Dietary Nanocurcumin Impacts Blood Biochemical Parameters and Works Synergistically with Florfenicol in African Catfish Challenged with Aeromonas veronii

Round 1

Reviewer 1 Report

Review: fishes-2410395

Dietary Nanocurcumin Impacts Blood Biochemical Parameters and Works Synergistically with Florfenicol in African Catfish Challenged with Aeromonas veronii.

 

Main comments

In this study, the authors aimed to present that incorporating nanocurcumin into the diet can have multiple benefits for the sustainable aquaculture of African catfish. Those included mitigating drug-induced damage to the liver and kidney tissues, augmenting the therapeutic efficacy of FFC in a synergistic manner, and providing protection against A. veronii infections. They thought that all results are supporting the nanocurcumin as an additive in feeds, which protect African catfish away from drug-induced damages and A. veronii infections. However, I still cannot recommend publication with current form of their manuscript for several reasons:

 

1. (major question)

In methods section, the author described their protocols of nanocurcumin preparing. Is the Cur/ D. dichotoma NPs used as an additive into the dietary feed in African catfish aquaculture? If yes, how to distinguish the effects from D. dichotoma powder or endophytic fungi?

According to the references provided from current manuscript, previous study (31) was producing endophytic fungi extracellular products from D. dichotoma powder to aid formation of ZnO nanoparticles. If this study utilize same method to create Cur/ D. dichotoma NPs-containing feeds, the antibacterial bioactivities could be derived from D. dichotoma powder, endophytic fungi biocompound or both, not just from curcumin only. Since lots of studies showed endophytes are rich source of natural products displaying broad spectrum of biological activities like anticancer, antibacterial, antiviral, immunomodulatory, antioxidan and anti-inflammatory. The authors should provide a detail information of “NCur”.

 

* 31. Kumar, R.V.; Vinoth, S.; V. Baskar, V.; Arun, M.; Gurusaravanan, P. Synthesis of zinc oxide nanoparticles mediated by Dictyota dichotoma endophytic fungi and its photocatalytic degradation of fast green dye and antibacterial applications, South African Journal of 656 Botany. 2022,151, 337-344. 657

* 32. Carvalho, D.M.; Takeuchi, K.P.; Geraldine, R. i Its in 660 Vitro Efficacy against Human Pathogens. 3 Biotech. 2015, 5, 991−997.

* 33. Pandit, R.S.; Gaikwad, S.C.; Agarkar, G.A.; Gade, A.K.; Rai, M. Curcumin Nanoparticles: Physico-Chemical Fabrication and Its in Vitro Efficacy against Human Pathogens. 3 Biotech. 2015, 5, 991−997.

 

2. I appreciate that English is not likely to be the first language of any of the authors. However, while parts of the manuscript are written clearly, unfortunately, there are sections that are very awkward and hard to understand.

 

 

 

 

Comments for author File: Comments.pdf

If possible, re-English editing of current manuscript by native English speakers or service will be better.

Author Response

Response to Reviewer 1

In this study, the authors aimed to present that incorporating nanocurcumin into the diet can have multiple benefits for the sustainable aquaculture of African catfish. Those included mitigating drug-induced damage to the liver and kidney tissues, augmenting the therapeutic efficacy of FFC in a synergistic manner, and providing protection against A. veronii infections. They thought that all results are supporting the nanocurcumin as an additive in feeds, which protect African catfish away from drug-induced damages and A. veronii infections. However, I still cannot recommend publication with current form of their manuscript for several reasons:

1. (major question)

In methods section, the author described their protocols of nanocurcumin preparing. Is the Cur/ D. dichotoma NPs used as an additive into the dietary feed in African catfish aquaculture? If yes, how to distinguish the effects from D. dichotoma powder or endophytic fungi?

