The Effects of Different Carbon Sources on Water Quality, Growth Performance, Hematology, Immune, and Antioxidant Status in Cultured Nile Tilapia with Biofloc Technology

Round 1

Reviewer 1 Report

The study of the present manuscript is very important for sustainable aquaculture, but there are some problems in this manuscript.

1.The experimental design was unreasonable, the control group should not be the flow-through system, it should be the system with biofloc technology and a common carbon source, and comparability among different groups could be achieved.
2. Why the fish groups of biofloc technology were fed at the 75% daily feeding rate? An adequate basis should be provided.

3. In the 2.8. Antioxidant parameters, only the CAT and SOD were determined, what about the GSH-Px, MAD, or T-AOC?  More antioxidant parameters should be determined.

4. Figure 1 to figure 9 were not suitable to publish in fishes. The related results should be presented in the tables.

5. In Table 4, is it right that the weight gain (%) is about 66%-98%? It seemed that the growth of tilapia could be not normal according the previous experiences.

6.The discussion was not enough to explain the relation between the different parameters and treatment groups. Why were those parameters affected by different carbon sources? The related reason should be clarified. 

It could not be considered for publication in Fishes at present status. Would you submit this manuscript to the Aquaculture Journal of MDPI? 

 

Author Response

The study of the present manuscript is very important for sustainable aquaculture, but there are some problems in this manuscript.

1. The experimental design was unreasonable, the control group should not be the flow-through system, it should be the system with biofloc technology and a common carbon source, and comparability among different groups could be achieved.

Author’s response: In order to clearly evaluate the effect of biofloc technology with different carbon sources, it was necessary to use the flow through system in case of control group. This is also done by other researchers (https://doi.org/10.1016/j.fsi.2017.03.025, https://doi.org/10.1016/j.aquaculture.2019.734639)

2. Why the fish groups of biofloc technology were fed at the 75% daily feeding rate? An adequate basis should be provided.

Author’s response: In biofloc treatment groups with different carbon sources, the fish also fed on microbial floc. So we assess the effect of carbon source on floc production which ultimately enhance the fish growth.

3. In the 2.8. Antioxidant parameters, only the CAT and SOD were determined, what about the GSH-Px, MAD, or T-AOC?  More antioxidant parameters should be determined.

Author’s response: Due to availability of resources, we currently focus on the two mostly determined antioxidant parameters. 

4. Figure 1 to figure 9 were not suitable to publish in fishes. The related results should be presented in the tables.

Author’s response: Figures were combined into single one.

5. In Table 4, is it right that the weight gain (%) is about 66%-98%? It seemed that the growth of tilapia could be not normal according the previous experiences.

Author’s response: This is possible because in other studies like Mansour and Esteban (https://doi.org/10.1016/j.fsi.2017.03.025); Bakshi et al. (https://doi.org/10.1111/are.13469) also revelaed the related results.

6. The discussion was not enough to explain the relation between the different parameters and treatment groups. Why were those parameters affected by different carbon sources? The related reason should be clarified. 

Author’s response: Clarified and improved 

Reviewer 2 Report

The authors Rind et al have investigated different carbon sources on Nile tilapia in biofloc system. As far as I am concerned, the manuscript is well written and  well structured. I appreciate the authors for taking time to write the manuscript neatly. But I have given some comments below to make some changes in the manuscript to improve the quality of it.

Line 23: Please give full form for BFT and also for other abbreviations used in abstract

Line 26: 260 Nile tilapia were distributed in 9 tanks at a density of 25 fish per tank. If you put 25 fish/tank and for 9 tanks the total would be 225 fish but here mentioned 260 fish. Please check the number of total fish or remove it from here.

Line 27-29: The authors needs to revise the sentence because it is unclear.

Please check table 3 temperature heading because it was in bold

Authors have to check the units throughout the manuscript. For example please check the units in Table 3.

Please give units in y axis for figures 1-9. My suggestion is instead of using 9 figures to depict the results. Why can’t authors combine these 9 figures in to two separate figures and giving Nitrite, nitrate etc. as figure panels a,b,c….. like that. Please consider this suggestion

Line 266: remove brackets

Line 281: globulin levels significantly higher or lower compared to the control group. Please check the line.

Line 289: Please check the abbreviation for tapioca flour

Instead of giving all the results as tables. Authors may prefer to give some data of tables as figures. Consider this suggestion.

Author Response

The authors Rind et al have investigated different carbon sources on Nile tilapia in biofloc system. As far as I am concerned, the manuscript is well written and well structured. I appreciate the authors for taking time to write the manuscript neatly. But I have given some comments below to make some changes in the manuscript to improve the quality of it.

Line 23: Please give full form for BFT and also for other abbreviations used in abstract

Author’s response: Corrected

Line 26: 260 Nile tilapia were distributed in 9 tanks at a density of 25 fish per tank. If you put 25 fish/tank and for 9 tanks the total would be 225 fish but here mentioned 260 fish. Please check the number of total fish or remove it from here.

Author’s response: Corrected

Line 27-29: The authors needs to revise the sentence because it is unclear.

Author’s response: Revised and improved

Please check table 3 temperature heading because it was in bold

Author’s response: Corrected

Authors have to check the units throughout the manuscript. For example please check the units in Table 3.

Author’s response: Units have been checked and corrected.

Please give units in y axis for figures 1-9. My suggestion is instead of using 9 figures to depict the results. Why can’t authors combine these 9 figures in to two separate figures and giving Nitrite, nitrate etc. as figure panels a,b,c….. like that. Please consider this suggestion

Author’s response: All the 9 figures has been combined as suggested.

Line 266: remove brackets

Author’s response: Removed

Line 281: globulin levels significantly higher or lower compared to the control group. Please check the line.

Author’s response: Corrected

Line 289: Please check the abbreviation for tapioca flour                                                       

Author’s response: Checked

Instead of giving all the results as tables. Authors may prefer to give some data of tables as figures. Consider this suggestion.

Author’s response: Possible tables has been converted into figures.

 

Reviewer 3 Report

Brief summary  

Following the COPE ethical guidelines for peer reviewers, I am pleased to submit the review report of the paper: Effects of different plant derived carbon sources (tapioca flour and sugarcane molasses) on water quality, growth performance, haematology, immune and antioxidant status of Oreochromis niloticus reared in biofloc cultured system.

