Induced Spawning of F1 Wreckfish (Hāpuku) Polyprion oxygeneios Using a Synthetic Agonist of Gonadotropin-Releasing Hormone

Round 1

Reviewer 1 Report

The manuscript describes a study on the use of hormonal therapies to induce the maturation and spawning of F1 generation female hapuku (Polyprion oxygeneios) and an effort to employ in vitro fertilization, since the fish do not mature and spawn spontaneously. The study demonstrated that it is possible to induce female F1 hapuku to undergo oocyte maturation, ovulation and spawning, and produce viable fertilized eggs, that produce large surviving 3-4 weeks after hatching.  In vitro fertilization was also effective in one female, producing a significant percentage of viable larvae at 28 days post hatching.  The results pave the way for the continued development of broodstock management methods for this fish with great aquaculture potential, and the eventual development of breeding selection programs.

The article is very well written, with adequate description of the Materials and Methods and Results.  Below are some specific comments and questions that I believe will enhance the quality of the manuscript.  My main suggestion is to (a) describe better the reproductive dysfunction of female F1 hapuku and (b) tone done the emphasis on closing the life cycle and F2 production.

In addition, but of lesser importance, the authors should make some reference to (a) why they used constant temperature in 2013, (b) why they switched the type of GnRHa implants used in 2014 and (c) the fact the fish continued spawning for 90 days after GnRHa treatment, presumably when the implants stopped release GnRHa.

Abstract

I find the reference to “completing the life-cycle”, “F2 progeny” and “a world first” a bit “unscientific”, and make the manuscript sound as an advertisement of a company.  The manuscript describes an experiment on the use of GnRHa to control reproduction in F1 wreckfish only.  It did not involve spawning of wild breeders, larval rearing, grow out or any of the other stages involved in the process of “closing the cycle” of an organism.  Also, no juveniles were produced at the end (if I am not wrong), only larvae at 25 dph.  So, although these authors have been involved in the process and have published other work towards this effort, I find the emphasis given in this article to “closing the life cycle” unwarranted.  Of course, this is a conclusion they can have in the discussion, but I suggest that the emphasis on closing the cycle and production of F2 is toned down a bit.  Also the reference to “a world first” is (in my opinion) again unnecessary.  Indeed this group is the first to succeed in this effort (a remarkable work indeed), but on the other hand, I do not know many other groups that are currently trying and failed.  So, I don't think it is necessary for the manuscript to try so much to praise the achievement.  It is apparent to anyone interested in this species, that this group has main a very significant contribution to the area, and this article is a proof of their huge investment and success!

Line 23.  Change “readily spawn” to “spawn readily”.  

Line 24.  The “significant reproductive dysfunction” must be defined here.  It must be clear what problem the work tried to solve.

Line 25 and throughout the ms (Title as well).  An analogue can be an agonist or an antagonist!  Only agonists have been used in fish to induce maturation.  So, please change the word “analogue” to “agonist” throughout the manuscript.

Introduction

Lines 49.  It is not clear how “manipulating spawning” can help in “ …maintaining a genetically diverse population”.  Please be explicit.

Line 51 -52.  The parenthesis should be place at the end of the phrase (after “not be overlooked”, where it make sense.  The reader must finish the sentence an then got back to read again to understand what is the point of the sentence.

Line 53.  The term “final oocyte maturation” is no longer used in the fish reproduction literature, because there is only one maturation (of the oocyte).  Please change to “oocyte maturation”.

Line 56. Missing text.  Perhaps “…oocyte maturation, ovulation and spawning.”

Line 70.  Delete the “(” after the word “matrix”.

Line 73.  Delete “Under appropriate conditions”.  The GnRHa always mimics GnRH, that is why it is called an agonist.

Line 77. Please add the scientific name for European sea bass (Dicentrarhus labrax)

Line 85. “…has been…”

Lines 89-90.  As mentioned earlier, the reproductive dysfunction is not clear, and should be qualified further.  Lack of spawning may be due to a) failure to spawn eggs that are ovulated, b) failure of oocytes to undergo maturation and ovulation or c) failure of the fish to undergo vitellogenesis (and thus ovulation and spawning).  Also, when you refer” to |poor quality gametes” you mean the males also?  Please be explicit.

