Effects of Di-(2-Ethylhexyl) Phthalate (DEHP) on Gamete Quality Parameters of Male Koi Carp (Cyprinus carpio)

Round 1

Reviewer 1 Report

 

The manuscript entitled “Effects of Di-(2-Ethylhexyl) Phthalate (DEHP) on Gamete Quality Parameters of Male Koi Carp (Cyprinus carpio)” aims to increase the knowledge on the effects of di-(2-ethylhexyl) phthalate (DEHP) exposure on male reproduction in fish. The study presents data on testis histoarchitecture and semen quality in male koi carp exposed to nominal 1, 10, and 100μg/L DEHP. The study is well organized in all its sections and in line with other similar studies on different species present in the literature and also presented by the same authors (Bisai et al., 2022, on common carp?). The introduction is streamlined and clear, the experimental protocol well explained, the data obtained presented clearly and adequately discussed. The manuscript may be published with minor revisions and an English language check, as detailed below.

Title

One question to the Authors. I know that the koi carp is a domesticated ornamental variety of the common carp: what is the criterion by which Cyprinus carpio is defined as common carp or koi carp? Are the same fish as in Bisai et al., 2022? Isn't there any way to clarify this aspect? I know that sometimes  it is referred to as Cyprinus rubrofuscus "koi".

Abstract

Sentence “Di-(2-ethylhexyl) phthalate also known as DEHP is a frequently used plasticizer chemical and considered emerging environmental pollutant is reported to have severe impacts on animals.“ should be modified as “Di-(2-ethylhexyl) phthalate, also known as DEHP, is a frequently used plasticizer chemical considered emerging environmental pollutant and reported to have severe impacts on animals.”

“……….in the GSI of 10 and 100 µg/l DEHP treated males.” Exposed males is better

“…decreased and suppressed” “and” is not possible

“In the treatment group, sperm……..” please delate “In the treatment group,” and start directly with “Sperm motility……”

“The biochemical compositions of the seminal fluid were estimated…..” should be changed as “The biochemical composition of the seminal fluid was estimated….”

Introduction

“This compound is not chemically bound to plastics and frequently leaches out in a liquid of these products into the surrounding environment [4,5] and therefore responsible for contaminating those which are in close contact [6].” Please differentiate a little bit this sentence. It is the same as in Bisai et al., 2022.

Material and Methods

Page 3, Line 5. In which solvent has been diluted the DEHP? The control group was treated with the xxx% of the same solvent alone? It is evident from the text, but it is better to declare it.

Did the authors carry out a check to get an idea of the real concentration?

Page 3 Line 13. The temperature is a little bit lower, but the water quality parameters reported here are exactly the same as those in Bisai et al., 2022. How is it possible?

Page 3. “After the post exposure of 60 days….” Please delete “post”

Page 3. “Using a rotary microtome (Leica RM 2025, Leica Biosystems, Germany), paraffin fixed tissues were sectioned at a ……….” This sentence should be reworded as follows: “After being fixed in Bouin’s solution for 24hrs, paraffin embedded tissues were sectioned at a thickness of 5µm, using a rotary microtome (Leica RM 2025, Leica Biosystems, Germany).”

Page 3. “The concentrations of DEHP were determined from the pooled samples of liver and testes of each experimental group….”. One question for the Authors: why pool liver and testis for DEHP detection? It would have been much more interesting to have the two results separated and to know the direct effect of the presence of DEHP in the testis.  

Page 5. “…….equilibrated with room temperature…….” better “at room temperature”

Results

Page 6, first paragraph. “60 days exposure of DEHP, significantly decreased the GSI of medium (10 µg/l) and higher concentration (100 µg/l) of DEHP treated males, but not in lower concentration (1µg/l) of DEHP treated males in comparison with the corresponding control males (Figure 1). ……………………..” Should be simplified as follows: “GSI significantly decreased in males exposed for 60 days to the medium (10 µg/l) and highest concentration (100 µg/l) of DEHP, but not in lower concentration (1µg/l), in comparison with control males (Figure 1). In control group, the GSI was 12.84 ± 0.54 (%), while in 100 µg/l DEHP treated group the value was found to be 6.86 ± 0.56 (%).” Here GSI after 10 µg/l DEHP exposure is missing……

Page 7, L3-5. “The testes of medium concentration (10 µg/l), showed significant changes like presence of large number of inflammatory cells and large number of intratubular vacuoles (Figure 2c).” should be simplified as follows:  “At the medium concentration (10 µg/l), increasing numbers of inflammatory cells and intratubular vacuoles (Figure 2c) were observed.”

