Changes in Plasma and Ovarian Steroid Hormone Level in Wild Female Blue Tang Fish Paracanthurus hepatus during a Reproductive Cycle

Round 1

Reviewer 1 Report

Manuscript: Animals 592258

The manuscript titled “Changes in plasma and ovarian steroid hormone level in wild female blue tang fish Paracanthurus hepatus during a reproductive cycle” by Huynh Minh Sang and co-authors reports the plasma and ovarian profiles of the major sex steroids during a complete seasonal cycle of a fish species with commercial interest. The aim of the manuscript is to collect data on the endocrine control of reproduction of blue tang fish as a critical step in order to achieve the reproduction of this species in captivity. For this reason the findings reported in the present manuscript are clearly interesting and necessary.

Although, in my opinion, the manuscript presents several imprecisions in the form and content and English employed is often confusing.

Here I list my mayor concerns:

Authors sate they use commercial ELISA kit to determine plasma and ovarian levels of different sex steroids, but they don’t even mention the name of the commercial kits. Have these kits already been used in blue tang fish? Do the authors know the detection limits of each for each hormone? Are the antibodies specifically designed against the blue tang sequences? Have authors or others checked the potential cross reactivity with other hormone in their fish species? All these questions are really important to address to know about the precision of the reported hormone levels. Statistically differences between experimental groups have been identified by using ANOVA test. Have authors test whether they data set respected the assumptions of ANOVA test? In lines 144 -145 authors write:” The GSI increased from March to August” but figure 5 suggest that GSI measured in these time point is actually constant. Have author performed a statistical analysis to check this.

This is also true for all data concerning the annual profile of the tested sex steroids: statistical analysis of this data set is required to check differences of sex steroid levels along the annual cycle.

In my opinion the discussion is really confusing since it’s not clear when authors discuss their own data reported in the present manuscript and whether they speculate on data published from other studies. References to such other studies is often missing. Line 172: Authors use two acronyms, i.e. GTH and GVBD, without defining their abbreviations. Lines 226-228: “However, in blue tang- a multiple spawner, asynchronous ovary coexists several types of oocytes during spawning season; the synthesis and utilization of hormones could be complicated because several processes relating to reproduction may occur at the same time.” I don’ understand what the authors mean. Complicated for what? In other multiple spawners the hormonal stimulation of final oocytes maturation and spawning works. In the simple abstract there is no reference to the objectives and the rational of the study.

 

Please correct the following imprecisions: Line 215: “that Brain-Pituitary-Gonadal axis was becoming inactive” In my opinion, in the introduction the term “endocrine” should be often changed with “endocrinology”. Line 66: “Fecundity ranged from 1.527 – to 20.618 ovaries/” ?? Line 72-75: “to investigate the changes in plasma…” plasma and ovary! Line 83: “total number of 15 fish”. Are they actually 15 females or how many females have you used in the study? Line 87: please correct the syntax. I’d not use the future in materials and methods section when describing the methods that have been already performed. Line 124: please correct “different oocytes” to “different oocyte maturational stages”

Author Response

Point 1: Authors sate they use commercial ELISA kit to determine plasma and ovarian levels of different sex steroids, but they don’t even mention the name of the commercial kits. Have these kits already been used in blue tang fish? Do the authors know the detection limits of each for each hormone? Are the antibodies specifically designed against the blue tang sequences? Have authors or others checked the potential cross reactivity with other hormone in their fish species? All these questions are really important to address to know about the precision of the reported hormone levels. Statistically differences between experimental groups have been identified by using ANOVA test. Have authors test whether they data set respected the assumptions of ANOVA test? In lines 144 -145 authors write:” The GSI increased from March to August” but figure 5 suggest that GSI measured in these time point is actually constant. Have author performed a statistical analysis to check this.

Response 1: We have added information on the ELISA kits to determine these steroids in fish. The parameters of the assay are described by manufacturers.

We have performed the ANOVA test.

Lines 144 -145: We rewrite the sentence and corrected it.

Point 2: This is also true for all data concerning the annual profile of the tested sex steroids: statistical analysis of this data set is required to check differences of sex steroid levels along the annual cycle.

Response 2: We have tested statistical analysis of hormone data during the annual cycle.

Point 3: In my opinion the discussion is really confusing since it’s not clear when authors discuss their own data reported in the present manuscript and whether they speculate on data published from other studies. References to such other studies is often missing. Line 172: Authors use two acronyms, i.e. GTH and GVBD, without defining their abbreviations. Lines 226-228: “However, in blue tang- a multiple spawner, asynchronous ovary coexists several types of oocytes during spawning season; the synthesis and utilization of hormones could be complicated because several processes relating to reproduction may occur at the same time.” I don’ understand what the authors mean. Complicated for what? In other multiple spawners the hormonal stimulation of final oocytes maturation and spawning works. In the simple abstract there is no reference to the objectives and the rational of the study.