Results

Lines 96.  As the experiment started with the production (spawning) of eggs, I suggest you first talk about egg production and not F2 production.  To talk about production of F2, the eggs must be first produced, fertilized, hatched, the larvae reared and weaned juveniles swimming in the tanks. So, it sounds awkward that the authors jump to the production of F2 even before any fertilized eggs or hatcher larvae were produced.

Line 118.  I suggest you mention again that these were the eggs from the in vitrofertilization trials.  It is important to be clear, so please repeat.

Line 122.  Correct the way the citation is presented (not just [23])!

Discussion

Lines 204-207.  As mentioned earlier, the reproductive dysfunction that hapuku exhibit in captivity must be referred to explicitly, as well as the reference to “poor quality” gametes.  From the results presented, it seems that the females do undergo vitellogenesis, but is not clear to what extent. It is not clear if the fish complete vitellogenesis and then become arrested.  So, the authors must describe better what is the reproduction problem with F1 hapuku, and also what issues exist in regards to gamete (eggs and/or spermatozoa) quality. As mentioned earlier, lack of spawning may be due to a) failure to spawn eggs that are ovulated, b) failure of oocytes to undergo maturation and ovulation or c) failure of the fish to complete vitellogenesis (and thus ovulation and spawning).

Line 209-210.  The present manuscript achieved one aim, which is the induction of spawning and production of fertilized eggs and viable embryos from F1 breeders.  This result certainly contributes to the closing of the lifecycle of the species, but it was notan achievement of this manuscript.  Also, without the production of large number of juveniles (the ms refers to 28 dph larvae), again reference to production of F2 must be toned down.

Line 220.  In relation to my questions above, how do the author know that the fish reached the “end of vitellogenesis”?  Where do they base this conclusion?  Given that the fish took 2-3 weeks to begin spawning after the GnRHa treatment in 2014, and it required additional GnRHa treatments after 1-month stimulation with chol/cell implants, perhaps the fish did not reach the conclusion of vitellogenenesis before being induced with the GnRHa therapies?

Line 222.  The authors must be careful to the terminology they use and conclusion they reach. It is known that LH release induces oocyte maturation, which then is followed by ovulation and then hopefully spawning.  I not believe that it sufficiently demonstrated that it is involved in “successful spawning behaviours”!

Line 227.  Similar to above, the authors must be careful about the terminology they use and the conclusion. “Spawning” must be restricted to mean the “release of gametes”, not the whole process of reproduction or maturation.  The prevention of “natural spawning”, may be due to failure to complete vitellogenesis, or failure to undergo maturation and ovulation, or simply failure to undergo spawning.  The authors should refer to the exact failure they believe is happening in hapuku, not only the end result (failure to spawn).

Line 231. Change “communally” to “communal”.

Line 261-263.  This has been examined with some success in Senegalese sole and it may indeed prove helpful in hapuku, given the low maternal contribution in the examined spawns. Of course this may be also indicative of a strong male hierarchy, which is common in many fish and would expect it exists in hapuku as well.  Check the literature for the first option.

Lines 287-304.  Join the two concluding paragraphs, starting with the second one first (reproductive dysfunctions first and then the successful solution).  As mentioned earlier, I suggest the author tone down the reference to F2 production and closing of the lifecycle.

Materials and Methods

Line 311. Better refer to “reproductive cycle, not “spawning cycle”.

Lines 317-318.  Why were the fish exposed to constant temperature?  Is this natural for this species?  Also, since the VT worked for the wild-caught fish, why this was tried for the F1 breeders?

Line 323 and elsewhere.  Why “c.f.” is written here?  This does not seem an appropriate citation method.

Line 325 and elsewhere.  Citation correct in this way (without Author?)