Page 8, final part. “Semen volumes were significantly decreased in order of increasing concentration of DEHP as compared to control group (Figure 4). The semen volume was 1.28 ± 0.02 ml in control group. However, decreased volume of semen (0.84 ± 0.01 ml) was recorded in 100 µg/l DEHP treated group. There ……..” should be modified as follows: “Semen volumes were significantly decreased at the increasing of DEHP concentrations, as compared to control group (Figure 4). The semen volume was 1.28 ± 0.02 ml in control group, while x.xx±x.xx ml, , x.xx±x.xx ml and x.xx±x.xx ml were recorded in 1 µg/l, 10 µg/l and 100 µg/l DEHP, respectively.” If you enter a numerical value, you must enter those of all the experimental groups.

Page 9, below. “Different doses of DEHP exposed fishes showed significant decreases in sperm motility as well as sperm motility durations as compared to control group (Figure 6).” Are these decreases dose-dependent? In the following sentence, instead of "However" is better "while", and this applies to all However up to the end of the text.

Page 12 “However, the level was remained unchanged in 1 µg/l DEHP treated group compare to control group (Figure 10a).” two very small oversights: was remained and compare, please delete was and change compare to compared.

Discussion

Page 13, first sentence. “The aim of this study was to explore the effects of DEHP exposure on gamete quality parameters of male fishes with environmentally relevant concentrations.” Should be better: “The aim of this study was to explore the effects on gamete quality parameters in C. carpio males after 60 days exposure to environmentally relevant concentrations of DEHP.”

Page 14. “The fishes of DEHP exposed groups also showed significant dose-dependent decreases in case of sperm density.” Please change “in case of sperm density.” To “of sperm density.”

Page 14. “No such work so far has been done on this aspect in case of fish and to best of our knowledge, …….” Should be modified to “In our knowledge, this is the first study………”

Page 14. “In the present study, we analysed………..of reproductive success of any teleost including fish.” This last parte of the sentence should be modified to “……of reproductive success in teleosts”.

Page 15, first line. Please change reduced to reduce.

Tables. Table 1 and 2 are ok, but the Primers are Table 1 and Biochemical parameters Table 2 (please check the text, too)

Figure 2. The use of coloured arrows is confusing since the same kind of arrow points different things in the pictures (ie. in a. red arrow indicates intact seminiferous tubules, in b. inflammatory cells, in c. vacuolization and in d. necrosis.  Authors should use a unique way to highlight the same morphological aspect and modify the caption accordingly.

 

 

Some minor oversights

Author Response

The manuscript entitled “Effects of Di-(2-Ethylhexyl) Phthalate (DEHP) on Gamete Quality Parameters of Male Koi Carp (Cyprinus carpio)” aims to increase the knowledge on the effects of di-(2-ethylhexyl) phthalate (DEHP) exposure on male reproduction in fish. The study presents data on testis histoarchitecture and semen quality in male koi carp exposed to nominal 1, 10 and 100μg/L DEHP. The study is well organized in all its sections and in line with other similar studies on different species present in the literature and also presented by the same authors (Bisai et al., 2022, on common carp?). The introduction is streamlined and clear, the experimental protocol well explained, the data obtained presented clearly and adequately discussed. The manuscript may be published with minor revisions and an English language check, as detailed below.

