The Effects of Replacing Fish Meal Protein with Black Soldier Fly Meal and Sodium Butyrate Supplementation on the Growth Performance, Lipid Peroxidation, and Intestinal Villi Status of Jade Perch, Scortum barcoo Fingerlings
Round 1
Reviewer 1 Report
Title "The effects of replacing fish meal protein with black soldier fly meal and sodium butyrate supplementation on the growth performance, lipid peroxidation, and intestinal villi status of the Jade perch, Scortum barcoo fingerlings" is is an original scientific article containing all the necessary attributes of the feeding test and its evaluation.
The topic is not new, but in this case it is not so important because, as the authors themselves state, aquaculture is the fastest growing sector of agriculture and the search for new sources of protein for fish is still very important. From the description of the methodology and then the results, it is clear that the authors have spent a lot of work and time on the practical part of it. I recommend the manuscript for publication after minor modifications.
Abstract - doesn´t need any change
Intoduction
Line 51 - authors are writing about the increasing price of soybeans, but citations are from year 2010 and 2012 (cit. 6 and7). There should be some more actual.
L 64 - inadequately cited. Are 70% of the nutrients really not used by the fish and excreted into the environment? Citation 11 is not telling it.
Materials and Methods
L 149 - how long was the adaptation period with feeding the commercial diet?
L 175 - why only three individuals from each tank, i.e. 9 from one group? It is not sufficient number for statistical evaluation.
L 221 - the same three individuals described above? I hope so. So I don´t understand why do you have only five tubes with blood?
Results
Result section is described well but the quality od tables is very poor.
The tables do not show in some rows where one value ends and another begins. It would be a good idea to modify them so that they are clear. I don't know if it's really necessary to have p-values there. The expression of statistical significance with indices is sufficient.
Discussion
L 424-432 - the same text as in introduction L 71-75 - I recommend to erase it in discussion
L 437 - description hoe the flies were bred. This should be part of the methods - whre have the ingredients of the feed come from. So erase it here and add it in the methods section.
L 557 and 562 - there are two citations (57 and 58) dealing with enzyme activity, but in mice and rats. It is not appropriate to compare the results of fish with warm-blooded animals. I recommend finding citations related to fish.
Conclusions
There is a summary of the effect of the addition of butyric acid, but there is no summary of the effect of changing the protein source. The authors should briefly add the effect of fly protein on production parameters of Jade perch.
Recommendation
I recommend to authors to increase the data base in future research. The sampling of nine fish is not ideal and the effect of additives is not sufficiently demonstrated in such a small sample ("n").
Author Response
Dear Reviewer
Thanks very much for taking your time to review this manuscript. I really appreciate all your comments and suggestions! Please find my itemized responses in below and my revisions/corrections in the re-submitted files. Thanks again!
Review 1
Line 51 - authors are writing about the increasing price of soybeans, but citations are from year 2010 and 2012 (cit. 6 and7). There should be some more actual.
A: cit. 6: Prudêncio da Silva, V.; van der Werf, H.M.G.; Spies, A.; Roberto Soares, S. Variability in environmental impacts of Brazilian soybean according to crop production and transport scenarios. Journal of Environmental Management, 2010, 91, 1831-1839. https://doi.org/10.1016/j.jenvman.2010.04.001 change as Parolini, M.; Ganzaroli, A.; Bacenetti, J. Earthworm as an alternative protein source in poultry and fish farming: Current applications and future perspectives. Science of The Total Environment 2020, 734, 139460. https://doi.org/10.1016/j.scitotenv.2020.139460
cit. 7: Olsen, R.L.; Hasan, M.R.A. limited supply of fishmeal: impact on future increases in global aquaculture production. Trends in Food Science and Technology, 2012 27, 120-128. https://doi.org/10.1016/j.tifs.2012.06.003 change as FAO. The United Nations Food and Agriculture Organization, Market report, 2023, Fishmeal and fish oil: Peru halts most fishing, Chinese demand wanes.
L 64 - inadequately cited. Are 70% of the nutrients really not used by the fish and excreted into the environment? Citation 11 is not telling it.
A: The sentence has been corrected as “However, numerous reports indicate that as high as 7% to 68% of the protein ingested by aquatic organisms are not absorbed ”. And deleted sentence of “and are instead excreted into the aquatic environment, leading to an increase in the feed cost and contamination of the environment.”
Accord cit.11. Faecal to ingested nutrient ratios in salmonids for nutrients from different sources of protein are 7% to 68%.
Materials and Methods
L 149 - how long was the adaptation period with feeding the commercial diet?
A: The sentence has been corrected as “In the raising period, the fries were fed with commercial feed (Marine fish larvae feed No. 3, Shye Yih Feeding Co., Ltd.) once per day for one week.”
L 175 - why only three individuals from each tank, i.e. 9 from one group? It is not sufficient number for statistical evaluation.
A: Due to some mistake in sampling process for hematological analysis. I will be cautious for sampling and increase the data base in future research.
The sentence has been corrected as “Three individuals were taken from each bucket (nine in each group) and anesthetized with 1% tricaine methane sulfonate (MS222, Sigma-Aldrich, U.S.).”
After feed trial, there are three replications form each experiment diet for hematological analysis.
L 221 - the same three individuals described above? I hope so. So I don´t understand why do you have only five tubes with blood?
A: In L221, the sentence of “three individuals were removed from each of three buckets (nine fish each group) for hematological analysis.” Is same described above. And the sentence has been corrected as “Blood was collected from the caudal vein, and eighteen tubes of about 1 ml whole blood for each tube were collected from each group and examined by a blood analyzer (Sysmex XP-300, San Tung Instrument Co., Ltd. Japan).”
