Effect of Replacing Fishmeal with Plukenetia volubilis Cake on Growth, Digestive Enzymes, and Body Composition in Whiteleg Shrimp (Litopenaeus vannamei)
, Roberto A. Santacruz-Reyes 1, MarÃa Isabel Saez 2, Dmitri Fabrikov 2, Fernando G. Barroso 2 and MarÃa del Carmen Vargas-GarcÃa 2Round 1
Reviewer 1 Report
Abstract
Line 16-18 rephrase
Line 19-22 separate into two sentences
Introduction
Line 36 change the word
Line 39 plural
Material and Method
Line 101 change the word
Line 121 add an article
Results
Line 196 change the spelling
Line 198 change the word
Line 203 plural
Line 213 plural
Line 219 plural
Discussion
Line 237 add an article
Line 249 change the spelling
Line 253 add an article
Line 285 plural
Line 302 change an article
References
Line 409 change the spelling
Comments for author File: 
Comments.pdf
Author Response
Thank for your comment
Author Response File: Author Response.pdf
Reviewer 2 Report
The MS is focused on the replacement of fish meal with P. volubilis cake in diets intended for whiteleg shrimp. The use of this ingredient in aquafeed formulation sounds innovative and results are promising.
Here the comments divided per section
Abstract
The abstract is not complete since it starts in the affiliations section
Authors can remove p<0.05 in this section
Line 24: fish meal
24: …shrimp feeding. However, it should be…
25: …replacement percentages, and to identify the adequate…
Introduction
31: I would not refer to shrimp as a species
34: please remove the sentence: “This means..absolute terms”
35: I would say fish/crustacean feed
40: please substitute “plant based” with “derived from plants”
41: “animal-derived sources”
41: please remove “compared to fish meal”
42: what do the authors mean with “improved composition consistency”?
43: the nutritional properties of soybean meal in aquafeed formulation are quite controversial and depend on the aquatic species analysed. Recent papers on L. vannameii show positive effects on growth and gut health with fish meal replacement with soy-related ingredients. However, this ingredient can cause severe negative effects on gut health of carnivorous fish. For that reason, I would specify in the introduction this aspect, especially because a few lines after, the authors state that the search of alternative ingredients is a necessity only in light to reduce deforestation, water consumption ect. This is absolutely correct, but the problems related to the use of plant-derived ingredients include also the adverse effects on different animals. I would stress more this aspect (I suggest these recent papers: https://doi.org/10.1016/j.aquaculture.2019.02.011; https://doi.org/10.1016/j.aquaculture.2019.734630; http://dx.doi.org/10.1016/j.aquaculture.2022.738137)
49: “research goal for aquatic animals’ nutrition”, not only fish
55-57: This sentence is related to the beneficial effects on humans, but it must be better contextualized. PUFA are important firstly for growth, welfare, and the biochemical composition of the edible parts of aquatic farmed animals. Providing aquafeeds with a proper amount of PUFA is the main goal to preserve these aspects and to obtain a product able to transfer PUFA to human nutrition.
62: “…as a nutritional supplement.” In which field? Animal nutrition?
66: after the reference 21, I think that some names are missing
Methods
88: kg-1
Experimental diets are characterized also by a reduction of soy meal (which is the second major ingredient used; in the introduction section, authors should emphasized this aspect since it seems that fish meal is the main ingredient used), and an increase in squid meal with the increasing replacement of fish meal with sacha inchi. Authors should justify in the methods these changes.
116: 15% (the same for 25 or 50 %) of sacha inchi cake respect to fish meal (please correct along the text)
117: “values are presented as mean…”(please correct along the text)
135: authors should add a reference after “standard methos”
On how many samples the proximate composition and the fatty acids analyses were conducted? What do the authors mean with abdomen samples? Please specify
159: remove ) after reference 29
162: µ-1
183: this paragraph should be moved before the 2.2 one
Results
190: remove bold form table 4
192. “replacement respect to fish meal”
Some of the growth performances showed in the table 4 are not mentioned in the methods.