Title

One question to the Authors. I know that the koi carp is a domesticated ornamental variety of the common carp: what is the criterion by which Cyprinus carpio is defined as common carp or koi carp? Are the same fish as in Bisai et al., 2022? Isn't there any way to clarify this aspect? I know that sometimes it is referred to as Cyprinus rubrofuscus "koi"

• We agree with reviewer’s comment. There are some confusions between common carp and koi carp. However, as per the reviewer, koi carp is a domesticated ornamental variety of the common carp. In our previous study (Bisai et al., 2022), we have used same species for different experiment named as common carp. But, some recent publication of International Journal (DOI as following) considered koi carp as Cyprinus carpio.

https://doi.org/10.1016/j.aquaculture.2022.738926

https://doi.org/10.1016/j.aquaculture.2022.739030

https://doi.org/10.1016/j.cbpb.2022.110814

https://doi.org/10.1186/s12917-023-03621-4

https://doi.org/10.1111/jfd.13781

In this study mature male koi carps (Figure is given below) were obtained from a local fish dealer and during the expression study of reproductive related genes, the cytochrome c oxidase subunit I (COX1) gene (widely used for species identification) of the koi carp was amplified and found 100% similarity with Cyprinus carpio. The sequence has been submitted to NCBI GenBank and the Accession number is OP984825. Therefore, in the present study we named koi carp as Cyprinus carpio.

 

 Abstract

Sentence “Di-(2-ethylhexyl) phthalate also known as DEHP is a frequently used plasticizer chemical and considered emerging environmental pollutant is reported to have severe impacts on animals”. should be modified as “Di-(2-ethylhexyl) phthalate, also known as DEHP, is a frequently used plasticizer chemical considered emerging environmental pollutant and reported to have severe impacts on animals.”

 

• As per the reviewer’s suggestion, we have modified the sentence accordingly in revised manuscript.

“……….in the GSI of 10 and 100 µg/l DEHP treated males.” Exposed males is better

• As suggested, we have changed it in revised manuscript.

“…decreased and suppressed” “and” is not possible

• We agreed with the reviewer’s suggestion. The change was made in revised manuscript.

“In the treatment group, sperm……..” please delate “In the treatment group,” and start directly with “Sperm motility……”

• As suggested, we have modified the sentence in revised manuscript.

“The biochemical compositions of the seminal fluid were estimated…..” should be changed as “The biochemical composition of the seminal fluid was estimated….”

• As per the suggestion, we have modified the sentence accordingly in revised manuscript.

 

Introduction

“This compound is not chemically bound to plastics and frequently leaches out in a liquid of these products into the surrounding environment [4,5] and therefore responsible for contaminating those which are in close contact [6].” Please differentiate a little bit this sentence. It is the same as in Bisai et al., 2022.

 

• We are sorry for the mistake. The sentence has been modified in revised manuscript.

 

Material and Methods

Page 3, Line 5. In which solvent has been diluted the DEHP? The control group was treated with the xxx% of the same solvent alone? It is evident from the text, but it is better to declare it.

 

• We agree with the reviewer’s comment, but it is mentioned previously in the sub-section 2.1. Chemicals Used (the first sub-section of Materials and Methods).

Did the authors carry out a check to get an idea of the real concentration?

• As per the reviewer query, there were three DEHP treated group and one control group. Three different concentrations of DEHP were 1, 10 and 100 µg/l, respectively. DEHP was dissolved in acetone (100 g/l) to create the stock solution and each group including control group contained 0.001% (v/v) acetone as a solvent. We have detected the DEHP in liver and testis tissue of each treated group by GC-MS technology but not from the water samples of glass tank.

Page 3 Line 13. The temperature is a little bit lower, but the water quality parameters reported here are exactly the same as those in Bisai et al., 2022. How is it possible?

• We agree with the reviewer’s comment. Present study and previously work published by Bisai et al., 2022, set the experiment at the same time and place. The water source was also similar. However, previous experimental study was carried out for 30 days and present study was carried out for 60 days. Therefore, only water temperature was little bit changed and other water quality parameters were similar.

Page 3. “After the post exposure of 60 days….” Please delete “post”

• As suggested by the reviewer, the change was made in revised manuscript.

Page 3. “Using a rotary microtome (Leica RM 2025, Leica Biosystems, Germany), paraffin fixed tissues were sectioned at a ……….” This sentence should be reworded as follows: “After being fixed in Bouin’s solution for 24hrs, paraffin embedded tissues were sectioned at a thickness of 5µm, using a rotary microtome (Leica RM 2025, Leica Biosystems, Germany).”

• As per the suggestion, we have modified the sentence in revised manuscript.

