Optimization of Live Prey Enrichment Media for Rearing Juvenile Short-Snouted Seahorse, Hippocampus hippocampus

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Abbreviations (DHA/EPA) in the abstract need further explanation in context also you must add the keys of tables

Please delete “were tested” in line 16

You mentioned that the "Results showed that juvenile seahorses fed on copepods attained a significantly higher (P<0.05) growth performance (5.1 mg d-1) than fish-fed diets with 5% and 10% copepod inclusion" I can’t understand what you mean, I think that the experimental diet need more clarifications in this context (Line 14-16).

You referred that “feeding copepods during the first 7 DPP promotes maturation of the digestive tract of juvenile seahorses” the manuscript did not contained any histological parameters related to this point.

You must add the  results related to feed utilization such as feed intake, feed conversion ratio, as well as the efficiency of protein if available

In Table 5; survival rate over time should estimate according to Kaplan- Meier methodology.

Please add the standard error of the values of LG and WG to obtain the significant differences as there were few differences between values however, there were significant differences.

Line 391-392, I can’t understand what you mean please clarify.

Figure 3. I think you must add  the linear regression equation to obtain the changes in growth and length over time or calculate the average daily gain and after that delete this figure

Author Response

Modifications suggested by the reviewer are marked in yellow in the manuscript.

Comment: “Abbreviations (DHA/EPA) in the abstract need further explanation in context also you must add the keys of tables)

Answer: Further explanation is now provided.

Comment: “Please delete “were tested” in line 16”

Answer: Deletion made.

Comment: “You mentioned that the "Results showed that juvenile seahorses fed on copepods attained a significantly higher (P<0.05) growth performance (5.1 mg d-1) than fish-fed diets with 5% and 10% copepod inclusion" I can’t understand what you mean, I think that the experimental diet need more clarifications in this context (Line 14-16).”

Answer: Sentences were revised to improve clarity.

Comment: “You referred that “feeding copepods during the first 7 DPP promotes maturation of the digestive tract of juvenile seahorses” the manuscript did not contained any histological parameters related to this point.”

Answer: We have re-phrased the sentence. The explanation here is that when juvenile seahorses are expelled from the male's pouch, their digestive tract is fully developed; however, they lack a developed gut biome and/or enzyme activity, which is acquired through the ingestion of natural prey.

Comment: “You must add the results related to feed utilization such as feed intake, feed conversion ratio, as well as the efficiency of protein if available”

Answer: While we acknowledge that this information would be valuable for a more comprehensive characterization of the growth performance of juvenile H. hippocampus, this experiment began with 2.4 mg fish that were fed live copepods and Artemia. Consequently, it was impossible to weigh the live diet before it was fed to the fish, as they only accept live feeds at this stage. Being unable to do so invalidates the possibility of obtaining additional information on feed utilization. Regarding protein efficiency, this would necessitate the sacrifice of all sampled fish. However, not only are these fish protected species, but also unnecessarily killing them is unethical. The primary objective of this study is to detail the feeding protocol for fish intended for the ornamental trade market, where they are sold live, and their protein content is not a significant concern.

Comment: “In Table 5; survival rate over time should estimate according to Kaplan- Meier methodology.”

Answer: We tested that option, but expressing the survival rate over time according to the Kaplan-Meier methodology would result in a rather confusing figure, as the survival decreasing steps would overlap between diets. Therefore, we chose to maintain the most relevant information, which is the final survival rate.

Comment: “Please add the standard error of the values of LG and WG to obtain the significant differences as there were few differences between values however, there were significant differences.”

Answer: the LG and WG are presented as cm d-1 and mg d-1, therefore those are absolute values without st error.

Comment: “Line 391-392, I can’t understand what you mean please clarify.”

Answer: Sentence was re-written to improve clarity.

Comment: “Figure 3. I think you must add the linear regression equation to obtain the changes in growth and length over time or calculate the average daily gain and after that delete this figure”

Answer: The reviewer presents two options for assessing changes in growth and length over time: using a linear regression equation or calculating the average daily gain. Since the average daily gain has already been calculated and presented in Table 5, and including linear regressions would result in an excessive amount of redundant information, we have chosen to maintain the information as it is.