Please use superscript letters for statistical differences in table 4 (and in the other tables)
Use the same name for the group (in the text is T 25, in the table is D25)
199: “among the different dietary treatments”
204: the name of the 3.2 paragraph is in the table caption
204: p<0.05. please check along the text
Line 205: this sentence should be reported in the methods instead of results
224: was found
224. it is not an inclusion level, but a fish meal replacement
227: please remove p>0.05
Discussion
241: “…out by XXX et al. [34],…”. Please add the name of the author
241: 10% replacement respect to ??
243. “compared to the control group”
245: add the common names.
247: ”…fed D25. This could be related…”
249-253: it is not a proper discussion of the result obtained. How the lower chymotrypsin activity an be related to the sgr?
257: abdomen and edible parts according to the methods section
259-267: the protein content in the edible part of shrimps fed the experimental diets decreased compared to those fed the control one. This is an important result that should be emphasized and coupled with a possible explanation
269: abdomen, edible parts, muscle. Please refer to the sample used for proximate composition and fatty acid analyses with the same name. It is quite confusing
278 add ] after 34
Conclusions
320: “[9]. However, in the present study,…”.
It is not true that the fish meal replacement with sacha inchi did not compromise the nutritional feature of shrimps, since a reduction in the protein content in the edible parts was detected in shrimps fed all the experimental diets, except for control.
References
References must be arranged according to the mdpi style. Please check the list
Author Response
thank for your comments
Author Response File: Author Response.pdf
Reviewer 3 Report
In view of the increase of the cost of animal protein ingredients in Aquaculture nutrition. This research conducted was a good attempt for replacing fish meal with Sacha inchi cake. However, I have a few queries to the authors which is mentioned below:
1. The authors had not mentioned the total proximate composition of the Plukenetia volubilis in the manuscript. However, different protein percentages i.e., 27 % in line 51, 59% in line 64 and 51.12 % in line 263. Whether the authors had done the proximate analysis or the values given are from previous references or works. If so details may be incorporated.
2. The process of feed ingredient preparation does not mention any cooking or steaming of the ingredients. The addition of vitamin mineral mix has not been mentioned in the procedure. The details of the entire feed preparation has be given.
3. Extruder pellet machine is used for preparation of the feed. Extruder pelletizer is used mainly for floating feed which is used for fish. As shrimps are bottom feeders they are given sinking feed which is done by steam pelleting. The authors may quote the probable reason for using the floating feed even though it is not applicable for the experimental shrimp.
4. The pellet size used in the study is 4mm diameter which is reported to be used from 3.41 g (stocking size after acclimatization of 3 weeks) to 14.25-16.72g ( 10 weeks or 70 days). The maximum pellet size used for commercial shrimp culture is 2.2-2.8 mm and 3.0 mm is used in extreme cases. The pellet size is determined based on size of the mouth of the shrimp. The pellet size for the stocking size to the end of the experiment will be in the range of 1.4-2.5 mm maximum. The authors may explain the reason for using the large pellet size in the study.
5. The authors had mentioned that they had prepared iso-lipidic diet (line 88) with crude lipid of 90 g kg-1. However, the proximate composition results of the feed had shown that the lipid content in the range of 83.8-87.9 g kg-1 of total lipid. The authors may specify how the diets can be termed as iso-lipidic with a wide range in the lipid content.
6. The authors had compared the amino acid composition of the sacha inchi cake and fish meal in table 1. However, it is clearly indicated in the composition that except two essential amino acids Arginine and Phenylalanine, all the essential amino acids are reported to be less in sacha inchi cake. How does the authors justify the loss/ deficiency in the essential amino acids in the diets with 50 % fish meal replacement.
7. The authors had conducted the study in cages fixed in the ponds. The exact measurements (length, width and height) of the cages and mesh size of the cages were not mentioned. The nature of the cage (floating or fixed) were not given and position of the cage (near bottom or bottom attached) were not detailed. The authors may please specify the rationale behind using the cages for the present study.
8. The experiment organism, shrimp is a bottom feeder and is not suitable for rearing in the cages. Authors may please specify what are the management methods used for the culture of shrimps in cages.