Line 366.  What was the oocyte diameter of “late vitellogenic” females? How do the authors know that these were “late vitellogenic oocytes”?  How many of the examined fish were considered to be “immature”? Based on the data from Table 1 and Figure 1, it is clear that oocytes in maturation are about 1.24 mm (Oocytes of this size on 11/11/2013 completed maturation in 24 h and ovulated). But what is the maximum diameter reached during vitellogenesis?  This information is important, in order to know at what oocyte diameter fish should be given a hormone treatment to induce maturation, ovulation and spawning.

Line 366 and 375. Change EVac to EVAc.  Why the change in “implants” from the cholesterol/cellulose ones used in 2013?

Line 370.  Change “reflects” to “reflect”.

Lines 459-461.  I disagree with the use of volume for eggs, and the relative fecundity should be show (eggs should per kg body weight of the females in the stock), even if the authors are not sure how many females participated.  In any case, this has been examined in some spawns and it appears (as expected) that not all females participated.  So, I do not see the reason for expressing the egg production as volume.

Author Response

Response to Reviewer 1 Comments

Comments and Suggestions for Authors

The manuscript describes a study on the use of hormonal therapies to induce the maturation and spawning of F1 generation female hapuku (Polyprion oxygeneios) and an effort to employ in vitro fertilization, since the fish do not mature and spawn spontaneously. The study demonstrated that it is possible to induce female F1 hapuku to undergo oocyte maturation, ovulation and spawning, and produce viable fertilized eggs, that produce large surviving 3-4 weeks after hatching.  In vitro fertilization was also effective in one female, producing a significant percentage of viable larvae at 28 days post hatching.  The results pave the way for the continued development of broodstock management methods for this fish with great aquaculture potential, and the eventual development of breeding selection programs.

The article is very well written, with adequate description of the Materials and Methods and Results.  Below are some specific comments and questions that I believe will enhance the quality of the manuscript.  My main suggestion is to (a) describe better the reproductive dysfunction of female F1 hapuku and (b) tone done the emphasis on closing the life cycle and F2 production.

In addition, but of lesser importance, the authors should make some reference to (a) why they used constant temperature in 2013, (b) why they switched the type of GnRHa implants used in 2014 and (c) the fact the fish continued spawning for 90 days after GnRHa treatment, presumably when the implants stopped release GnRHa.

Abstract

I find the reference to “completing the life-cycle”, “F2 progeny” and “a world first” a bit “unscientific”, and make the manuscript sound as an advertisement of a company.  The manuscript describes an experiment on the use of GnRHa to control reproduction in F1 wreckfish only.  It did not involve spawning of wild breeders, larval rearing, grow out or any of the other stages involved in the process of “closing the cycle” of an organism.  Also, no juveniles were produced at the end (if I am not wrong), only larvae at 25 dph.  So, although these authors have been involved in the process and have published other work towards this effort, I find the emphasis given in this article to “closing the life cycle” unwarranted.  Of course, this is a conclusion they can have in the discussion, but I suggest that the emphasis on closing the cycle and production of F2 is toned down a bit.  Also the reference to “a world first” is (in my opinion) again unnecessary.  Indeed this group is the first to succeed in this effort (a remarkable work indeed), but on the other hand, I do not know many other groups that are currently trying and failed.  So, I don't think it is necessary for the manuscript to try so much to praise the achievement.  It is apparent to anyone interested in this species, that this group has main a very significant contribution to the area, and this article is a proof of their huge investment and success!

First and foremost, the authors would like to thank the reviewer for the very thorough feedback. After reading the comments from the reviewer, it appears our manuscript needs to more clearly communicate that the F2 offspring produced by strip-spawning and in vitro fertilisation did survive beyond 25 days post-hatching to become sub-adults for eventual breeding purposes. In order to communicate this more clearly the results section has been amended (Lines 132-136):

“While no F2 were produced during communal spawning events when blank- and GnRHa-implanted individuals were co-housed (data not shown), surviving F2 from eggs fertilized in vitro were successfully raised to sub-adults using grow-out conditions as previously reported [22,25].”