I have read it carefully in its entirety, thanking the authors for their effort and dedication in its elaboration.

The Manuscript has as objective:

Effect of different carbon sources (sugarcane molasses and tapioca flour) on water characteristics, growth, haematology and non specific immune and antioxidant status of Oreochromis niloticus fingerlings.

The work is very complete, since it implies for its development a multidisciplinary team, due to the variables of study that they worked on, which includes productive aspects, blood study, antioxidant immune defenses in tilapia under BFT conditions with different carbon sources.

The work contains methodological elements that should be clarified: therefore, I have determined to suggest accepting the manuscript after major revisions.

 Areas of weakness: Methodological inaccuracies:

The authors claim to have performed a randomized experiment with three treatments, however, it seems confusing, as their study focuses on 2 carbon sources, and a free flowing water control, the correct in my opinion is:

T1: SM + BFT

T2: TF + BFT

Control: FT / 3% water replacement

All in triplicate, which is correct.

The water quality variables should be expressed correctly, as appropriate: ammonia (NH4+-N), nitrite (NO2--N) and nitrate (NO3--N) and explain in detail their operationalization. With is recommended in the body of the text.

Due to the weight of the Tilapias they are not fry, they are juveniles.  This aspect should be corrected in the text. And it is an important delimitation of the work.

 

The instruments used for the realization of the work should be described appropriately. Following the authors' guidelines.  Example: (YSI-650 Inc., Yellow Spring Instruments, Yellow Springs, OH, USA)

Specific comments:

Specific comments are included in the body of the text.

I suggest the authors to investigate which carbon sources have been studied with Tilapia with BFT, the variables they propose and write it in the introduction, (the current one is very brief), in order to justify and detect gaps in knowledge and thus strengthen their work. Some examples are:

https://doi.org/10.3390/ani11010184

https://doi.org/10.1016/j.fsi.2023.108805 

https://doi.org/10.3390/ani11072035

https://doi.org/10.1016/j.aquaculture.2022.738021

https://doi.org/10.1016/j.fsi.2022.09.031

The search string was:

https://scholar.google.es/scholar?hl=es&as_sdt=0%2C5&q=Biofloc+technology+and+immune+response+tilapia&oq=Biofloc+technology+and+immune+response+tilapia&oq=Biofloc+technology+and+immune+response+tilapia.

Authors are strongly encouraged to make their methodology clear and replicable, hence the importance of reviewing the empirical evidence.

Is the manuscript clear, relevant to the field, and presented in a well-structured manner?

Unfortunately, the methodology is not clear; in the body of the text I indicate observations that can be improved.

Are the references cited mostly recent (within the last 5 years) and relevant publications?

They are appropriate references, however they should focus on the ample existing evidence of carbon sources used in BFT in Tilapia, it is not necessary to analyze other species.

Does it include an excessive number of author citations?

No.

Is the manuscript scientifically sound and is the experimental design appropriate to test the hypothesis?

Should be improved from the introduction to the conclusion, the experiment is too short. The experimental design seems to have confused a control with a treatment. Paragraph 108.

Authors should argue for using Duncan's multiple range test. It is a stepwise comparison method. It controls the error rate by using, for the set of separated means r steps apart, a significance level aD = 1 - ( 1 -a/ . The more steps there are between two means, the greater the minimum difference at which these means will be considered to differ significantly. The method is more powerful than the Student-Neuwman-Keuls method, but it does not adequately protect type I error. It is also unreliable when the number of replicates is not equal in the treatments being compared, so its use is not generally recommended.

Are the results of the manuscript reproducible according to the details provided in the methods section?

No, the experimental protocol is not clear, especially the time required to set up the system with BFT. Dominated by bacteria these do not dominate the system as quickly as normally thought, this part was not reported.

Are the figures/tables/pictures/schemes appropriate, do they show the data correctly, are they easy to interpret and understand?

The tables are ok, the graphs should be improved in their visual quality, the behavior is strange, in my opinion it does not represent, a system with BFT. The curves of the variables are very stable during the whole test, which is not normal, I invite the authors to review the behavior of a system with BFT.

Are the data interpreted adequately and consistently throughout the manuscript?

They should be more organized, when presenting their results and discussion. Since the discussion is in another section, and they are not separated into subheadings.

Are the conclusions consistent with the evidence and arguments presented?

Should they be improved, recommendations are in the body of the text.

 

Evaluate ethics statements and data availability statements to ensure that they are adequate.

Without are stated in the manuscript.

General Comments (Included in the body of the text)

The importance and strength of the proposed hypotheses: Not presented.

 

The appropriateness and feasibility of the experimental and analytical methodology: Should be improved.

 

Whether sufficient detail has been provided to replicate the proposed experimental procedures and analysis; Unfortunately in the implementation of the BFT it was not well detailed.

  Whether there is sufficient outcome-neutral hypothesis testing, including positive controls and quality controls: Not stated

Comments for author File: Comments.zip

Author Response

Following the COPE ethical guidelines for peer reviewers, I am pleased to submit the review report of the paper: Effects of different plant derived carbon sources (tapioca flour and sugarcane molasses) on water quality, growth performance, haematology, immune and antioxidant status of Oreochromis niloticus reared in biofloc cultured system.

I have read it carefully in its entirety, thanking the authors for their effort and dedication in its elaboration.

The Manuscript has as objective:

Effect of different carbon sources (sugarcane molasses and tapioca flour) on water characteristics, growth, haematology and non specific immune and antioxidant status of Oreochromis niloticus fingerlings.

The work is very complete, since it implies for its development a multidisciplinary team, due to the variables of study that they worked on, which includes productive aspects, blood study, antioxidant immune defenses in tilapia under BFT conditions with different carbon sources.

The work contains methodological elements that should be clarified: therefore, I have determined to suggest accepting the manuscript after major revisions.

 Areas of weakness: Methodological inaccuracies:

The authors claim to have performed a randomized experiment with three treatments, however, it seems confusing, as their study focuses on 2 carbon sources, and a free flowing water control, the correct in my opinion is:

T1: SM + BFT

T2: TF + BFT

Control: FT / 3% water replacement

All in triplicate, which is correct.