According to the references provided from current manuscript, previous study (31) was producing endophytic fungi extracellular products from D. dichotoma powder to aid formation of ZnO nanoparticles. If this study utilize same method to create Cur/ D. dichotoma NPs-containing feeds, the antibacterial bioactivities could be derived from D. dichotoma powder, endophytic fungi biocompound or both, not just from curcumin only. Since lots of studies showed endophytes are rich source of natural products displaying broad spectrum of biological activities like anticancer, antibacterial, antiviral, immunomodulatory, antioxidan and anti-inflammatory. The authors should provide a detail information of “NCur”.

We sincerely appreciate and thank the reviewer for their valuable comment, and we acknowledge the validity of the point raised. In response to the concern, we would like to provide further clarification. Seaweed extracts have indeed been extensively utilized in the synthesis of various nanoparticles. Numerous studies (Kumar et al. 2022; Kumar and Sudha 2013) have demonstrated that the substance derived from seaweed extract does not have an impact on the properties of the resulting nanoparticles. The role of the seaweed extract is primarily confined to facilitating the synthesis process of the nanoparticles, rather than directly influencing their downstream effects. Biological materials are actually preferred for the synthesis of nanoparticles, as they are eco-friendly and compatible with pharmaceuticals (SenthilKannan et al. 2021). In our study, we used the extract of Dictyota dichotoma as a reducing and capping agent for curcumin nanoparticle isolation similar to previous research (Vivek et al. 2011). Dictyota dichotoma contains sulfated polysaccharides and phenolic compounds that possess excellent reducing and capping abilities. These compounds have the capacity to effectively reduce metal ions and stabilize the resulting nanoparticles during synthesis.

In our manuscript, we presented a concise overview of the experimental procedures, as our primary focus was not on the synthesis of curcumin nanoparticles per se, considering the various methods available for nanoparticle synthesis. Nonetheless, in response to the reviewer's request, we are pleased to provide a more detailed description of the steps involved in the synthesis process:

• One gram of D. dichotoma powder was dissolved in 100 mL of distilled water at 100°C for 1 h in an Erlenmeyer flask.
• The extract was filtered using Rotilabo® Tyb 601P filter paper.
• The filtrate was heat sterilized by autoclaving at 121°C for 15 min. Subjecting the extract to high pressure and temperatures deactivates the bioactive compounds in D. dichotoma extract.
• The sterilized filtrate was stored in a refrigerator at 4°C until use.
• To exclude any antibacterial activity of the D. dichotoma extract against A. veronii, we used the disk-diffusion susceptibility. We loaded 15µL of the extract on a blank antimicrobial susceptibility disk and placed the disk on a tryptic soy agar plate inoculated with A. veronii. After incubation for 48 hours, there was no zone of growth inhibition around the disk, indicating no antibacterial activity.
• Curcumin powder (0.5 g) was dissolved in 150 mL of distilled water under continuous stirring at 45°C and the aqueous solution of curcumin was added in a dropwise manner to a 100 mL aliquot of D. dichotoma extract in a conical flask.
• The color changed from bright yellow to orange within 1 h with precipitation of Cur/ D. dichotoma NPs.
• To separate the curcumin nanoparticles from the mixture, the solution was centrifuged at 6000 rpm/min for 10 min followed by extracting the pellets.
• Then, curcumin nanoparticles were rinsed in sterile distilled water to remove any traces of algal extract and any impurities from the nanoparticles after their synthesis. By rinsing the nanoparticles in water, we removed D. dichotoma residual material, unbound reactants, or byproducts that may have been present. Subsequently, the suspension was centrifuged to separate the purified nanoparticles from the rinsing solution.
• Finally, the purified curcumin nanoparticles were dried (heated to 90°C). Purified curcumin nanoparticles were added to the fish feed.

We hope this clarification provides a better understanding of the role of seaweed extracts in nanoparticle synthesis and adequately addresses the concern raised by the reviewer. We have modified the text under section “2.2. Preparation of nanocurcumin” to reflect the missing steps. We remain committed to providing a comprehensive and accurate representation of our research findings. References used for this response are listed below.

Vivek, M., Kumar, P.S., Steffi, S. and Sudha, S., 2011. Biogenic silver nanoparticles by Gelidiella acerosa extract and their antifungal effects. Avicenna Journal of Medical Biotechnology, 3(3), p.143.