As requested by the reviewer, reference to findings being ‘a world first’ has now been omitted from the manuscript (Line 41) as the intention of this manuscript was not to be an advertisement for a company. To add, reference to ‘life cycle completion’ has removed from the title of the manuscript and rephrased to “Induced Spawning of F1 Wreckfish (Hāpuku) Polyprion oxygeneios using a Synthetic Agonist of Gonadotropin-releasing Hormone” (Lines 1-5)

Line 23.  Change “readily spawn” to “spawn readily”.  

Response: “readily spawn” has been changed to “spawn readily” as requested by the reviewer (Lines 24):

Line 24.  The “significant reproductive dysfunction” must be defined here.  It must be clear what problem the work tried to solve.

In order to define the “significant reproductive dysfunction”, text has been replaced to the following (Lines 24-26):

“Wild-caught hāpuku (Polyprion oxygeneios) spawn readily in captivity, but although first filial (F1) hāpuku complete vitellogenesis, females fail to undergo oocyte maturation and spawn or produce poor quality eggs.”

Line 25 and throughout the ms (Title as well).  An analogue can be an agonist or an antagonist!  Only agonists have been used in fish to induce maturation.  So, please change the word “analogue” to “agonist” throughout the manuscript.

As requested by the reviewer, the term “analogue” has been replaced with “agonist” in the title and throughout the manuscript as follows:

Lines 24-28: “Wild-caught hāpuku (Polyprion oxygeneios) spawn readily in captivity, but although first filial (F1) hāpuku complete vitellogenesis, females fail to undergo oocyte maturation and spawn or produce poor quality eggs. This study investigated whether administration of a synthetic agonist of gonadotropin-releasing hormone (GnRHa) could improve F1 hāpuku spawning and complete the life-cycle in captivity.”

Lines 66-70: “The development and use of synthetic agonists of gonadotropin-releasing hormone (GnRHa) have proven useful to mitigate reproductive dysfunctions in multiple cultured species. Examples include Atlantic bluefin tuna (Thunnus thynnus thynnus) [13], longfin yellowtail (Seriola rivoliana) [14], meagre (Argyrosomus regius) [15], blue mackerel (Scomber australasicus) and Eastern little tuna (Euthynnus affinis) [16].”

Introduction

Lines 49.  It is not clear how “manipulating spawning” can help in “ …maintaining a genetically diverse population”.  Please be explicit.

Response: In order to make the sentence clear, the text has been amended to state: (Lines 49-52):

“Of equal importance is the ability to induce spawning of broodstock to produce large numbers of high quality seed on demand [4]”

Line 51 -52.  The parenthesis should be place at the end of the phrase (after “not be overlooked”, where it make sense.  The reader must finish the sentence an then got back to read again to understand what is the point of the sentence.

Response: Parenthesis have been placed at the end of the phrase as requested by the reviewer (Lines 53-56).  

Line 53.  The term “final oocyte maturation” is no longer used in the fish reproduction literature, because there is only one maturation (of the oocyte).  Please change to “oocyte maturation”.

Response: The term “final oocyte maturation” has been replaced with “oocyte maturation” throughout the manuscript as requested by the reviewer. Maturation has also been added to the list of key words as well as hapuku (the alternative way to spell the species name) (Lines 43-44).  

Line 56. Missing text.  Perhaps “…oocyte maturation, ovulation and spawning.”

Response: The missing text “oocyte maturation, ovulation and spawning” has been added to the manuscript (Lines 55-56). 

Line 70.  Delete the “(” after the word “matrix”.

Response: “(” after the word “matrix” has been deleted (Line 73). 

Line 73.  Delete “Under appropriate conditions”.  The GnRHa always mimics GnRH, that is why it is called an agonist.

Response: “Under natural conditions” has been deleted as suggested by the reviewer (Line 58). 

Line 77. Please add the scientific name for European sea bass (Dicentrarhus labrax)

Response: The scientific name (Dicentrarhus labrax) has been added for European sea bass (Line 80). 