Author’s response: Corrected as suggested

The water quality variables should be expressed correctly, as appropriate: ammonia (NH4+-N), nitrite (NO2--N) and nitrate (NO3--N) and explain in detail their operationalization. With is recommended in the body of the text.

Author’s response: The changes has been made.

Due to the weight of the Tilapias they are not fry, they are juveniles.  This aspect should be corrected in the text. And it is an important delimitation of the work.

Author’s response: Corrected, Juviniles or fingerlings are typically about the size of human finger.

 

 

Round 2

Reviewer 1 Report

1. The experimental design was unreasonable, the control group should not be the flow-through system, it should be the system with biofloc technology and a common carbon source, and comparability among different groups could be achieved.

Author’s response: In order to clearly evaluate the effect of biofloc technology with different carbon sources, it was necessary to use the flow through system in case of control group. This is also done by other researchers (https://doi.org/10.1016/j.fsi.2017.03.025, https://doi.org/10.1016/j.aquaculture.2019.734639)

Re: I still think that it would be more reasonable to use a standard carbon source in the control group. You should cited the above reference in the trial design of your manuscript to explain why the control group was the one with the flow through system.

2. Why the fish groups of biofloc technology were fed at the 75% daily feeding rate? An adequate basis should be provided.

Author’s response: In biofloc treatment groups with different carbon sources, the fish also fed on microbial floc. So we assess the effect of carbon source on floc production which ultimately enhance the fish growth.

Re: I know that fish might eat microbial floc, but why is it 75%, not 80% or 60% or other levels?

3. In the 2.8. Antioxidant parameters, only the CAT and SOD were determined, what about the GSH-Px, MAD, or T-AOC? More antioxidant parameters should be determined.

Author’s response: Due to availability of resources, we currently focus on the two mostly determined antioxidant parameters.

Re: There is nothing to say about this response.

4. Figure 1 to figure 9 were not suitable to publish in fishes. The related results should be presented in the tables.

Author’s response: Figures were combined into single one.

Re: the author did not understand the meaning of the suggestion. The figure is not necessary, those results should not be presented in the figures, they could be summarized in a table. I think the figures were not fit the requirement of Fishes.

5. In Table 4, is it right that the weight gain (%) is about 66%-98%? It seemed that the growth of tilapia could be not normal according the previous experiences.

Author’s response: This is possible because in other studies like Mansour and Esteban (https://doi.org/10.1016/j.fsi.2017.03.025); Bakshi et al. (https://doi.org/10.1111/are.13469) also revelaed the related results.

Re: for the most of similar tilapia study, the weight gain (%) is much higher than that in your manuscript, some reason should be explained in the discussion in detail. there might be some be problem in your trial, and the feeding condition might be not normal.

 

6. The discussion was not enough to explain the relation between the different parameters and treatment groups. Why were those parameters affected by different carbon sources? The related reason should be clarified.

Author’s response: Clarified and improved

Re: it is not well discussed that the reason why different carbon sources could affect the growth, haematological parameters, non-specific immune parameters, and antioxidant enzymatic activities. Each of those parameters should be explained thoroughly.

Author Response

1. The experimental design was unreasonable, the control group should not be the flow-through system, it should be the system with biofloc technology and a common carbon source, and comparability among different groups could be achieved.

Author’s response: In order to clearly evaluate the effect of biofloc technology with different carbon sources, it was necessary to use the flow through system in case of control group. This is also done by other researchers (https://doi.org/10.1016/j.fsi.2017.03.025, https://doi.org/10.1016/j.aquaculture.2019.734639)

Re: I still think that it would be more reasonable to use a standard carbon source in the control group. You should cited the above reference in the trial design of your manuscript to explain why the control group was the one with the flow through system.

Author’s response: No standard source of carbon has been reported yet in any study. The reference has been cited.

1. Why the fish groups of biofloc technology were fed at the 75% daily feeding rate? An adequate basis should be provided.

Author’s response: In biofloc treatment groups with different carbon sources, the fish also fed on microbial floc. So we assess the effect of carbon source on floc production which ultimately enhance the fish growth.

Re: I know that fish might eat microbial floc, but why is it 75%, not 80% or 60% or other levels?

Author’s response: In the biofloc treatments, feeding was 25% less than the control group. In these treatments, feeding was considered 75% artificial diet plus 25% produced biofloc in the rearing tanks. Reference has been added.

1. In the 2.8. Antioxidant parameters, only the CAT and SOD were determined, what about the GSH-Px, MAD, or T-AOC? More antioxidant parameters should be determined.

Author’s response: Due to availability of resources, we currently focus on the two mostly determined antioxidant parameters.

Re: There is nothing to say about this response.

1. Figure 1 to figure 9 were not suitable to publish in fishes. The related results should be presented in the tables.

Author’s response: Figures were combined into single one.

Re: the author did not understand the meaning of the suggestion. The figure is not necessary, those results should not be presented in the figures, they could be summarized in a table. I think the figures were not fit the requirement of Fishes.

Author’s response: Dear reviewer, we respect your suggestion, but in table 3, the same result of water quality parameters is already mentioned. The figures illustrate the weekly changes in parameters.

1. In Table 4, is it right that the weight gain (%) is about 66%-98%? It seemed that the growth of tilapia could be not normal according the previous experiences.

Author’s response: This is possible because in other studies like Mansour and Esteban (https://doi.org/10.1016/j.fsi.2017.03.025); Bakshi et al. (https://doi.org/10.1111/are.13469) also revelaed the related results.

Re: for the most of similar tilapia study, the weight gain (%) is much higher than that in your manuscript, some reason should be explained in the discussion in detail. there might be some be problem in your trial, and the feeding condition might be not normal.

 Author’s response: The lower weight gain was recorded only in control group as reported in several studies, but in BFT treatment groups the weight was found higher due to extra protein source in the form of microbial floc. The more detail has been added.

1. The discussion was not enough to explain the relation between the different parameters and treatment groups. Why were those parameters affected by different carbon sources? The related reason should be clarified.

Author’s response: Clarified and improved

Re: it is not well discussed that the reason why different carbon sources could affect the growth, haematological parameters, non-specific immune parameters, and antioxidant enzymatic activities. Each of those parameters should be explained thoroughly.