Kumar, R.V., Vinoth, S., Baskar, V., Arun, M. and Gurusaravanan, P., 2022. Synthesis of zinc oxide nanoparticles mediated by dictyota dichotoma endophytic fungi and its photocatalytic degradation of fast green dye and antibacterial applications. South African Journal of Botany, 151, pp.337-344.

Kumar, P.S. and Sudha, S., 2013. Biosynthesis of silver nanoparticles from Dictyota bartayresiana extract and their antifungal activity. Nano Biomedicine & Engineering, 5(2). DOI:10.5101/nbe.v5i2.p72-75

SenthilKannan, K., Flora, G., Gunasekaran, S., Ali, H.A.J., Vimalan, M. and Balasubramanian, S., 2021. Extraction of Silver Nanoparticles (Ag-NPs) by Green Synthesis from Aqueous Extract of Seaweeds and their consequences on HeLa Cell Line and their utility on soil by spectroscopic tools. Nanotechnology for Advances in Medical Microbiology, pp.119-138.

 

2. I appreciate that English is not likely to be the first language of any of the authors. However, while parts of the manuscript are written clearly, unfortunately, there are sections that are very awkward and hard to understand.

We would like to emphasize that the manuscript has been thoroughly reviewed by two other professional reviewers and the Editor/Editorial office of the journal, and no concerns were raised regarding the English language. However, we carefully reviewed the entire manuscript, please see the new version with track changes. We believe that this indicates a high level of attention to detail in terms of grammar and language usage. It is important to avoid making assumptions about authors' language proficiency based on their names or affiliations “unconscious biases”. English may not be our first language, but we have made considerable efforts to ensure the clarity and coherence of the manuscript. We are more than willing to rephrase and clarify any sections to improve the overall readability of the manuscript. We value your input and are committed to delivering a high-quality manuscript. Thank you for your understanding.

Reviewer 2 Report

The work contains important information for solving the antibiotic resistance problem in the diffrent fish species in aquaculture. However, some parts of the manuscript are not clearly described and require additional information and explanations. I suggest minor revisions.

1.       Line 157. Where were the fish during the period of acclimation before being distributed to research groups in 160 l aquariums?

2.       Lines 159 and 179. Did the fact that during the acclimation period the fish were fed once a day, and in the experimental period the fish received food twice a day affect the studied biochemical parameters? Why?

3.       Line 162: "Dissolved oxygen saturation (DO)" is expressed in %, whereas in line 181 "DO" is expressed in other units (7.5 ± 0.4 mg l-1). It may be useful to keep the same units in the text (mg l-1).

4.       Line 167: When describing the experimental conditions (chapter 2.4.1), I would like to know if the nitrogen content of the water was controlled. Nitrogen is one of the most important hydrochemical parameters in fish farming.

5.       Line 184. Why, after the experimental period was completed, only four randomly selected fish from each group were netted out of their holding tanks for blood sampling? What explanation for such a small sample?

6.       Line 186. It would be good to know how blood was collected from the caudal veins: was by sticking a needle into a vein, or a tail cut.

7.       Line 297. I suggest that the authors use a less complicated system for reading significant differences in Figures 4-6, and describe in more detail which letter indicates a significant difference in which group.

8.       Lines 418-420. It would be nice if you could add some references.

Author Response

Response to Reviewer 2

The work contains important information for solving the antibiotic resistance problem in the diffrent fish species in aquaculture. However, some parts of the manuscript are not clearly described and require additional information and explanations. I suggest minor revisions.

We value the reviewer's input and are committed to addressing any issues or concerns they have raised. Please, see below a detailed “point by point” response to each point.

1. Line 157. Where were the fish during the period of acclimation before being distributed to research groups in 160 l aquariums?