Page 3. “The concentrations of DEHP were determined from the pooled samples of liver and testes of each experimental group….”. One question for the Authors: why pool liver and testis for DEHP detection? It would have been much more interesting to have the two results separated and to know the direct effect of the presence of DEHP in the testis.

• Thank you for your valuable suggestion. Unfortunately, we could not perform the experiment separately in this study.

DEHP is considered as estrogenic compound and exposure of this chemical showed vitellogenin activity in male fishes. So, there is relation between liver and testis tissue and liver tissue plays a primary role in the metabolism and excretion of xenobiotic compounds like DEHP. Thus, we have pooled the samples of liver and testes for quantification of DEHP.

 

Page 5. “…….equilibrated with room temperature…….” better “at room temperature”

• As suggested, we have changed it in revised manuscript.

 

Results

Page 6, first paragraph. “60 days exposure of DEHP, significantly decreased the GSI of medium (10 µg/l) and higher concentration (100 µg/l) of DEHP treated males, but not in lower concentration (1µg/l) of DEHP treated males in comparison with the corresponding control males (Figure 1). ……………………..” Should be simplified as follows: “GSI significantly decreased in males exposed for 60 days to the medium (10 µg/l) and highest concentration (100 µg/l) of DEHP, but not in lower concentration (1µg/l), in comparison with control males (Figure 1). In control group, the GSI was 12.84 ± 0.54 (%), while in 100 µg/l DEHP treated group the value was found to be 6.86 ± 0.56 (%).” Here GSI after 10 µg/l DEHP exposure is missing……

• As suggested by the reviewer, the changes were incorporate in revised manuscript. The GSI of 10 µg/l DEHP exposed group was also included.

Page 7, L3-5. “The testes of medium concentration (10 µg/l), showed significant changes like presence of large number of inflammatory cells and large number of intratubular vacuoles (Figure 2c).” should be simplified as follows:  “At the medium concentration (10 µg/l), increasing numbers of inflammatory cells and intratubular vacuoles (Figure 2c) were observed.”

• As per the suggestion, we have modified the sentence in revised manuscript.

Page 8, final part. “Semen volumes were significantly decreased in order of increasing concentration of DEHP as compared to control group (Figure 4). The semen volume was 1.28 ± 0.02 ml in control group. However, decreased volume of semen (0.84 ± 0.01 ml) was recorded in 100 µg/l DEHP treated group. There ……..” should be modified as follows: “Semen volumes were significantly decreased at the increasing of DEHP concentrations, as compared to control group (Figure 4). The semen volume was 1.28 ± 0.02 ml in control group, while x.xx±x.xx ml, , x.xx±x.xx ml and x.xx±x.xx ml were recorded in 1 µg/l, 10 µg/l and 100 µg/l DEHP, respectively.” If you enter a numerical value, you must enter those of all the experimental groups.

• As suggested by the reviewer, the changes were incorporate in revised manuscript.

Page 9, below. “Different doses of DEHP exposed fishes showed significant decreases in sperm motility as well as sperm motility durations as compared to control group (Figure 6).” Are these decreases dose-dependent? In the following sentence, instead of "However" is better "while", and this applies to all However up to the end of the text.

• As questioned by the reviewer, decreases in sperm motility as well as sperm motility durations are dose-dependent and as per the suggestion, we have replaced ‘however’ with ‘while’ in the revised manuscript.

Page 12 “However, the level was remained unchanged in 1 µg/l DEHP treated group compare to control group (Figure 10a).” two very small oversights: was remained and compare, please delete was and change compare to compared.

• As suggested by the reviewer, the changes were done in revised manuscript.

Discussion

Page 13, first sentence. “The aim of this study was to explore the effects of DEHP exposure on gamete quality parameters of male fishes with environmentally relevant concentrations.” Should be better: “The aim of this study was to explore the effects on gamete quality parameters in C. carpio males after 60 days exposure to environmentally relevant concentrations of DEHP.”

• As per the suggestion of the reviewer, we have modified the sentence in revised manuscript.

Page 14. “The fishes of DEHP exposed groups also showed significant dose-dependent decreases in case of sperm density.” Please change “in case of sperm density.” To “of sperm density.”

• As suggested by the reviewer, we have modified the sentence in revised manuscript.