Author’s response: Biofloc have probiotic properties due to addition of carbon sources and is a source of different essential nutrients and enhance microbial floc which significantly affect the different parameters. Improved and discussed with reason.

 

Author Response File: Author Response.pdf

Reviewer 3 Report

Brief summary Round 2

Following the COPE ethical guidelines for peer reviewers, I am pleased to submit the review report of the paper: Effects of different plant-derived carbon sources (tapioca flour and sugarcane molasses) on water quality, growth performance, hematology, immune and antioxidant status of Oreochromis niloticus reared in biofloc cultured system. 

I have read it carefully in its entirety, thanking the authors for their effort and dedication in its elaboration. 

Authors' Responses to the Reviewer 

1.-It is advisable to address the summary briefly: Field of knowledge, the Problem and objective, knowledge gaps, methodology, Results, and Conclusion. 

Author's response: The suggested information has been added and the abstract is overall improved.

Reviewer's comments: 

Thank you for your attention, The suggestion has partially been addressed, I recommend in summary correcting: 

Based on the principle of nutrient recycling with the addition of a carbon source to give dominance to heterotrophic microorganisms. 

With a seeding density of organisms per 25 organisms per m3.

Nile tilapia with an initial body weight of 47.0 ± 1.3 g

Water quality parameters were found to be adequate for Tilapia production.

In the abstract change the word Fish to Tilapia, fish there are many, and your research focuses on Tilapia, therefore, use the appropriate word. 

The conclusion states: Improvement........., however, you should try to show an indicator as a function of the variables, the effect of the ...........  

Example: The effect of tapioca meal as a carbon source in the biofloc technology improved water quality by xx%, etc etc etc. 

An example summary for your support: 

The biofloc technology system (BFT) is considered one of the sustainable aquaculture systems, which is based on the principle of nutrient recycling with the addition of a carbon source to give dominance to heterotrophic microorganisms, derived that aquaculture generates waste by the metabolism of cultured organisms, carbon sources are a fundamental part for the development of this technology and evidence indicates that the most used in molasses , Therefore, the objective of this study was to evaluate the effect of sugar cane molasses and tapioca flour as a carbon source on water quality, growth, hematology, immune status and non-specific antioxidant status of Oreochromis juveniles. Methodologically, the experiment was carried out for 10 weeks, with 225 juvenile Nile tilapia with an initial body weight of 47.0 ± 1.3 g that were randomly distributed in 09 tanks (1000 L) with a stocking density of 25 tilapia per tank, the treatments were: BFT + SM (S molasses) and BFT + TF tapioca flour (TF) and a control with no carbon source added.  The control group was fed 100% feed, while the BFT experimental groups were fed microbial flocs along with 100% feed fed with microbial flocs and 75% feed. 

The results revealed that water quality parameters were affected by the carbon sources, but were adequate for normal fish welfare, the bio floc volume was higher (place Data) with the TF carbon source. Growth performance, such as growth performance, including weight gain, survival, and improved feed conversion ratio (FCR), was recorded in BFT + TF. (place Data).

Significant improvements were observed. (place Data) significant improvements in WBCs, HCT, HB, lymphocytes, plasma proteins, albumin, and non-specific immune factors (lysozyme activity, immunoglobulin levels and ACH50) in Tilapia reared in bio floc with tapioca meal as carbon source compared to control and sugar cane molasses, an increase (Data) in catalase (CAT) and superoxide dismutase (SOD) was observed in Tilapia. 

In conclusion, the use of tapioca meal can be used as a carbon source in biofloc technology was found to affect improving water quality, growth, hematology, and immunity of juvenile Tilapia growth, hematology, and immune and antioxidant status. 

Note for clarification: 

There is a difference in the abstract and methodology section here:  

BFT + SM ( S Molasses,) at 100% balanced feed and flocs. 

BFT + SM ( S Molasses,) at 75% feed and flocs 

BFT + TF tapioca meal (TF) at 100% of feed and flocs 

BFT + TF tapioca meal (TF) at 75% feed and flocs 

If so, there are not two treatments. It would be four, correct, and clarify in both sections.

 

2.-Line 28: Well, there is a saving in the supply of commercial feed. There is a savings in the supply of commercial feed, what percentage of protein was used in the commercial diet? With BFT the FCRs should be close to 1 to 1. And the reported FCRs are higher. 

Author's response: The protein percentage was 30%. It is not essential that FCR should be close to 1. In several studies, the FCR in BFT was found higher. Check the latest high impact studies (https://doi.org/10.1111/are.13469, https://doi.org/10.1016/j.aquaculture.2019.734639, https://doi.org/10.1016/j.fsi.2017.03.025).

 

Reviewer: 

Thank you very much for your answer, however, of the articles you mention, unfortunately only one of them refers to Nilotic tilapia, an aspect that is not correct to compare between species, in the initial stages of tilapia with BFT until the end of cycle the FCR should be close to 1.  

The results of Effects of carbon sources and plant protein levels in a biofloc system on growth performance and the immune and antioxidant status of Nile tilapia (Oreochromis niloticus) https://doi.org/10.1016/j.fsi.2017.03.025,is a clear example of the methodology that is appropriate to follow, and very important for discussion.

I recommend you look for articles with BFT/ molasses/ tilapia/ with longer experimental time and you will get a better idea of what I am referring to in terms of FCR. 

 

A clarification, the concept of high impact is not appropriate, the correct is to state, that this research was published in scientific journals included in the JCR and with an impact factor.  

 

The high impact in our field is relative, in the recent JCR listing the CA A Cancer Journal for clinicians its JIF is 254.7 and Fish & Shellfish Immunology has a JIF of 4.7, therefore it is not necessary to state a high impact factor. Each area of knowledge is handled differently.

 

Key contribution: In juvenile Tilapia, specify at laboratory level, it is not possible to generalize since the experiment was only 70 days. 2.5 months, that is less than half of a culture cycle. 

Author's response: 

Although the experiment was 70 days, overall the performance of fish was recorded better in BFT with TF source. In several other studies (https://doi.org/10.1016/j.fsi.2017.03.025, https://doi.org/10.1111/are.13469) the experimental period of fingerling tilapia was 10 weeks or less and they conclude and generalize their experiment

Reviewer: 

Thank you very much for your answer, however, my comments are suggestion based on evidence, in my previous report I send scientifically correct age states, and my duty to guide authors how to improve their work, for the above contribution states FISH Cultured (very general) fish there are many. 