After the fish were delivered from the commercial catfish farm to our holding facility in the Department of fish diseases, Faculty of Veterinary Medicine at South Valley University, the fish were placed in our fish quarantine room in large fiberglass tanks - 270 gal - dimensions 72 x 36 x 24-in for acclimation. The fish were monitored for two weeks period for the development of any clinical signs of disease. Prior to enrollment in the study, ten fish were randomly sampled, screened, and confirmed negative for A. veronii via gyrB PCR as described under 2.5.1. The following text was added to section 2.3. - Fish acclimation and husbandry conditions “fish were held under quarantine in a separate room in a fiberglass tank - 270 gallons - dimensions 72 x 36 x 24-in for acclimation to the laboratory conditions for two weeks prior to allocation to study groups in 160 L tanks. During this acclimation period, the fish were monitored for the development of any clinical signs of disease. Ten catfish were randomly sampled and confirmed negative for A. veronii via gyrB PCR as indicated below.”

2. Lines 159 and 179. Did the fact that during the acclimation period the fish were fed once a day, and in the experimental period the fish received food twice a day affect the studied biochemical parameters? Why?

We do not think so. The feeding rate throughout the experiment remained consistent, with a feeding rate of 5% of the fish's body weight. To optimize the consumption of the ration and minimize the loss of substances into the water (potential diffusion of florfenicol and nanocurcumin from the feed pellets into the water), we decided to split the feeding into two meals per day during the experimental period. This approach aimed to ensure that the fish would consume the provided ration more efficiently. Additionally, throughout the study, a control group was included as a reference point providing a baseline for evaluation.

3. Line 162: "Dissolved oxygen saturation (DO)" is expressed in %, whereas in line 181 "DO" is expressed in other units (7.5 ± 0.4 mg l-1). It may be useful to keep the same units in the text (mg l-1).

We changed the dissolved oxygen units to mg l-1 as the reviewer suggested. See section 2.3. Fish acclimation and husbandry conditions

4. Line 167: When describing the experimental conditions (chapter 2.4.1), I would like to know if the nitrogen content of the water was controlled. Nitrogen is one of the most important hydrochemical parameters in fish farming.

We agree with the reviewer’s point regarding the importance of nitrogen in fish farming. However, given the specific design of our experiment, which involved multiple treatment groups and controls, we employed a flow-through tank system rather than a recirculating aquaculture system (RAS). In the flow-through system, water continuously flows through the tanks at a high rate, equivalent to approximately one tank exchange per hour. This rapid water flow prevents the accumulation of nitrogen compounds in the tanks. To ensure accurate monitoring, we regularly measured the levels of total ammonia nitrogen and un-ionized ammonia (NH3) throughout the study. However, the recorded readings were consistently negligible, ensuring that the experimental conditions remained within optimal ranges for the well-being of the fish.

5. Line 184. Why, after the experimental period was completed, only four randomly selected fish from each group were netted out of their holding tanks for blood sampling? What explanation for such a small sample?

The selection of four fish from each group for blood sampling was based on statistical considerations. While the sample size may appear small, it is important to note that it is sufficient for maintaining statistical power. This was also chosen to minimize the number of animals sacrificed, as we are required to use the least sample size by our Institutional Animal Care & Research Ethics Committee. In our study, four fish from each group were deemed appropriate to achieve statistically significant results. While three is often considered the minimum sample size for certain statistical analyses, having four individuals from each group provided an additional level of reliability and enhanced the robustness of the findings.

 

6. Line 186. It would be good to know how blood was collected from the caudal veins: was by sticking a needle into a vein, or a tail cut.

The blood sample was taken using a needle attached to a vacutainer system inserted into the musculature tail at the ventral surface of the fish. We made this clear in the text according to the reviewer’s recommendation. The modified text now reads as follows “Blood was collected from the caudal veins of sacrificed C. gariepinus using a needle attached to vacutainer plain blood collection tubes.”

 

7. Line 297. I suggest that the authors use a less complicated system for reading significant differences in Figures 4-6, and describe in more detail which letter indicates a significant difference in which group.

Thank you for your comment regarding the presentation of significant differences in Figures 4-6. We appreciate your suggestion for using a less complicated system. This letter labeling method is widely recognized and provides a concise and clear way to present significant differences, making it accessible to readers with different levels of statistical knowledge. However, we improved the system for reading significant differences in Figures 4-6 as you recommended. We believe that the system we employed to indicate significance is now straightforward. Bars labeled with different (or not sharing) the same lowercase letters are significantly different, and if two bars share the same letter, it indicates that they are not significantly different from each other.