Response 3:  We rewrite some discussions

Line 172: we clarify GTH and GVBD.

Lines 226-228: We rewrite the sentences.

Point 4: Please correct the following imprecisions: Line 215: “that Brain-Pituitary-Gonadal axis was becoming inactive” In my opinion, in the introduction the term “endocrine” should be often changed with “endocrinology”. Line 66: “Fecundity ranged from 1.527 – to 20.618 ovaries/” ?? Line 72-75: “to investigate the changes in plasma…” plasma and ovary! Line 83: “total number of 15 fish”. Are they actually 15 females or how many females have you used in the study? Line 87: please correct the syntax. I’d not use the future in materials and methods section when describing the methods that have been already performed. Line 124: please correct “different oocytes” to “different oocyte maturational stages” 

Response 4:

Line 215: “Brain-Pituitary-Gonadal axis was becoming inactive” was changed with “activity of the hypothalamic-pituitary-gonadal axis was becoming diminishing”

“endocrine” was changed with “endocrinology”

Line 66: We checked Fecundity. It is true.

Line 72-75: We added “and ovary”.

We clarified the number of females used in each month.

In materials and methods section we used the past tense.

Line 124: “different oocytes” was changed with “different oocyte maturational stages” 

Reviewer 2 Report

In this study plasma and ovarian steroid levels was studied over the yearly cycle and at different stages of ovarian maturation in female blue tang, Paracanthrurus hepatus.Levels in ovaries and plasma followed similar patterns. The levels of testosterone (T) and estradiol (E) peaked at stage II and the progestin DHP at stage IV. This is new for this species, but not surprising as it follows the usual pattern in fishes.

 

Comments:

 

There is a statement in the Simple summary and in the M & M that fish with a total length > 15 mm were studied. This seems very strange to me. How could blood samples be taken from specimens even close to that size limit? On line 66 it is stated the minimum size for maturity was 149.2 mm, so why was such small fish at all involved? Of course, the mature fish are clearly > 15 mm, but a limit only makes sense if it is much higher.

 

Material and methods

 

How were the fish collected in the field? Under what conditions were they kept in captivity? How were fish anaesthesized for the sampling? Until information on this is given, the ethical aspects are entirely unsettled.

 

Information about what ELISAs were used, manufacturer, cross reactivity etc. should be given.

 

 

 

Fig. 1. This is not really relevant for the science, but why are the borders in the map drawn so that they look like barbed wire?

 

Table 1. What does “snake-eyes” mean?

 

Table 1. Where fish in Stage 1 used for anything? If so, how was sex determined?

 

Figures 2-4, 5-8. n-values for the different categories/months should be given. If n is the same for all parameters, it is enough to show it only on one.

 

Fig 5. Average GSI is only 0.6-0.7% at most, which is really very little for adult females. The fish that I mostly work with have peak levels around 20%. How come it is so low? Are there masses of immature fish that takes down the average? I would also like to see GSI values for the Maturity stages.

 

Sometimes the English is strange. 

Author Response

Point 1: There is a statement in the Simple summary and in the M & M that fish with a total length > 15 mm were studied. This seems very strange to me. How could blood samples be taken from specimens even close to that size limit? On line 66 it is stated the minimum size for maturity was 149.2 mm, so why was such small fish at all involved? Of course, the mature fish are clearly > 15 mm, but a limit only makes sense if it is much higher.

Response 1: Actually we could collect about 0.5 – 1 mL blood from the fish with total length about 15 mm.

line 66: In our data we collected the fish with that sizes. That is random data.

Point 2: Material and methods

How were the fish collected in the field? Under what conditions were they kept in captivity? How were fish anaesthesized for the sampling? Until information on this is given, the ethical aspects are entirely unsettled.

Response 2: We described in detail in the manuscript

Point 3: Information about what ELISAs were used, manufacturer, cross reactivity etc. should be given.

Response 3: We have added information of the elisa kits to determine these steroids in fish. The parameters of the assay are described by manufacturers.

Point 4: Fig. 1. This is not really relevant for the science, but why are the borders in the map drawn so that they look like barbed wire?

Response 4: We added more information.

Point 5: Table 1. What does “snake-eyes” mean?

Response 5: “snake-eyes” was changed with “naked-eyes”

Point 6: Table 1. Where fish in Stage 1 used for anything? If so, how was sex determined?

Response 6: Actually, fish in Stage 1 is impossible to be distinguished between the sexes. We did not use these fishes because the present study refers only female fish.

Point 7: Figures 2-4, 5-8. n-values for the different categories/months should be given. If n is the same for all parameters, it is enough to show it only on one.

Response 7: We added n-values

Point 8: Fig 5. Average GSI is only 0.6-0.7% at most, which is really very little for adult females. The fish that I mostly work with have peak levels around 20%. How come it is so low? Are there masses of immature fish that takes down the average? I would also like to see GSI values for the Maturity stages.

Response 8: GSI is only 0.6-0.7%. It is true data. Please see in the attached file of GSI of female.