The correct in my opinion could be: 

Tapioca meal effect as a carbon source improved water quality parameters, growth, blood profile, immunity and antioxidant status in juvenile Tilapia cultured biofloc technology with tapioca meal.

 

4.-Line 65: They are the most studied, due to their worldwide economic importance. search reference. 

Author's Response: Added

 

Reviewer 

Thank you very much for your reply, suggestion taken care of.

 

5.-Line 76: https://scholar.google.es/scholar?hl=es&as_sdt=0%2C5&q=Biofloc+technology+and+immune+response+tilapia&oq=Biofloc+technology+and+immune+response+tilapia&oq=Biofloc+technology+and+immune+response+tila 

This statement is not very precise. 

Author's Response: Removed

Reviewer 

It was just for consultation, and to improve with more evidence your work, the search string in Google academic was the keywords.

 

6.-Line 78: Because they are contradictory, it is better to state, the results differ due to ........ 

Author's response: Sentence structure has been changed

Reviewer 

Suggestion addressed.

7.-Line 85: Reference 

Author's Response: Added

Reviewer 

Suggestion addressed.

 

8.-Line 86: Yes, there is research, you should investigate a little more, or some author claims that. 

I have attached CVS files for your analysis. 

Author's Response: Sentence has been changed 

 

Reviewer 

Suggestion addressed.

 

9.-line 88: The objective must coincide with the one written in the abstract. 

Author's response: Corrected 

Reviewer 

Suggestion addressed.

 

10.-Line 110: But it is observed that they started with 47.0 grams and 1.3 of SD with BFT, which should be their initial weight, the pre-fattening stage should not be considered in the calculations. 

Author's response: Corrected

Reviewer 

Suggestion addressed.

 

11.-line 129: Type of aeration, blower, what power in HP? 8.5 g/lL salinity in Tilapia should have an effect to consider and should be discussed. Why measure salinity if it is fresh water? or indicate the source water with its physicochemical analysis.

 

Author's response: Type of aeration has been added, Tilapia can tolerate high salinity levels and this is also reported in different studies that the current conc. level does not affect fish. Salinity is an important parameter in BFT that should be monitored and raw salt in BFT minimizes the risk of infection by fungi and external bacteria, and also prevents nitrite toxicity.

 

Reviewer 

Partially addressed. Compressor capacity was not included in the methodology. 

"A continuous aeration system was installed in the tank and the temperature of the culture water was maintained using an electric heater at 27.0 ± 1°C"

Please mention this is important.

On the other hand, what you state regarding salinities should always be answered with evidence. The water source and water analyses were not included.  Please report them.

 

12 Table 2: add the Carbon-Nitrogen Ratio of the external sources during the experiment. the initial, the final and how did you determine to add it to the system during the 70 days? what was the protocol? 

Author's response: 

The carbon sources were added once a day based on the calculation as described by Avnimelech (2009). The C: N ratio was considered 15:1. The carbon sources were mixed in a beaker with the water tank and distributed evenly over the tank surface immediately after feeding. The addition of carbon sources to one gram of feed depends on the assimilation and assumption of nitrogen in the system. The quantity of carbon sources daily added is given in Table 2 and their ratio is also added.

Reviewer 

Suggestion taken care of.

 

13.- Correct the way of reporting the instruments according to this guide: 

The water quality of the breeding water, DO, pH, water temperature, and electrical conductivity (EC) were measured for a week using a multi-item water quality meter (YSI-650 Inc., Yellow Spring Instruments, Yellow Springs, OH, USA) It is also recommended to review the authors' guide. 

https://doi.org/10.3390/fishes8080422 

Author's response: Corrected and improved

Reviewer 

Suggestion addressed.

 

14.-Figure 7: The system was at low load, 25 org/m3, with an assumed final weight of 500 grams, 12.5 kg/m3 to reach commercial size, which is good for aquaculturists but not the purpose of the BFT (semi-intense, low for the experimental size). It is necessary to increase the carrying capacity of the system, the graphs should be improved and the data should be reviewed, If the behavior is not consistent with those reported in the literature, the authors should review, discuss, and analyze the data with more evidence. The behavior of the physicochemical parameters is not observed in the ammonium peaks versus the control, due to the turnover which is a natural behavior. The maturation time of the system is not observed in the graphs, I regret to note that in my opinion, the data are not appropriate for the behavior of a culture system with BFT.

Author's response: The data has been reviewed and found correct. The ammonia level reported in the current study is also consistent with other studies DOI 10.2478/aoas-2022-0025)

Reviewer 

Suggestions can be improved

Dear authors, the ammonia levels are consistent with a mature system in BFT... but because of the low loading and level of the experiment both in the control and the two treatments in three treatments the water quality values are optimal for juvenile Tilapia development, 

Normally it should be noted, that each time a replacement is performed in the Control: FT / 30% water replacement, its periodicity is not indicated. It is here where the ammonia peaks should be observed and this is where the control with the BFT comes in... this is what I am trying to make you understand, but such a short study cannot be observed.

 

Alkalinity was not reported, if possible.

The authors should discuss versus optimal levels in water quality for tilapia production with respect to the results of their research, and clarify the effect of TSS and care for it as a recommendation. 

I recommend reading: 

The basics of bio-flocs technology: The added value for aquaculture.

Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2008.02.019

 

15.-Figure 8: TSS levels are high in contrast to the control, something is going on, the data is better in the control than with BFT. 

Why use BFT, if the other variables are within range? and salinity and TSS are elevated. 

Author's response: 

Normally TSS level in BFT is high due to addition of carbon sources and different particulate organic matters. Also the salinity level also increased in different studies. See 3 

the high quality JCR papers (https://doi.org/10.1007/s10499-020-00627-9, DOI:10.2478/aoas-2022-0025)

Reviewer 

Suggestion taken care of, include it in the manuscript.

To facilitate the review process.  When sending references to respond to a reviewer I recommend writing what is stated in the paper, it is not appropriate, read your manuscript, and that you send a DOI as justification for the reviewer to read another manuscript, it is much better:

According to Khanjani, MH, Alizadeh, M. & Sharifinia (2021) and Suleiman, A. & Abdel-Tawwab, M. (2022) and answer the question with available empirical evidence.