Reviewer 2 Report

The present study assesses the more appropriate feeding protocol for Hippocampus hippocampus at early stages. It mainly focuses on the fatty acid profile of the experimental diets since it is a key factor for marine fish larvae reared in captivity. All the manuscript is held in two datasets: fatty acids profile of the diets and growth performance of the individuals. There are several points that must be addressed before considering the manuscript for publication. 

- The role of aquaculture for the species conservation is repeated several times along the introduction (L 39-44, L55-58, L59-61). Please, simplify. 

- L128: The authors refer to the newborns as juveniles, however, there is some discrepancy about this issue. As they well mentioned in the discussion, newborns present limited digestive capacity and an undeveloped digestive tract, which undergoes developmental changes during ontogeny. The same has been described for osteological and morphological aspects (Novelli et al., 2017). Thus, evidence indicates the existence of a larval stage in seahorse species. What is the authors' argument for considering newborns as juveniles?

Novelli B, Otero-Ferrer F, Socorro JA, Caballero MJ, Segade-Botella A, Molina Domínguez L., 2017. Fish Physiol Biochem. 43, 833-848. doi: 10.1007/s10695-017-0339-2

- Why Artemia was enriched for 24h? Standard enrichment protocols last for 12h, since after that period it is considered that enrichment can be lost. 

- L238: "To analyse the nutritional quality of prey and the dietary treatments used in the experimental trial, samples of natural copepods (Cop), unenriched Artemia (ArtRef), 24h enriched Artemia plus 5% copepods (ArtDHA/EPA5%) and enriched Artemia plus 10%..." Please, include also ArtDHA/EPA (enriched Artemia without copepods)

- Results describing dietary treatments' proximate analysis and fatty acid composition refer to higher/lower levels in the different variables, but no statistics are shown. Are the differences statistically significant? Please, include in the tables and graphs the statistical results as done in table 5.

- For the fatty acids profile, please include absolute values (amount of FA per amount of sample) instead of relative values (%). Relative values may cause confusion since the change in a single FA alters the relative abundance of all the rest. That is, an increase in the relative abundance of a specific FA may not reflect an absolute increase of this FA but a decrease in others.  

- Which are the units for the FA in Table 2?

- L346-349: What do authors mean with "were present on the expanses of copepods inclusion"? ArtDHA/EPA also increased DHA content, so Artemia enrichment also enhanced DHA. Are differences in DHA content significant between dietary treatments?

- Figure 1 represents data that is already shown in Table 3, please avoid repetition of the results (The same for figure 3 and Table 5).

- L427: change "this" by "Thus"

- L434: Both, protein and lipid content were calculated on dry matter basis, so differences should not be attributed to differences in moisture.

- The fact that copepods presented less lipids content but more gross energy may suggest higher carbohydrate content. Could this be due to copepods' exoskeleton?

- L455-470 in the discussion is a description of the results. Please avoid repetition.

- L487: In the preliminary trial survival was 0%, however in the second trial survival with ArtDHA/EPA treatment was 88%. How do authors explain this difference?

- Please, include an abbreviation list for Table 5.

Minor editing of English language required

Author Response

Modifications suggested by the reviewer are marked in chartreuse green in the manuscript.

“The present study assesses the more appropriate feeding protocol for Hippocampus hippocampus at early stages. It mainly focuses on the fatty acid profile of the experimental diets since it is a key factor for marine fish larvae reared in captivity. All the manuscript is held in two datasets: fatty acids profile of the diets and growth performance of the individuals. There are several points that must be addressed before considering the manuscript for publication.”

Comment: “- The role of aquaculture for the species conservation is repeated several times along the introduction (L 39-44, L55-58, L59-61). Please, simplify.”

Answer: We do acknowledge the point raised by the reviewer; therefore, the sentences between L55-58 were deleted. As for the remaining sentences, we believe they are complementary and help to explain the purpose of seahorse aquaculture. The first sentence refers to the conservation problems to which most seahorse species are exposed, while the second emphasizes the significance of seahorse aquaculture in their conservation.

- L128: The authors refer to the newborns as juveniles, however, there is some discrepancy about this issue. As they well mentioned in the discussion, newborns present limited digestive capacity and an undeveloped digestive tract, which undergoes developmental changes during ontogeny. The same has been described for osteological and morphological aspects (Novelli et al., 2017). Thus, evidence indicates the existence of a larval stage in seahorse species. What is the authors' argument for considering newborns as juveniles?