8. Lines 418-420. It would be nice if you could add some references.

We appreciate the reviewer's suggestion regarding the inclusion of additional references in our manuscript. We acknowledge the importance of providing appropriate references to support the information presented. However, we would like to clarify that we have already included a comprehensive list of 88 references that cover the relevant literature on the topic. These references encompass a wide range of studies and provide a robust foundation for our research's scientific background, methodology, and findings. We believe that the current set of references provides substantial support for the claims and statements made throughout the text. We are also open to considering specific references suggested by the reviewer if they address any specific gaps or provide significant additional value to our work. Once again, we express our gratitude to the reviewer for their valuable feedback.

Reviewer 3 Report

The purpose of this study was to investigate the potential effects of feeding nanocurcumin (150 ppm) and florfenicol (10 and 30 mg/kg.BW) separately and combined for 15 days on serum biochemical indices and histological architecture of liver and kidney tissues of C. gariepinus. In addition, a challenge experiment was conducted to assess resistance of C. gariepinus to Aeromonas veronii infection. Flufenicol has been widely used. Although the innovation of this paper needs to be improved, it still has some reference for the development of aquaculture.

(1) The fish were fed once a day (5% of the fish’s body weight) with commercial feed pellets purchased from Skretting Animal Nutrition (30% protein). The author should give the basic feed formula for the experiment, and also include the data of fat level, energy level and so on.

(2) 2.4.2. Analysis of serum biochemical indices. Add kit model

(3) Didn't the author analyze the homogeneity of variance and so on? What does the author do with the data if the variance is uneven?

Author Response

Response to Reviewer 3

We thank the reviewer for his valuable comments.

(1) The fish were fed once a day (5% of the fish’s body weight) with commercial feed pellets purchased from Skretting Animal Nutrition (30% protein). The author should give the basic feed formula for the experiment, and also include the data of fat level, energy level and so on.

The basal feed ingredients were: soybean meal cake 46%, wheat bran, yellow corn, distiller's dried grains with solubles (DDGS), high-fat fish meal 65%, choline chloride, sodium chloride, DL-methionine, monocalcium phosphate, vitamin and mineral pre-mix. These commercial feed pellets contained 30% crude protein, 5.22 % crude fiber, 6 % crude lipid, 9.5 % Ash and gross energy 3900 kCal were obtained from (Skretting Company, Egypt).

The protein, fiber, lipid, ash, and gross energy levels of the basal diet used for feeding the fish during this experiment and were added to the text according to the reviewer’s recommendation. See the materials and methods section, 2.3. Fish acclimation and husbandry conditions

(2) 2.4.2. Analysis of serum biochemical indices. Add kit model

The catalog number of the kits used for serum biochemical indices was added in the revised version of the manuscript as you suggested. Total protein & albumin (Biomed Diagnostics, Badr city, Egypt, Cat. NO. TP 2020 and AB 1010, respectively); alanine aminotransferase (ALT) (Cat. NO. 292005), aspartate aminotransferase (AST) (Cat. NO. 291005), and alkaline phosphatase (ALP) (Cat. NO. 215001), urea (Cat. NO. 318002) and creatinine (Cat. NO. 234001) (Spectrum Diagnostics, Egypt).

(3) Didn't the author analyze the homogeneity of variance and so on? What does the author do with the data if the variance is uneven?

The current data was validated for normality and homogeneity of variance by the Shapiro-Wilk and Kolmogorov–Smirnov normality tests. When the data was uneven, it was transformed into a logarithmic form or reciprocals. This was indicated in the revised manuscript, under section” 2.6. Statistical analysis”.

Round 2

Reviewer 3 Report

• The author has revised the paper very well and sent you the acceptance criteria

 

 

Author Response

We express our gratitude to the reviewer for providing us with their insightful comments and suggestions throughout the review process. Thank you.