  

Figure 9: Data within appropriate limits for Tilapia. 

Author's response: The data of biofloc volume was found suitable for tilapia.

Reviewer 

Suggestion addressed

 It is correct, but they should put the evidence when answering, values less than 50 is appropriate, but they should look for the reference.

 

16.-Table 4: FCR levels are high, not consistent with Biofloc technology, do you have an analysis of the bacterial community, microscopic photographs, if copepods, nematodes, etc. were established? 

Author's response:

 In BFT,FCR doesn't need tod be lower or close to 1. In several studies,s as I mentioned above, the result was found consistent.

Reviewer 

Suggestion partially addressed

You do not answer the question properly, please look it up, many studies (which ones ?) should place the evidence when answering, I attach support of the evidence from review articles that I recommend for your work and future experiments.

 

10.1111/raq.12352

10.1016/j.aquaculture.2022.738021

10.1111/raq.12713

10.1111/raq.12649

10.1111/raq.12781

10.1016/j.sciaf.2021.e01053

10.1007/s10499-023-01162-z

10.3389/fmars.2022.975042

10.1016/j.aaf.2023.07.006

10.1111/raq.12617

10.1515/bot-2020-0007

10.1016/j.scitotenv.2023.165044

10.1111/raq.12412

10.1111/raq.12494

10.1007/s11356-023-25601-9

10.2478/aoas-2023-0044

10.1016/j.aaf.2022.08.010

10.1016/j.cofs.2021.01.001

10.1016/j.aqrep.2022.101300

10.1111/raq.12520

10.1111/raq.12379

10.1111/are.13728

10.1016/j.fsi.2023.108805

10.1007/s11356-022-22371-8

10.1016/j.heliyon.2021.e08283

10.33073/PJM-2020-049

10.1007/s10499-016-0108-8

10.3390/w14244019

10.1111/raq.12431

10.3109/07388551.2016.1144043

10.1016/j.margen.2022.100967

10.3390/su13137255

10.1016/j.micres.2022.127239

10.1111/raq.12744

10.1061/(ASCE)EE.1943-7870.0001850

10.1016/j.jenvman.2023.118237

10.1111/raq.12770

10.3390/antiox12020398

10.1080/01904167.2020.1739308

10.1016/j.aaf.2020.05.005

10.1016/j.tplants.2019.06.005

10.1007/s10499-017-0209-z

10.3389/fmicb.2021.741164

10.1021/es300110x

10.1016/j.aquaculture.2019.734675

10.1007/s10499-023-01091-x

10.1016/j.fsi.2023.108796

10.3389/fnut.2021.791738

10.1007/s10499-021-00681-x

10.1007/s10499-021-00781-8

10.1016/j.jenvman.2021.112271

10.1111/raq.12758

10.1016/j.aquaculture.2012.04.046

10.1111/raq.12408

10.1111/raq.12285

 

17.-line 342: the authors do not speak of costs, therefore, there are no elements for discussion. 

Author's response: Removed

Reviewer 

Suggestion addressed

18.-line 385: With the results found for 10 weeks, do you really consider that there is evidence for fish farmers to adopt this carbon source? 

Author's response: 

This study provides new insight for future studies that can consider using TF as a carbon source on a larger scale with a long duration. This will enable us to understand better TF's effect on fish health and final yield.

 

Reviewer 

Suggestion addressed, they do not answer the question, but address a future line of research that should be included in the manuscript with the limitations of the study.

 

 

19.-33. Reference: There is no rule regarding references, but for the study variables, I suggest adding as much evidence as possible of the effects of carbon sources with respect to the study variables, for a proper discussion. 

What future lines of research do these authors propose? 

To take elements from the reviews for their introduction and discussion. 

this is their theoretical evidence 

Author's response: Changes has been made as suggested 

 

It is suggested that in future studies a mixture of simple and complex carbon sources in different ratios will be used and evaluated on performance of the Nile tilapia.

 

Reviewer 

The questions were not answered, Suggestion partially addressed. I suggest including them in the text.

Finally, I recommend the authors to include the text of responses to reviewers in future revisions:

1.-Write the reviewer suggestions:  

2.-write the change in manuscript, because the original lines change.

3.-Add text in the manuscript. 

Success in your work. 

The graphs should be made with another graph manager of better quality.

 

 

 

Author Response

Reviewer comments: 

1.-It is advisable to address the summary briefly: Field of knowledge, the Problem and objective, knowledge gaps, methodology, Results, and Conclusion. 

Thank you for your attention, The suggestion has partially been addressed, I recommend in summary correcting: 

Based on the principle of nutrient recycling with the addition of a carbon source to give dominance to heterotrophic microorganisms. 

With a seeding density of organisms per 25 organisms per m3.

Nile tilapia with an initial body weight of 47.0 ± 1.3 g

Water quality parameters were found to be adequate for Tilapia production.

In the abstract change the word Fish to Tilapia, fish there are many, and your research focuses on Tilapia, therefore, use the appropriate word. 

The conclusion states: Improvement........., however, you should try to show an indicator as a function of the variables, the effect of the ...........  

Example: The effect of tapioca meal as a carbon source in the biofloc technology improved water quality by xx%, etc etc etc. 

An example summary for your support: 

The biofloc technology system (BFT) is considered one of the sustainable aquaculture systems, which is based on the principle of nutrient recycling with the addition of a carbon source to give dominance to heterotrophic microorganisms, derived that aquaculture generates waste by the metabolism of cultured organisms, carbon sources are a fundamental part for the development of this technology and evidence indicates that the most used in molasses , Therefore, the objective of this study was to evaluate the effect of sugar cane molasses and tapioca flour as a carbon source on water quality, growth, hematology, immune status and non-specific antioxidant status of Oreochromis juveniles. Methodologically, the experiment was carried out for 10 weeks, with 225 juvenile Nile tilapia with an initial body weight of 47.0 ± 1.3 g that were randomly distributed in 09 tanks (1000 L) with a stocking density of 25 tilapia per tank, the treatments were: BFT + SM (S molasses) and BFT + TF tapioca flour (TF) and a control with no carbon source added.  The control group was fed 100% feed, while the BFT experimental groups were fed microbial flocs along with 100% feed fed with microbial flocs and 75% feed. 