Page 14. “No such work so far has been done on this aspect in case of fish and to best of our knowledge, …….” Should be modified to “In our knowledge, this is the first study………”

• As per the suggestion of the reviewer, we have modified the sentence in revised manuscript.

Page 14. “In the present study, we analysed………..of reproductive success of any teleost including fish.” This last part of the sentence should be modified to “……of reproductive success in teleosts”.

• As suggested, we have modified the sentence in revised manuscript.

Page 15, first line. Please change reduced to reduce.

• As suggested by the reviewer, we have modified the word in revised manuscript.

Tables. Table 1 and 2 are ok, but the Primers are Table 1 and Biochemical parameters Table 2 (please check the text, too)

• We are sorry for the mistake. The numbering of the Table 1 and 2 has been corrected in revised manuscript.

Figure 2. The use of coloured arrows is confusing since the same kind of arrow points different things in the pictures (ie. in a. red arrow indicates intact seminiferous tubules, in b. inflammatory cells, in c. vacuolization and in d. necrosis.  Authors should use a unique way to highlight the same morphological aspect and modify the caption accordingly.

• Thank you for your suggestion. We have modified the Figure 2. The caption of the figure was also changed accordingly.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments about the manuscript:

“Effects of Di-(2-Ethylhexyl) Phthalate (DEHP) on Gamete Quality Parameters of Male Koi Carp (Cyprinus carpio)”

Di-(2-ethylhexyl) phthalate, (DEHP), is a frequently used substance considered to be an environmental pollutant with serious impacts on animals. Its effects on fish reproduction are still little known. The aim of this study was to assess the quality of mature male koi carp (Cyprinus carpio) gametes exposed to several concentrations of DEHP. After 60 days of exposure to the pollutant, significant effects were demonstrated: decrease in GSI, altered histological structure of the gonads, decrease or even suppression of sperm production, decrease in sperm motility, spermatocrit and sperm density. The ionic and biochemical compositions of seminal fluid also underwent changes. The mRNA expression levels of several reproductive-related genes were altered in a dose-dependent manner. The authors concluded that this study confirmed the negative impact of DEHP on koi carp by contributing to a decline in the quality of male gametes.

This well-conducted work brings useful elements to the knowledge of the ecotoxicological effects of a widely used molecule but with obvious polluting effects. This work deserves to be published after however some corrections in order to improve the manuscript. Here are some remarks.

Page 3, line 3. “and 12 numbers of fish were kept in each tank.”: suggestion: write “and 12 fish were kept in each tank”. Were all the fish adults? What was the number of males and females (all males, I assume?), please specify.

Page 3, paragraph 2, line 7. Write “Hematoxylin and Eosin stain (H & E stain) was used to stain” instead of “Hematoxylin and Eosin stain (H & E stain) were used to stain”.

Page 3, paragraph 4, line 2. Write “and the volume was recorded” instead of “and the volume were recorded”

Page 4, paragraphs 2 and 4, page 5, paragraph 2: use g-number instead of RPM for centrifuge speed (RPM is related to centrifuge model, g-number is general, applicable to all models).

Page 5, Paragraph 4, line 1 “Following the manufacturer’s instructions” is not enough for a scientific article: please briefly describe the method used.

Page 5, paragraph 4. Same: “ in accordance with manufacturer” is not enough for a scientific article: please briefly describe the method used.

Page 5, paragraph 4: table 2 is placed before table 1 (found on page 11): move table 1 to place it before table 2, or change the numbering: table 1 becomes table 2 and table 2 becomes table 1.

Pages 7, 8, figure 2:

-          add a scale bar on pictures,

-          It would be clearer if the different parts were labelled with a letter or group of letter (example: ST for seminiferous tubules, C1 for primary spermatocytes...) than with arrows (some color of arrows indicate different things on the picture).

Pages 12-13, figure 10: There is certainly a software problem: part of figure 10 is hidden by page 13.

Page 13, discussion, line 5. “in male D. rerio exposed to 0.5 µg/l of DEHP for 180 days [32]”. In the text, I did not find Ref [31]. Please, chek.

Supplementary: As in the main text, use the g-number for the centrifuge speed.