Novelli B, Otero-Ferrer F, Socorro JA, Caballero MJ, Segade-Botella A, Molina Domínguez L., 2017. Fish Physiol Biochem. 43, 833-848. doi: 10.1007/s10695-017-0339-2

Comment: The study by Nouvelli et al. 2017 marked the first attempt to address a potential discrepancy in the terminology used to describe seahorse newborns, specifically whether they should be classified as larvae or juveniles. In terms of fish physiology, most newborn fish are considered larvae because they undergo various sequential morphological changes until they reach a morphology similar to that of adults. A classic example are the flatfish, which undergo several morphological changes, including eye migration.

Conversely, newborn seahorses, upon parturition, already resemble small replicas of adults, with only slight allometric differences. Furthermore, their digestive tracts are fully formed and ready to ingest food, and in most species, they no longer have a visible yolk sac. This differs from many fish larvae, where the mouth remains closed upon hatching, the digestive tract is undeveloped, and the yolk sac remains attached, allowing them to feed endogenously throughout metamorphosis.

Nouvelli et al. 2017 based their assumption on the observation of remnants of yolk sac in newborn seahorses, suggesting they should be considered larvae. However, this observation was made in fish hatched in the southern distribution of the species, where warmer water temperatures promote faster development compared to the northern population. In the northern population, the yolk sac is completely consumed during ontogenic development inside the male's pouch. Therefore, this morphological difference between populations, while not commonly described for other species, is not sufficient to categorize newborn seahorses as larvae rather than juveniles.

Regarding limited digestive capacity, this constraint is observed in captive-bred juvenile seahorses. Just like in all fish, the digestive tract undergoes microbiome and enzymatic maturation during the initial feedings with natural prey. This maturation does not occur in captivity when fish are fed partially or fully sterile diets like Artemia and artificial enrichments. Thus, when it is mentioned that the digestive tract is not mature at hatching, it pertains to its microbiome and enzymatic activity, not its morphology.

Top of Form

Comment: “- Why Artemia was enriched for 24h? Standard enrichment protocols last for 12h, since after that period it is considered that enrichment can be lost.”

Answer: In this study, we used AR Artemia. AR Artemia is characterized by the fact that the nauplii have a smaller size upon hatching, which is more suitable for some seahorse species, including H. hippocampus. Since AR Artemia is smaller upon hatching, the nauplii have their mouths closed, and they only open them after 8 hours. Therefore, the actual enrichment period is 16 hours, which is similar to the time period pointed out by the reviewer and is the full process that takes 24h.

Comment: - L238: "To analyse the nutritional quality of prey and the dietary treatments used in the experimental trial, samples of natural copepods (Cop), unenriched Artemia (ArtRef), 24h enriched Artemia plus 5% copepods (ArtDHA/EPA5%) and enriched Artemia plus 10%..." Please, include also ArtDHA/EPA (enriched Artemia without copepods)

Answer: Thanks to point this flaw, it’s now added to the text.

Comment: “- Results describing dietary treatments' proximate analysis and fatty acid composition refer to higher/lower levels in the different variables, but no statistics are shown. Are the differences statistically significant? Please, include in the tables and graphs the statistical results as done in table 5.”

Answer: This information is now provided in the tables.

- For the fatty acids profile, please include absolute values (amount of FA per amount of sample) instead of relative values (%). Relative values may cause confusion since the change in a single FA alters the relative abundance of all the rest. That is, an increase in the relative abundance of a specific FA may not reflect an absolute increase of this FA but a decrease in others. 

Comment: As described, the legend in Figure 3 could potentially cause a misunderstanding of how the data is presented and may have been the reason for this comment. In our study, the fatty acid composition is expressed as a percentage (%) of the total identified fatty acids in the diets, which we believe is the most accurate way to present this type of information. It is also the most common method for expressing the fatty acid composition of any diet, enabling easy comparisons between the fatty acid profiles of diets used in different studies. The table legend has been revised accordingly.

Comment “- Which are the units for the FA in Table 2?”

Answer: Units are now included in the legend (% total FA).

- L346-349: What do authors mean with "were present on the expanses of copepods inclusion"? ArtDHA/EPA also increased DHA content, so Artemia enrichment also enhanced DHA. Are differences in DHA content significant between dietary treatments?

Comment: The reviewer is correct. The sentence was wrongly addressed and is now corrected.