The results revealed that water quality parameters were affected by the carbon sources, but were adequate for normal fish welfare, the bio floc volume was higher (place Data) with the TF carbon source. Growth performance, such as growth performance, including weight gain, survival, and improved feed conversion ratio (FCR), was recorded in BFT + TF. (place Data).

Significant improvements were observed. (place Data) significant improvements in WBCs, HCT, HB, lymphocytes, plasma proteins, albumin, and non-specific immune factors (lysozyme activity, immunoglobulin levels and ACH50) in Tilapia reared in bio floc with tapioca meal as carbon source compared to control and sugar cane molasses, an increase (Data) in catalase (CAT) and superoxide dismutase (SOD) was observed in Tilapia. 

In conclusion, the use of tapioca meal can be used as a carbon source in biofloc technology was found to affect improving water quality, growth, hematology, and immunity of juvenile Tilapia growth, hematology, and immune and antioxidant status. 

Note for clarification: 

There is a difference in the abstract and methodology section here:  

BFT + SM ( S Molasses,) at 100% balanced feed and flocs. 

BFT + SM ( S Molasses,) at 75% feed and flocs 

BFT + TF tapioca meal (TF) at 100% of feed and flocs 

BFT + TF tapioca meal (TF) at 75% feed and flocs 

If so, there are not two treatments. It would be four, correct, and clarify in both sections.

Author’s response: The summary has been corrected as suggested by the reviewer, I really appreciate the reviewer suggestion and support. Further, there were two treatments and is corrected in abstract and methodology.

Thank you very much for your answer, however, of the articles you mention, unfortunately only one of them refers to Nilotic tilapia, an aspect that is not correct to compare between species, in the initial stages of tilapia with BFT until the end of cycle the FCR should be close to 1.  

The results of Effects of carbon sources and plant protein levels in a biofloc system on growth performance and the immune and antioxidant status of Nile tilapia (Oreochromis niloticus) https://doi.org/10.1016/j.fsi.2017.03.025,is a clear example of the methodology that is appropriate to follow, and very important for discussion.

I recommend you look for articles with BFT/ molasses/ tilapia/ with longer experimental time and you will get a better idea of what I am referring to in terms of FCR. 

A clarification, the concept of high impact is not appropriate, the correct is to state, that this research was published in scientific journals included in the JCR and with an impact factor.  

The high impact in our field is relative, in the recent JCR listing the CA A Cancer Journal for clinicians its JIF is 254.7 and Fish & Shellfish Immunology has a JIF of 4.7, therefore it is not necessary to state a high impact factor. Each area of knowledge is handled differently.

Author’s response: The suggested article has been followed. In future long term studies will be consider. Thanks for your guidance.

Key contribution: In juvenile Tilapia, specify at laboratory level, it is not possible to generalize since the experiment was only 70 days. 2.5 months, that is less than half of a culture cycle. 

Thank you very much for your answer, however, my comments are suggestion based on evidence, in my previous report I send scientifically correct age states, and my duty to guide authors how to improve their work, for the above contribution states FISH Cultured (very general) fish there are many. 

The correct in my opinion could be: 

Tapioca meal effect as a carbon source improved water quality parameters, growth, blood profile, immunity and antioxidant status in juvenile Tilapia cultured biofloc technology with tapioca meal

Author’s response: Corrected as suggested.

11.-line 129: Type of aeration, blower, what power in HP? 8.5 g/lL salinity in Tilapia should have an effect to consider and should be discussed. Why measure salinity if it is fresh water? or indicate the source water with its physicochemical analysis.

Partially addressed. Compressor capacity was not included in the methodology. 

"A continuous aeration system was installed in the tank and the temperature of the culture water was maintained using an electric heater at 27.0 ± 1°C"

Please mention this is important.

On the other hand, what you state regarding salinities should always be answered with evidence. The water source and water analyses were not included.  Please report them.

Author’s response: A continuous aeration system (1.5 hp blower) was installed in the tank and the temperature of the culture water was maintained using an electric heater at 27.0 ± 1°C.

Water source has been added. (Well water at salinity 15 ppt was used).

14.-Figure 7: The system was at low load, 25 org/m3, with an assumed final weight of 500 grams, 12.5 kg/m3 to reach commercial size, which is good for aquaculturists but not the purpose of the BFT (semi-intense, low for the experimental size). It is necessary to increase the carrying capacity of the system, the graphs should be improved and the data should be reviewed, If the behavior is not consistent with those reported in the literature, the authors should review, discuss, and analyze the data with more evidence. The behavior of the physicochemical parameters is not observed in the ammonium peaks versus the control, due to the turnover which is a natural behavior. The maturation time of the system is not observed in the graphs, I regret to note that in my opinion, the data are not appropriate for the behavior of a culture system with BFT.

Suggestions can be improved

Dear authors, the ammonia levels are consistent with a mature system in BFT... but because of the low loading and level of the experiment both in the control and the two treatments in three treatments the water quality values are optimal for juvenile Tilapia development, 

Normally it should be noted, that each time a replacement is performed in the Control: FT / 30% water replacement, its periodicity is not indicated. It is here where the ammonia peaks should be observed and this is where the control with the BFT comes in... this is what I am trying to make you understand, but such a short study cannot be observed.

Author’s response: The changes has been made as suggested.

Alkalinity was not reported, if possible.

The authors should discuss versus optimal levels in water quality for tilapia production with respect to the results of their research, and clarify the effect of TSS and care for it as a recommendation. 

I recommend reading: 

The basics of bio-flocs technology: The added value for aquaculture.

Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2008.02.019

Author’s response: Alkalinity was not measured. The optimum levels of water quality for nile tilapia has been discussed and compare with results of the current study. The suggested article was helpful.

To facilitate the review process.  When sending references to respond to a reviewer I recommend writing what is stated in the paper, it is not appropriate, read your manuscript, and that you send a DOI as justification for the reviewer to read another manuscript, it is much better:

According to Khanjani, MH, Alizadeh, M. & Sharifinia (2021) and Suleiman, A. & Abdel-Tawwab, M. (2022) and answer the question with available empirical evidence.