Line 85. “…has been…”

Response: “is” has been replaced with “has been” as suggested by the reviewer (Line 88). 

Lines 89-90.  As mentioned earlier, the reproductive dysfunction is not clear, and should be qualified further.  Lack of spawning may be due to a) failure to spawn eggs that are ovulated, b) failure of oocytes to undergo maturation and ovulation or c) failure of the fish to undergo vitellogenesis (and thus ovulation and spawning).  Also, when you refer” to |poor quality gametes” you mean the males also?  Please be explicit.

Response: In order to be more explicit around the type of reproductive dysfunction observed in F1 hāpuku and clarify that broodstock to complete vitellogenesis, text has been extended as follows (Lines 88-96):

“NIWA’s primary source of hāpuku eggs has been from wild-caught captivity-acclimated (F0) broodstock, which predictably spawn large quantities of eggs (70-80% fertilization rate) from late August through to December without any hormonal intervention [22]. However, despite first filial (F1) hāpuku completing vitellogenesis when maintained under similar environmental conditions to their parents [23], females have exhibited reproductive dysfunctions, including the complete failure to spawn or the production of poor quality eggs in low quantities, at least within the first six years of life. Lack of spawning may be due to a) failure of post-vitellogenic oocytes to undergo maturation and ovulation or b) failure to spawn eggs that are ovulated.”

For addition information, this paragraph has also been supported with a reference from our previous research that concluded that F1 hāpuku complete vitellogenesis and produce milt in captivity.

[23] Wylie, M.J.; Setiawan, A.N.; Irvine, G.W.; Symonds, J.E.; Elizur, A.; Dos Santos, M.; Lokman, P.M. Ovarian development of captive F1 wreckfish (hāpuku) Polyprion oxygeneios under constant and varying temperature regimes—Implications for broodstock management. Gen. Comp. Endocrinol. 2018, 257, 86–96.

Results

Lines 96.  As the experiment started with the production (spawning) of eggs, I suggest you first talk about egg production and not F2 production.  To talk about production of F2, the eggs must be first produced, fertilized, hatched, the larvae reared and weaned juveniles swimming in the tanks. So, it sounds awkward that the authors jump to the production of F2 even before any fertilized eggs or hatcher larvae were produced.

Response: As recommended by the reviewer, the order of the results has been changed to describe the production of eggs first –followed by the production of F2 (Lines 102-136).

Line 118.  I suggest you mention again that these were the eggs from the in vitro fertilization trials.  It is important to be clear, so please repeat.

Response: In order to be clear that embryonic development was described in eggs fertilised in vitro, the text has been amended as suggested by the reviewer (Line 126):

“Embryonic development of surviving F2 was documented from eggs fertilized in vitro (Figure 2).”

Line 122.  Correct the way the citation is presented (not just [23])!

Response: The citation has been corrected in the text as follows (Line 131):

“Embryonic development was consistent with that previously described for hāpuku [24] and the survival rate of F2 offspring (ca. 12,000 eggs) was 1.4% at 28 days post-hatching.”

Discussion

Lines 204-207.  As mentioned earlier, the reproductive dysfunction that hapuku exhibit in captivity must be referred to explicitly, as well as the reference to “poor quality” gametes.  From the results presented, it seems that the females do undergo vitellogenesis, but is not clear to what extent. It is not clear if the fish complete vitellogenesis and then become arrested.  So, the authors must describe better what is the reproduction problem with F1 hapuku, and also what issues exist in regards to gamete (eggs and/or spermatozoa) quality. As mentioned earlier, lack of spawning may be due to a) failure to spawn eggs that are ovulated, b) failure of oocytes to undergo maturation and ovulation or c) failure of the fish to complete vitellogenesis (and thus ovulation and spawning).

Response: As suggested by the reviewer, the reproductive dysfunctions of F1 hapuku have been described in more detail in the introduction – with the accompanying reference to highlight that females complete vitellogenesis in captivity (Lines 88-96). The type of reproductive dysfunction is then also described again in the discussion of the manuscript (Lines 223-228).