Author Response

“Effects of Di-(2-Ethylhexyl) Phthalate (DEHP) on Gamete Quality Parameters of Male Koi Carp (Cyprinus carpio)”

Di-(2-ethylhexyl) phthalate, (DEHP), is a frequently used substance considered to be an environmental pollutant with serious impacts on animals. Its effects on fish reproduction are still little known. The aim of this study was to assess the quality of mature male koi carp (Cyprinus carpio) gametes exposed to several concentrations of DEHP. After 60 days of exposure to the pollutant, significant effects were demonstrated: decrease in GSI, altered histological structure of the gonads, decrease or even suppression of sperm production, decrease in sperm motility, spermatocrit and sperm density. The ionic and biochemical compositions of seminal fluid also underwent changes. The mRNA expression levels of several reproductive-related genes were altered in a dose-dependent manner. The authors concluded that this study confirmed the negative impact of DEHP on koi carp by contributing to a decline in the quality of male gametes.

This well-conducted work brings useful elements to the knowledge of the ecotoxicological effects of a widely used molecule but with obvious polluting effects. This work deserves to be published after however some corrections in order to improve the manuscript. Here are some remarks.

Page 3, line 3. “and 12 numbers of fish were kept in each tank.”: suggestion: write “and 12 fish were kept in each tank”. Were all the fish adults? What was the number of males and females (all males, I assume?), please specify.

• As suggested by the reviewer, the changes were made in revised manuscript. As questioned by the reviewer, all the fish were adult and only males were used in this study and this is included in revised manuscript.

Page 3, paragraph 2, line 7. Write “Hematoxylin and Eosin stain (H & E stain) was used to stain” instead of “Hematoxylin and Eosin stain (H & E stain) were used to stain”.

• As per the suggestion, the change was incorporate in revised manuscript.

Page 3, paragraph 4, line 2. Write “and the volume was recorded” instead of “and the volume were recorded”

• As suggested by the reviewer, the change was made in revised manuscript.

Page 4, paragraphs 2 and 4, page 5, paragraph 2: use g-number instead of RPM for centrifuge speed (RPM is related to centrifuge model, g-number is general, applicable to all models).

• As suggested by the reviewer, the changes have been done in revised manuscript.

Page 5, Paragraph 4, line 1 “Following the manufacturer’s instructions” is not enough for a scientific article: please briefly describe the method used.

• As per the suggestion of reviewer, the method is briefly described in revised manuscript.

Page 5, paragraph 4. Same: “in accordance with manufacturer” is not enough for a scientific article: please briefly describe the method used.

• As suggested by the reviewer, the method is briefly described in revised manuscript.

Page 5, paragraph 4: table 2 is placed before table 1 (found on page 11): move table 1 to place it before table 2, or change the numbering: table 1 becomes table 2 and table 2 becomes table 1.

• We are sorry for the mistake. The numbering of the Table 1 and 2 has been corrected in revised manuscript.

Pages 7, 8, figure 2:

-          add a scale bar on pictures

• As per the suggestion of the reviewer, scale bar is included in histological photographs of revised manuscript.

-          It would be clearer if the different parts were labelled with a letter or group of letters (example: ST for seminiferous tubules, C1 for primary spermatocytes...) than with arrows (some color of arrows indicate different things on the picture).

• Thank you for your suggestion. We have modified the Figure 2.

Pages 12-13, figure 10: There is certainly a software problem: part of figure 10 is hidden by page 13.

• We are sorry for it. The problem has been rectified in revised manuscript.

Page 13, discussion, line 5. “in male D. rerio exposed to 0.5 µg/l of DEHP for 180 days [32]”. In the text, I did not find Ref [31]. Please, chek.

• As per the query of the reviewer, the Ref [31] (changed to [36] in revised manuscript) is in Table 1 (source of 18S rRNA primer). The Ref no has been changed as one of the reviewers has suggested to include some input in Introduction section to improve the manuscript. 

Supplementary: As in the main text, use the g-number for the centrifuge speed.

• As suggested by the reviewer, the change has been done in supplementary file.