9. The experiment was conducted in duplicate and there were no enough replicated which may lead to error maximization
10. The authors had used the feeding rate of 7% which is feasible for the initial stages of the experiment, however it is not applicable as the shrimp grows in size (feeding rate should be reduced as the size increases).
11. The authors had just mentioned the water quality parameters like temperature, salinity and pH. However, another important parameter for the aquatic organism, Dissolved oxygen level was not mentioned in the study. The fluctuation in the water quality parameters is inevitable through out the study especially in ponds. However, no such results are presented in the given study.
12. The stocking density reported in the study is 15 shrimps/ 1m3 cage which is a very low stocking density when cultured in ponds. However, only maximum of 0.19 g of daily growth is recorded which is low as per the stocking density of the study.
13. The FCR reported in the study is quite high. The maximum FCR of the shrimp culture is 1.1-1.5 and higher values were reported to be uneconomical. The values reported in the study are in the range of 3.69-4.72 which are quite high. Whether the authors had taken any measures to record the uneaten/wasted feed and used it in the calculation of FCR.
14. The body composition of the shrimps reported that the shrimp protein percentage is highest in control (86.14) and lowest in 50 % fish meal replacement treatment (75.05) which is showing that replacement of sacha inchi cake had reduced the protein content of the shrimp thereby affecting its nutritional quality. How can the authors justify this reduction in nutritional quality by 50 % replacement of fish meal with sacha inchi cake.
15. The authors had not done the proximate composition of the fish meal (line 264-267) and mentioned the values reported in the references and the same had been used for feed formulation. How can the authors confirm that the fish meal proximate composition remains the same as reported in the references.
16. The authors are well aware of the presence of the anti-nutritional factors/components in the sacha inchi cake and it would have been better if some analysis related to that is reported.
Author Response
thank for your comments
Author Response File: Author Response.pdf
Reviewer 4 Report
The manuscripts described the outcomes of an experiment carried out in shrimp L. vannamei which were housed in cages within a pond and fed 4 experimental diets. These diets had reduced inclusions of fishmeal/soybean meal with increasing inclusions of Plukenetia volubilis cake. The statistical robustness of the treatment effect is questionable due to the low number of replicate cages. Justification for this sample number is required. Incorrect interpretation of the growth results was evident in the results section. Further comments are detailed below:
Abstract.
Line 15. Some description of P. volubilis is required since it is not a commonly known plant. Including the common name would be useful.
Line 17. Give a value for the prawn weight and abdomen weight that were described as ‘high’.
Line16-23. The reporting of insignificant results is distracting when reported in the abstract with very little room for explanation. Please be more clear and stick to the significant or most meaning results from the trial.
Line 24. ‘Interesting’ is too generic a term and can be interpreted in many ways. Be more direct in the descriptions being provided. The reader is not able to gain much from the current abstract.
Intro
Line 35. More clarification is required regarding the statement that FM use is not sustainable because the next sentences contradict this when FM use is reducing. There are other reviews which show that FM can be a sustainable protein source if used strategically, which is what this article is also suggesting. You also talk about the unsustainability of soybean meal but do not say that its use is unsustainable, the same way you talk about FM. A balanced argument is needed.
Line 50. What kind of volumes of Plukenetia volubilis are produced? Where is it used and produced? Is there enough cake available to satisfy shrimp production? The reader needs more justification for its use.
Table 2. Please provide details of the vitamin, mineral mix used.
Line 124. Have you completed a trial with this number of replications before (2 cages with 15 shrimp/cage)? Is this enough number of reps? What kind of mean difference would you need to obtain a significant difference between treatments? This website can be used to do a sample size analysis: https://statulator.com/
Line 132. How were the abdomen samples taken?
Results
Line 190. Why did you say there were no statistical difference when there was a statistical difference for final weight (p = 0.031) where the D-50 performed better than the control? Therefore the interpretation of the results is incorrect here.
Line 196. Again for HP weight, there was a difference between treatments, not as similar as you say.
Line 198. Another wrong statement here. FCR is significantly different where p = 0.042 which is less then 0.05.