Please see comments below regarding evidence that F1 hāpuku complete vitellogenesis in captivity.

To highlight the need for future investigations into the quality of sperm in hāpuku “milt quality” has been added to the discussion (Lines 290).

Line 209-210.  The present manuscript achieved one aim, which is the induction of spawning and production of fertilized eggs and viable embryos from F1 breeders.  This result certainly contributes to the closing of the lifecycle of the species, but it was notan achievement of this manuscript.  Also, without the production of large number of juveniles (the ms refers to 28 dph larvae), again reference to production of F2 must be toned down.

Response: As mentioned earlier, it appears our manuscript failed to communicate that the F2 offspring produced by strip-spawning and in vitro fertilisation survived beyond 25 days post-hatching to become sub-adults for eventual breeding purposes. To clarify this, the results section has been amended to the following (Lines 132-136):

“While no F2 were produced during communal spawning events when blank- and GnRHa-implanted individuals were co-housed (data not shown), surviving F2 from eggs fertilized in vitro were successfully raised to sub-adults using grow-out conditions as previously reported [22, 25].”

Line 220.  In relation to my questions above, how do the author know that the fish reached the “end of vitellogenesis”?  Where do they base this conclusion?  Given that the fish took 2-3 weeks to begin spawning after the GnRHa treatment in 2014, and it required additional GnRHa treatments after 1-month stimulation with chol/cell implants, perhaps the fish did not reach the conclusion of vitellogenenesis before being induced with the GnRHa therapies?

Response: While the authors appreciate that oogenesis is a dynamic process and that the classification of developmental stages is subjective, oocyte histology from previous research describing oogenesis in F1 hāpuku indicated that late-vitellogenic oocytes as those with diameters ≥ 0.9 mm while oocytes of ~ 1.2 mm were the largest size that oocytes attained prior to undergoing cytoplasmic maturation. Data from gonadal biopsies collected prior to the administration of any GnRHa or placebo suggest that females were near or had reached post-vitellogenesis. In 2013, oocyte diameters of ‘control’ females from the strip-spawning trial reached ~ 1.2 mm (Table 1). In 2014, oocyte diameters from gonad biopsies prior to the administration of GnRHa implants were of the same size. A posteriori genotyping information from 2014 later confirmed that fertilised eggs were produced by females with mean oocyte diameters ranging between 0.9 and 1.2 mm at the time of GnRHa implant administration (Lines 186; 217).   

Line 222.  The authors must be careful to the terminology they use and conclusion they reach. It is known that LH release induces oocyte maturation, which then is followed by ovulation and then hopefully spawning.  I not believe that it sufficiently demonstrated that it is involved in “successful spawning behaviours”!

Response: As a measure to avoid any controversial statements, any reference to Lh release being a requisite for “successful spawning behaviours” has been removed from the manuscript (Line 244). Accordingly, the manuscript has been rewritten to say:

“The dysfunctional release of Lh is a common issue exhibited by many finfish species under aquaculture conditions [5,6,9] and its release is a requisite for the initiation of oocyte maturation, ovulation and spawning [26].”

Line 227.  Similar to above, the authors must be careful about the terminology they use and the conclusion. “Spawning” must be restricted to mean the “release of gametes”, not the whole process of reproduction or maturation.  The prevention of “natural spawning”, may be due to failure to complete vitellogenesis, or failure to undergo maturation and ovulation, or simply failure to undergo spawning.  The authors should refer to the exact failure they believe is happening in hapuku, not only the end result (failure to spawn).

Response: As suggested by the reviewer, the manuscript has been edited accordingly to specify the exact failure the authors believe is happening in F1 hāpuku (Line 249-250):

“It remains unclear what key factors or cues are preventing natural oocyte maturation, ovulation and spawning in F1 hāpuku.”

Line 231. Change “communally” to “communal”.

Response: “communally” has been changed to “communal” as requested by the reviewer (Line 254).