Author’s response: Noted

16.-Table 4: FCR levels are high, not consistent with Biofloc technology, do you have an analysis of the bacterial community, microscopic photographs, if copepods, nematodes, etc. were established? 

Reviewer 

Suggestion partially addressed

You do not answer the question properly, please look it up, many studies (which ones ?) should place the evidence when answering, I attach support of the evidence from review articles that I recommend for your work and future experiments.

 

10.1111/raq.12352

10.1016/j.aquaculture.2022.738021

10.1111/raq.12713

10.1111/raq.12649

10.1111/raq.12781

10.1016/j.sciaf.2021.e01053

10.1007/s10499-023-01162-z

10.3389/fmars.2022.975042

10.1016/j.aaf.2023.07.006

10.1111/raq.12617

10.1515/bot-2020-0007

10.1016/j.scitotenv.2023.165044

10.1111/raq.12412

10.1111/raq.12494

10.1007/s11356-023-25601-9

10.2478/aoas-2023-0044

10.1016/j.aaf.2022.08.010

10.1016/j.cofs.2021.01.001

10.1016/j.aqrep.2022.101300

10.1111/raq.12520

10.1111/raq.12379

10.1111/are.13728

10.1016/j.fsi.2023.108805

10.1007/s11356-022-22371-8

10.1016/j.heliyon.2021.e08283

10.33073/PJM-2020-049

10.1007/s10499-016-0108-8

10.3390/w14244019

10.1111/raq.12431

10.3109/07388551.2016.1144043

10.1016/j.margen.2022.100967

10.3390/su13137255

10.1016/j.micres.2022.127239

10.1111/raq.12744

10.1061/(ASCE)EE.1943-7870.0001850

10.1016/j.jenvman.2023.118237

10.1111/raq.12770

10.3390/antiox12020398

10.1080/01904167.2020.1739308

10.1016/j.aaf.2020.05.005

10.1016/j.tplants.2019.06.005

10.1007/s10499-017-0209-z

10.3389/fmicb.2021.741164

10.1021/es300110x

10.1016/j.aquaculture.2019.734675

10.1007/s10499-023-01091-x

10.1016/j.fsi.2023.108796

10.3389/fnut.2021.791738

10.1007/s10499-021-00681-x

10.1007/s10499-021-00781-8

10.1016/j.jenvman.2021.112271

10.1111/raq.12758

10.1016/j.aquaculture.2012.04.046

10.1111/raq.12408

10.1111/raq.12285

 

Author’s response: Different studies have been studied and the FCR value close to 1 was recorded in the studies of longer duration. So, we discuss the values of FCR observed in different studies and compared with our study and explained the reason that this might be due to short study duration.

19.-33. Reference: There is no rule regarding references, but for the study variables, I suggest adding as much evidence as possible of the effects of carbon sources with respect to the study variables, for a proper discussion. 

What future lines of research do these authors propose? 

To take elements from the reviews for their introduction and discussion. 

this is their theoretical evidence 

Reviewer:

Suggestion partially addressed. I suggest including them in the text.

Author’s response: Different studies have been discussed and included them in text. Also compare with the values recorded in current study.

The graphs should be made with another graph manager of better quality.

Author’s response: Quality has been improved and regenerated.

 

 

Author Response File: Author Response.docx

Round 3

Reviewer 1 Report

This manuscript has been made some modification, and there were some problems in the revised manuscript.

 

1.In the introduction, the authors should review the progress studies of carbon sources application in the biofloc technology system in aquaculture, and tell the readers that why you choose those two carbon sources in present study.

 

2.The tilapia increased from 27.0 g/fish to 47.0 g/fish during the acclimatized (indoor) 14 days, the WGR was about 74%, while in the formal trial, the WGR of fish after 10 weeks was only 66% in the control group. why was there so great difference? It seemed that the fish were fed the same diet in the acclimatized 14 days and the formal trial period.

 

3.Please use the abbreviations (SM and TF) of sugarcane molasses and tapioca flour correctly. The other abbreviations should be also noticed.

 

4.The P value in the manuscript should be italic，please check it in the manuscript.

 

5.In your manuscript, following are the treatment protocols for the control and BFT groups:

T1: SM + BFT
T2: TF + BFT
Control: FT / 30% water replacement.

 

6.Please show those in the order, control, BFT + SM (not SM + BFT) and BFT +TF (not TF + BFT). The names of different groups should be consistent in the whole manuscript.

 

7.The tables in present manuscript should be reorganized according to the format of Fishes.

 

8.In the discussion part, please explain why the growth performance and other parameters in BFT +TF groups was higher than those of BFT + SM groups,

Author Response

1.In the introduction, the authors should review the progress studies of carbon sources application in the biofloc technology system in aquaculture, and tell the readers that why you choose those two carbon sources in present study.

Author’s response: Added to the introduction section

2.The tilapia increased from 27.0 g/fish to 47.0 g/fish during the acclimatized (indoor) 14 days, the WGR was about 74%, while in the formal trial, the WGR of fish after 10 weeks was only 66% in the control group. why was there so great difference? It seemed that the fish were fed the same diet in the acclimatized 14 days and the formal trial period.

Author’s response: This might be due to experimental duration and the water quality particularly salinity effect.

3.Please use the abbreviations (SM and TF) of sugarcane molasses and tapioca flour correctly. The other abbreviations should be also noticed.

Author’s response: Corrected

4.The P value in the manuscript should be italic，please check it in the manuscript.

Author’s response: Converted into italic 

5.In your manuscript, following are the treatment protocols for the control and BFT groups:

T1: SM + BFT
T2: TF + BFT
Control: FT / 30% water replacement.

Please show those in the order, control, BFT + SM (not SM + BFT) and BFT +TF (not TF + BFT). The names of different groups should be consistent in the whole manuscript.

Author’s response: Corrected in the whole manuscript

6.The tables in present manuscript should be reorganized according to the format of Fishes.

Author’s response: Reorganized according to the journal format 

7.In the discussion part, please explain why the growth performance and other parameters in BFT +TF groups was higher than those of BFT + SM groups,

Author’s response: BFT + TF shows better results, this might be because the tapioca contains more than 90% carbohydrate which is important for the growth of microbes. Thus provides extra protein source for fish. The details have been added.

 

Author Response File: Author Response.pdf