Reviewer 3 Report

In the article titled “Effects of Di-(2-Ethylhexyl) Phthalate (DEHP) on Gamete Quality Parameters of Male Koi Carp (Cyprinus carpio)” the authors evaluated gamete quality parameters of mature male koi carp (Cyprinus carpio) exposed to different concentration of DEHP (1, 10 and 100 µg/l).

 

I think that I can consider the possibility of publication after a major revision.

 

My suggestions are the following:

 

The abstract is too long:  the abstract must be  maximum 200 words

 

 μg/l: l must be changed in L in all manuscript

 

There are not lines in the pages, so it is difficult to indicate the corrections to made.

 

“Centrifugation at 10,000 RPM”: specify the type of centrifuge or express the velocity in g

 

μl must be μL   

 

 

The authors must better argue on the decline of male fertility worldwide. In the last decade environmental pollution has a strong impact on semen quality. The pollutants also produce inflammatory problems in the reproductive sphere.

 

 

The results must be commented on and an explanation must also be given for the effect of this substance on the alterations of the various parameters examined. A molecular mechanism must also be hypothesized

 

try to correlate the results obtained with each other

 

There is a very recent work on the evidence of microplastic in human semen. Read and quote the following work:

Raman Microspectroscopy evidence of microplastics in human semen. Sci Total Environ. 2023 Jul 31;901:165922. doi: 10.1016/j.scitotenv.2023.165922.

 

 

I believe that to make the article more appealing you should include a nice color image that summarizes the results, the molecular mechanism and the message the authors want to give

 

Define the limitations of this study

 Moderate editing of English language required

Author Response

In the article titled “Effects of Di-(2-Ethylhexyl) Phthalate (DEHP) on Gamete Quality Parameters of Male Koi Carp (Cyprinus carpio)” the authors evaluated gamete quality parameters of mature male koi carp (Cyprinus carpio) exposed to different concentration of DEHP (1, 10 and 100 µg/l).

 

I think that I can consider the possibility of publication after a major revision.

 

My suggestions are the following:

 

The abstract is too long: the abstract must be maximum 200 words

• As per the suggestion of the reviewer, we have shorted the abstract section in revised manuscript.

 

μg/l: l must be changed in L in all manuscript

• As suggested by the reviewer, changes have been made in revised manuscript.

 

There are not lines in the pages, so it is difficult to indicate the corrections to made.

• We are sorry for it. Line numbers are inserted in revised manuscript.

“Centrifugation at 10000 RPM”: specify the type of centrifuge or express the velocity in g

• As per the suggestion of the reviewer, the changes have been done in revised manuscript.

μl must be μL

• As suggested, the changes have been incorporated in revised manuscript.

The authors must better argue on the decline of male fertility worldwide. In the last decade environmental pollution has a strong impact on semen quality. The pollutants also produce inflammatory problems in the reproductive sphere.

• As per the suggestion of the reviewer, several sentences have been included accordingly in the Introduction section of the revised manuscript.

 

The results must be commented on and an explanation must also be given for the effect of this substance on the alterations of the various parameters examined. A molecular mechanism must also be hypothesized

 

• As per the reviewer, the explanation has been given in Discussion section and a hypothesis related to molecular mechanism also included in Introduction section of revised manuscript.

 

Try to correlate the results obtained with each other

• As suggested, we have modified the results section and a comparative explanation were provided.

 

There is a very recent work on the evidence of microplastic in human semen. Read and quote the following work:

Raman Microspectroscopy evidence of microplastics in human semen. Sci Total Environ. 2023 Jul 31;901:165922. doi: 10.1016/j.scitotenv.2023.165922.

• As suggested by the reviewer, several inputs have been taken from the above-mentioned work to our revised manuscript.

 

I believe that to make the article more appealing you should include a nice color image that summarizes the results, the molecular mechanism and the message the authors want to give

• Thank you for your suggestion. We have added a graphical abstract in revised manuscript.

 

Define the limitations of this study

• As suggested by the reviewer, several sentences have been included in revised manuscript.

 

Comments on the Quality of English Language

Moderate editing of English language required

• As per the suggestion of the reviewer, the proofreading was done by an English native speaker from Australia and all the changes have been incorporated in revised manuscript.

 

Round 2

Reviewer 3 Report

accept in the present form

 Minor editing of English language required