Line 261-263.  This has been examined with some success in Senegalese sole and it may indeed prove helpful in hāpuku, given the low maternal contribution in the examined spawns. Of course this may be also indicative of a strong male hierarchy, which is common in many fish and would expect it exists in hāpuku as well.  Check the literature for the first option.

Response: The authors appreciate the suggestion regarding the co-housing of different Senegalese sole year classes. As a result, the discussion has been extended to include the following (Lines 284-289):

“Hence, a measure to promote and/or improve spawning behavior in hāpuku may be to combine wild-caught spawning-experienced and captive spawning-naive F1 individuals. In Senegalese sole, combinations of wild-caught and captive-bred broodstock have been useful for the identification of behavioral dysfunctions in captive-bred males. Accordingly, F1 males failed to spawn with wild females, yet wild males and F1 females were able to spawn successfully [35].

Lines 287-304.  Join the two concluding paragraphs, starting with the second one first (reproductive dysfunctions first and then the successful solution).  As mentioned earlier, I suggest the author tone down the reference to F2 production and closing of the lifecycle.

Response: As recommended by the reviewer, the two concluding paragraphs have been combined starting with the reproductive dysfunctions first and then the successful solution (Lines 320-336).

Materials and Methods

Line 311. Better refer to “reproductive cycle, not “spawning cycle”.

Response: The term “spawning cycle” has been replaced with “reproductive cycle” as requested by the reviewer (Line 343).

Lines 317-318.  Why were the fish exposed to constant temperature?  Is this natural for this species?  Also, since the VT worked for the wild-caught fish, why this was tried for the F1 breeders?

Response: The rational for using a constant temperature has been added to the manuscript (Lines 350-352)

“The second temperature regime (n= 3 tanks) was maintained at a constant 17˚C (constant temperature, ‘CT group’). A constant temperature was selected on the basis of juvenile hāpuku growth performance [25] and its potential benefits for broodstock conditioning (e.g. ease of application and control) [23].”

Line 323 and elsewhere.  Why “c.f.” is written here?  This does not seem an appropriate citation method.

Response: “c.f.” and the citation have been removed from this area of text as it is unnecessary (Line 357).

Line 325 and elsewhere.  Citation correct in this way (without Author?)

Response: The citation style has been altered throughout the text to include the author at the end of the sentence (e.g. Lines 357-359).

Fish were left to spawn from August to October 2013 without any intervention. On 21 October 2013, when communal spawning was not evident, fish were anesthetized and the reproductive status of individuals was assessed as described previously [23].

Line 366.  What was the oocyte diameter of “late vitellogenic” females? How do the authors know that these were “late vitellogenic oocytes”?  How many of the examined fish were considered to be “immature”? Based on the data from Table 1 and Figure 1, it is clear that oocytes in maturation are about 1.24 mm (Oocytes of this size on 11/11/2013 completed maturation in 24 h and ovulated). But what is the maximum diameter reached during vitellogenesis?  This information is important, in order to know at what oocyte diameter fish should be given a hormone treatment to induce maturation, ovulation and spawning.

Response: Please see comments above regarding oocyte histology from previous research that indicates that oocytes of ~ 1.2 mm were the largest size that oocytes attained prior to undergoing cytoplasmic maturation.

The citation provided contains micrographs of oocyte cytology during key stages of oogenesis in F1 hāpuku.

Line 366 and 375. Change EVac to EVAc.  Why the change in “implants” from the cholesterol/cellulose ones used in 2013?

Response: The term “EVac” has been replaced with “EVAc” throughout the manuscript as requested by the reviewer. There is no specific reason for the change in implant type.

Line 370.  Change “reflects” to “reflect”.

Response: “reflects” has been changed to “reflect” as requested by the reviewer (Line 405).

Lines 459-461.  I disagree with the use of volume for eggs, and the relative fecundity should be show (eggs should per kg body weight of the females in the stock), even if the authors are not sure how many females participated.  In any case, this has been examined in some spawns and it appears (as expected) that not all females participated.  So, I do not see the reason for expressing the egg production as volume.