Comment: - Figure 1 represents data that is already shown in Table 3, please avoid repetition of the results (The same for figure 3 and Table 5).

Answer: We agree with reviewer, figures 1 and 3 were deleted.

Comment: - L427: change "this" by "Thus"

Answer: Corrected.

Comment: - L434: Both, protein and lipid content were calculated on dry matter basis, so differences should not be attributed to differences in moisture.

Answer: Reviewer is correct the sentence was deleted.

Comment: - The fact that copepods presented less lipids content but more gross energy may suggest higher carbohydrate content. Could this be due to copepods' exoskeleton?

Answer: Yes, we believe so, but to provide a sure answer to this question, further analysis had to be done, and as it could only be a guess that chitin is the main source of carbs in copepods exoskeletons, we left this eventual explanation out to avoid speculative explanations.

Comment: - L455-470 in the discussion is a description of the results. Please avoid repetition.

Answer: We agree, values were removed, and the sentences were re-written.

Comment: - L487: In the preliminary trial survival was 0%, however in the second trial survival with ArtDHA/EPA treatment was 88%. How do authors explain this difference?

Answer: Our explanation, as mentioned in the manuscript, is based on the fact that when this species is fed with Artemia immediately after parturition, and the Artemia is enriched with non-live supplements (e.g., microalgae concentrate rather than natural live microalgae), both the Artemia and the enrichment are sterile in terms of microbiome and/or relevant enzyme activity. When ingested, this fails to promote the maturation of the seahorse digestive tract microbiome necessary for a normal digestive process.

In contrast, when juvenile seahorses are fed a natural prey item (such as copepods), which inherently possess their own digestive microbiome, the juvenile seahorses benefit from it. This initiates the development of their microbiome, which in turn enables them to digest other diets, like the one used in this study, more effectively.

Comment: - Please, include an abbreviation list for Table 5.

Answer: Abbreviation were included as suggested.

Round 2

Reviewer 1 Report

You noted that the LG and WG are presented as cm d-1 and mg d-1, therefore those are absolute values without st error. I do not agree with you, as long as there is a standard error for body weight and length, then there is a possibility to calculate the weight and length gains, including the standard error. Please revise.

Author Response

Comment: "You noted that the LG and WG are presented as cm d-1 and mg d-1, therefore those are absolute values without st error. I do not agree with you, as long as there is a standard error for body weight and length, then there is a possibility to calculate the weight and length gains, including the standard error. Please revise."

Answer: The requested information was added to Table 5.

Reviewer 2 Report

The reviewer thanks the authors for the responses to the comments and the modifications made in the manuscript. However, there are minor corrections to be addressed before publication.

The incorporation of different letters indicating significant differences between treatments highlights the robustness of the results, however, letters are used in an inconsistent way. For example, in Table 1, for moisture "a" designates the highest value while for dry matter and gross protein "a" designates the lowest value. Please, in order to facilitate the interpretation of the results to the reader, be consistent with the use of the different letters, use "a" for the highest/lowest value and the consecutive letters in alphabetical order to the decreasing/increasing values.

Similarly, for gross energy, it appears "bc" group but there is no "c" group, and "ac" group is statistically inconsistent. In table 3, for C16:2ω4, "a" indicates the lowest value, "b" the highest value while "c" and "d" are intermediate values.

Statistical results are so confusing, please be consistent with the use of the different letters.

Author Response

Comment: The incorporation of different letters indicating significant differences between treatments highlights the robustness of the results, however, letters are used in an inconsistent way. For example, in Table 1, for moisture "a" designates the highest value while for dry matter and gross protein "a" designates the lowest value. Please, in order to facilitate the interpretation of the results to the reader, be consistent with the use of the different letters, use "a" for the highest/lowest value and the consecutive letters in alphabetical order to the decreasing/increasing values. Similarly, for gross energy, it appears "bc" group but there is no "c" group, and "ac" group is statistically inconsistent. In table 3, for C16:2ω4, "a" indicates the lowest value, "b" the highest value while "c" and "d" are intermediate values. Statistical results are so confusing, please be consistent with the use of the different letters."

Answer: The reviewer is correct; in the previous version, the different letters indicating significant differences between treatments were assigned in reference to the first-row value, and we agree that this is incorrect. Letters are now correctly assigned as suggested by the reviewer.