Line 242. There was a significant difference between the control and D-50 for final body weight
Line 292. How can you explain the reduced ability to digest protein, coupled with the lower protein composition but higher final weight gain and abdomen weight?
Line 293. What are the other anti-nutritional components present in Plukenetia volubilis and how should it’s inclusion rate be restricted in formulations?
Line 321. Protein content of shrimp was reduced with 50% replacement of FM with Plukenetia volubilis
Author Response
thank for your comments
Author Response File: Author Response.pdf
Round 2
Reviewer 3 Report
The revised manuscript seems to be fine. Please kindly provide economic feasibility of the usage of P. volubilis meal in the diet of L. vannamei.
Author Response
Due to the recent introduction of Sacha culture, it is difficult to establish the economic benefit of its introduction in aquaculture. The authors acknowledge that currently the production of sacha is not enough to be considered a global alternative to fishmeal, but its cultivation is increasing, as well as its applications, so it is foreseeable that in the future the hectares destined for this will increase. cultivation and a stabilization of prices that allow it to be considered as an alternative source of protein in animal feed.
Thank you for your help to improve the article
Reviewer 4 Report
Edits have been made to improve the manuscript but some of the previous comments need to be addressed further.
Line 11. The first sentence of the abstract repeats the title wording. This is not needed.
Line 210. Remove ‘although’. Describe the statistical difference in final weight before alluding to the trend between final weight, cephalothoraxe/abdomen weight and increasing sacha inchi.
Line 214. The HP weights are not similar between treatments because they are significantly different (P-value is < 0.05). You can say that the sacha inchi replacement diets were similar to the control diet but there was a difference between D-15 and D-25. This is a result that needs to be addressed.
Line 216. Again, you cannot say that there is no statistical difference in FCR because the p-value indicates that treatments are statistically different. The FCR between control and D-50 is different.
In this section, the interpretation of the statistical analysis needs improvement and the language used to express these results requires more accuracey.
Line 221. The interpretation of the results here are more accurate where the statistically different treatment is acknowledged
Author Response
Line 11. The first sentence of the abstract repeats the title wording. This is not needed.
The sentence was deleted.
Line 210. Remove ‘although’. Describe the statistical difference in final weight before alluding to the trend between final weight, cephalothoraxe/abdomen weight and increasing sacha inchi.
Done
Line 214. The HP weights are not similar between treatments because they are significantly different (P-value is < 0.05). You can say that the sacha inchi replacement diets were similar to the control diet but there was a difference between D-15 and D-25. This is a result that needs to be addressed.
Actually, we don´t know the reason for high hepatopancreas weight in D-25 group but we can not to relate with Sacha inclusion. This have been includ in the discussion (L 294-300)
The lowest SGR was found in the shrimp fed with D-25 diet. This could be related to the heavier hepatopancreas weight observed in this group, and could be provoked by hepatotoxicity, deriving to lower enzymes secretion and lower digestive efficiency as described in poultry fed with sacha inchi cake [35,36]. However, in shrimps fed with 50% fish meal replacement level (D-50), the SGR was higher than that observed for CT and D-25 groups. So, it is plausible to question if sacha inchi was responsible for the observed lower SGR obtained or heavier hepatopancreas weight in D-25 group. A decrease in chymotrypsin activity was observed in the D-25 group, which could be related with the lower SGR observed in this group.
Line 216. Again, you cannot say that there is no statistical difference in FCR because the p-value indicates that treatments are statistically different. The FCR between control and D-50 is different.
In the ms this difference is clarified in Material, and methods (L 241-242) and in discission (L 286)
“No statistical differences in FCR were observed among the different experimental diets except between control and D-50.”
“…weight by increasing cephalothorax and abdomen weight (Table 5) being those shrimps fed with D-50 which showed higher values of growth indices and better FCR.
In this section, the interpretation of the statistical analysis needs improvement and the language used to express these results requires more accuracey.
The section has been revised
Line 221. The interpretation of the results here are more accurate where the statistically different treatment is acknowledged
Thank you for your help to improve the article
Round 3
Reviewer 4 Report
The manuscript is suitable for publication.