Response: As suggested by the reviewer, egg production data has been presented as daily relative fecundity (number of eggs produced per kg^-1 of mean body weight of the females in each tank; Figures 3 and 4). Figure legends have been updated (Lines 173-180 and 202-213).

The materials and methods sections have also been updated to remove reference to the use of total volume for eggs (Lines 459-462):

4.7.1. Egg collection and assessment

External and in-tank passive egg collectors were checked three times a day (between 8-9 am, at noon and between 4-5 pm) for spawned eggs from July to December in both years. When spawning had occurred, eggs were collected for assessment and/or incubation. The total volume, floating fractions (buoyancy) and sinking fractions were estimated in a measuring cylinder. Sinking fractions of eggs from each spawn were discarded prior to incubation and excluded from further sampling. Estimates of egg production data are presented as daily relative fecundity (number of eggs produced per kg^-1 of mean body weight of the females in each tank for communal spawning events and number of eggs produced per kg^-1 of body weight of individual females for strip-spawning).

Reference to egg volumes throughout the manuscript have also been supported with relative fecundity estimates (e.g. lines 111-116):

One GnRHa-treated female from the VT group (Female B) produced transparent free-flowing ovulated eggs (93 mL; 1430 eggs kg ^-1) at strip-spawning (Figure 1c); these eggs had dramatically increased in size, from an average of 1.2 mm at the time of the first GnRHa injection, to 1.8 mm at stripping, 24 h later (Table 1). In contrast, only small volumes (18-34 mL; 224-488 eggs kg ^-1) of cloudy ovulated eggs were collected from the three other females (Females A, D, J), and these eggs did not show any hydration-associated increase in diameter (Table 1).

Reviewer 2 Report

Review for Journal: Fishes

manuscript nr: fishes-475713

Title: Completion of the Wreckfish (Hāpuku) Polyprion oxygeneios Lifecycle after Spawning Induction with Gonadotropin-releasing Hormone Analogue

The aim of this study was to investigate the possibility of successful reproducing of the F1 Wreckfish (Hāpuku) Polyprion oxygeneios in captivity after spawning induction with GnRH analogue. The spawning trials (2013 and 2014) were conducted on 5-6 years old fish.  It was shown that the most effective was  the use of implants (ethylene-vinyl acetate 32 copolymer matrix) with GnRHa. The highest tested dose 100 mg/kg^-1 of GnRHa stimulated oocyte maturation and ovulation in F1 hāpuku. Eggs release in treated females was detected from 12-15 days  post implantation up to 90 days during the spawning season. The successful production of F2 hāpuku viable larvae in captivity by strip-spawning and in vitro fertilization were achieved. The possibility to complete lifecycle of Polyprion oxygeneios in captivity was shown after spawning stimulation.  

Summing up, presented manuscript is well written and interesting. I would recommend it for publication by Fishes.

Author Response

Response to Reviewer 2 Comments

Comments and Suggestions for Authors

Title: Completion of the Wreckfish (Hāpuku) Polyprion oxygeneios Lifecycle after Spawning Induction with Gonadotropin-releasing Hormone Analogue

The aim of this study was to investigate the possibility of successful reproducing of the F1 Wreckfish (Hāpuku) Polyprion oxygeneios in captivity after spawning induction with GnRH analogue. The spawning trials (2013 and 2014) were conducted on 5-6 years old fish.  It was shown that the most effective was  the use of implants (ethylene-vinyl acetate 32 copolymer matrix) with GnRHa. The highest tested dose 100 mg/kg^-1 of GnRHa stimulated oocyte maturation and ovulation in F1 hāpuku. Eggs release in treated females was detected from 12-15 days  post implantation up to 90 days during the spawning season. The successful production of F2 hāpuku viable larvae in captivity by strip-spawning and in vitro fertilization were achieved. The possibility to complete lifecycle of Polyprion oxygeneios in captivity was shown after spawning stimulation.  

Summing up, presented manuscript is well written and interesting. I would recommend it for publication by Fishes.

Response: The authors would like to thank Reviewer 2 for such positive feedback.