Point 9: Sometimes the English is strange.

Response 9: We checked and revised

Author Response File: Author Response.pdf

Reviewer 3 Report

This paper provides some information for reproductive physiology of the blue tang fish. However there are several typographical errors.

Table 1: "snake-eyes" >> "naked eyes" ?

Line 173-174: "C-21 steroids or closely related C-21 steroids" >> "Some C-21 steroids"

Line 181: "Transitional fish" >> "In protandrous seabass, transitional fish"

 

Author Response

Point 1: This paper provides some information for reproductive physiology of the blue tang fish. However there are several typographical errors.

Table 1: "snake-eyes" >> "naked eyes" ?

Response 1: "snake-eyes" was changed with "naked eyes"

Point 2: Line 173-174: "C-21 steroids or closely related C-21 steroids" >> "Some C-21 steroids"

Response 2: "C-21 steroids or closely related C-21 steroids" was  changed with "Some C-21 steroids"

Point 3: Line 181: "Transitional fish" >> "In protandrous seabass, transitional fish"

Response 3: "Transitional fish" changed was with "In protandrous seabass, transitional fish"

Round 2

Reviewer 1 Report

In my opinion the authors have correctly address some of the points I have indicated. They have changed and corrected most (but not all) the imprecisions of the form and added missing information. Although in my opinion the authors have not answered to a couple of major points:

The authors state they have performed ANOVA test to the GSI data as suggested, although we still don't know whether data normality and homogeneity of variances have been check before applying the ANOVA test. Moreover, the caption of figure 2 should explain what box plots, colors and symbols means to make readers able to understand whether there is a statistical difference or not. The statistical significance of values of GSI should also be described in the text.

 

Authors state they” have tested statistical analysis of hormone data during the annual cycle”, but we cannot see it in the text nor in the figures 6-8. The reader should be able to understand if values are statistically different or not.

Author Response

Point 1: The authors state they have performed ANOVA test to the GSI data as suggested, although we still don't know whether data normality and homogeneity of variances have been check before applying the ANOVA test. Moreover, the caption of figure 2 should explain what box plots, colors and symbols means to make readers able to understand whether there is a statistical difference or not. The statistical significance of values of GSI should also be described in the text.

Response 1:

For ANOVA test, we inserted in section 2.3. Data analysis “All data were checked for normality and homogeneity of variances before applying the Analysis of variance (ANOVA) test. The normality data were subjected to the least significant difference (LSD) post hoc test were used to determine the significant difference between the level of steroid hormone at different ovarian development stages”.

In figure 2, we explained the box-whisker diagram of GSI.

The statistical significance of values of GSI also inputs to the text “The GSI varied by months during the year cycle, the rank of 0.08-0.96% and the average of 0.44 ±0.22%. The GSI was significantly increased from March to the period of April - July when ovary of fish was processed maturation and ovulation and then decreased sharply after August to October (p<0.05) (Fig. 2). The GSI was also significantly increased from maturity stage I to IV and decreased to stage V (p <0.05)”.

Point 2: Authors state they” have tested statistical analysis of hormone data during the annual cycle”, but we cannot see it in the text nor in the figures 6-8. The reader should be able to understand if values are statistically different or not. 

Response 2: We put the statistical significance in the text of section “3.3. Monthly variation in steroid hormone level of wild caught blue tang fish”

Reviewer 2 Report

After having looked on the revised version I have a few comments, the last one additional:

Line 81. It is still stated that the fish had a total length of > 15 mm. In the table in the point-by-point response the smallest individual is 108 mm. That is of course larger than 15 mm, but so much larger that the statement is utterly misleading.

Line 86: How where the fish kept in the aquaria?

Line 89. Anesthetic with ice water is not the best method, but OK for warm water fish, though I suspect that more blood could have been collected with other methods. I do not demand any comment of this in the MS.

Line 97 take away "alive". The fish in the picture is not alive.

Line 175-185. The account of steroid levels in the seabass are far too detailed. 

Author Response

Point 1: Line 81. It is still stated that the fish had a total length of > 15 mm. In the table in the point-by-point response the smallest individual is 108 mm. That is of course larger than 15 mm, but so much larger that the statement is utterly misleading.

Response 1: We change to “Alive blue tang fish (average length: 188.23 ± 33.34 mm)”

Point 2: Line 86: How where the fish kept in the aquaria?

Response 2: Please see the Fig. 1 with the aquarium tank

Point 3: Line 89. Anesthetic with ice water is not the best method, but OK for warm water fish, though I suspect that more blood could have been collected with other methods. I do not demand any comment of this in the MS.

Response 3: Thanks

Point 4: Line 97 take away "alive". The fish in the picture is not alive.

Response 4: We change another picture in figure 1, so we kept "alive"

Point 5: Line 175-185. The account of steroid levels in the seabass are far too detailed. 

Response 5: We rewrote the paragraph, please see in the text.