish-fed dietary ginger had a lower MDA level than the glyphosate intoxicated group after 2 and 4 weeks

Immune response

Fish exposed to glyphosate showed the lysozyme activity and total immunoglobulin level, while fish in the control group and fish intoxicated with glyphosate and fed ginger nanoparticles had the highest lysozyme activity and total immunoglobulin level. Further, fish-fed dietary ginger had higher lysozyme activity and total immunoglobulin levels than the glyphosate intoxicated group after 2 and 4 weeks.

Conclusion

In conclusion, glyphosate toxicity represents environmental agriculture- aquaculture risk resulting in the dysfunction of the hepato-renal tissues and impairment of the antioxidative and immune responses of Nile tilapia. However, dietary ginger and its nanoparticles markedly relived the toxic impacts of glyphosate, resulting in regulated hepato-renal tissues, antioxidative, and immune responses. Interestingly, ginger nanoparticles are superior to the standard ginger form in enhancing the antioxidative and immune responses of Nile tilapia exposed to glyphosate.

Reference:

Abdelmagid, A.D., Said, A.M., El-Gawad, A., Eman, A., Shalaby, S.A. and Dawood, M.A., 2022. Glyphosate-induced liver and kidney dysfunction, oxidative stress, immunosuppression in Nile tilapia, but ginger showed a protection role. Veterinary Research Communications, pp.1-11.