Result section is described well but the quality of tables is very poor.
The tables do not show in some rows where one value ends and another begins. It would be a good idea to modify them so that they are clear. I don't know if it's really necessary to have p-values there. The expression of statistical significance with indices is sufficient.
A: The Tables have been revised.
Discussion
L 424-432 - the same text as in introduction L 71-75 - I recommend to erase it in discussion
A:It is deleted.
L 437 - description hoe the flies were bred. This should be part of the methods - whre have the ingredients of the feed come from. So erase it here and add it in the methods section.
A: It is deleted. The sentence of “Approximately 210,000 eggs of black soldier fly were obtained from Kunyi Biotech (Chiayi County, Taiwan), which were reared at National Chiayi University (NCYU; Chiayi City, Taiwan). The eggs were hatched and grown in empty containers (140 cm × 100 cm × 50 cm) indoors, and the temperature was maintained at 22°C–25°C. Three days after hatching, the larvae were feed with soybean meal (crude protein: 38.4% and crude lipid: 17.26%; TTET Union Corporation, Tainan City, Taiwan). For formulating the diet for fish, black soldier flies in the pupal stage were dried and homogenized.” was added In Materials and Methods.
L 557 and 562 - there are two citations (57 and 58) dealing with enzyme activity, but in mice and rats. It is not appropriate to compare the results of fish with warm-blooded animals. I recommend finding citations related to fish.
A: Number cit. 57 was change cit 58. Yao, J.; Wang, J.Y.; Liu, L.; Li, Y.X.; Xun, A.Y.; Zeng, W. S.; Jia, C. H.; Wei, X. X.; Feng, J.L.; Zhao, L.; Wang, L. S. Anti-oxidant effects of resveratrol on mice with DSS-induced ulcerative colitis. Archives of Medical Research, 2010, 41, 288–294. https://doi.org/10.1016/j.arcmed.2010.05.002 change as Vinagre, C.; Madeira, D.; Narciso, L.; Cabral, H.N.; Diniz, M. Effect of temperature on oxidative stress in fish: Lipid peroxidation and catalase activity in the muscle of juvenile seabass, Dicentrarchus labrax. Ecological Indicators 2012, 23, 274-279.
Number cit. 58 was change cit 59. Lubkowska, A.; Bryczkowska, I.; Gutowska, I.; Rotter, I.; Marczuk, N.; Baranowska-Bosiacka, I.; Banfi, G. The effects of swimming training in cold water on antioxidant enzyme activity and lipid peroxidation in erythrocytes of male and female aged rats. International Journal of Environmental Research and Public Health, 2019, 16, 647. doi:10.3390/ijerph16040647 change as Slaninova, A.; Smutna, M.; Modar, H.; Svobodova, Z. A review: Oxidative stress in fish induced by pesticides. Neuroendocrinology Letters 2009, 30, 2-12.
Conclusions
There is a summary of the effect of the addition of butyric acid, but there is no summary of the effect of changing the protein source. The authors should briefly add the effect of fly protein on production parameters of Jade perch.
A: The sentence of “Complete substitution of FM with black soldier fly meal derived from flies fed with soybean meal considerably reduced the growth performance of jade perch fingerlings and changed their muscle protein contents and lipid contents. Black soldier fly meal supplemented with butyric acid may effectively partially substitute FM in the diet of the jade perch fingerlings without adversely affecting their growth performance.” was added.
Recommendation
I recommend to authors to increase the data base in future research. The sampling of nine fish is not ideal and the effect of additives is not sufficiently demonstrated in such a small sample ("n").
A: Thank you. I will increase the data base in future research.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
General comments
Well-structured work, with analyzes that justify the results. Authors need to decrease comparisons with other works. There is a need to further explain the results. The questions asked by the reviewer about the discussion of the manuscript go in this direction.
Discussion
Line 429: “Black soldier fly is a sustainable feed ingredient option rich in micronutrients” Why the use Black soldier fly is a sustainable? Explain in the manuscript.
Line 443-456 Can the Black soldier fly have antinutrients? Can the quitine cause prejudice in the absorption of nutrients? Explain in the manuscript.
Lines 466-468 “Ref. [38] noted that a black soldier fly meal content higher than 5.3% in feed caused damage to fish intestinal villi, created vacuoles in the hepatopancreas, and reduced lipid accumulation in tissues”. Why? Explain in the manuscript.
Lines 493-511 It is unclear how butyric acid promotes an increase in villous height. "Ref. [44], namely that butyric acid promoted nutrient digestion and absorption by increasing the number of epithelial cells and reducing the pH level in the fish intestine". How does it promote cell growth? Could the better health of the intestinal epithelium be due to the antioxidant action of butyric acid? Explain in the manuscript.
Lines 537-538 "Ref. [52] added 2 g/kg of butyric acid to the feed of largemouth bass (Micropterus salmoide) with an initial weight of 12.3 g and noted increased ALT and AST activities in the liver". The increase in transaminases (ALT and AST) in the liver may be due to increased protein synthesis and not due to liver damage. The increase in transaminases in the blood, indicates liver damage. Check the information in the manuscript.
Lines 555-556 “Butyric acid is an effective immunity booster that removes harmful reactive oxygen species (ROS) from the cellular environment, thereby reducing oxidative stress [55,56]” How? The butyric acid is eletrons donator? It is unclear from the manuscript discussion how butyric acid acts to enhance antioxidant response. Explain in the manuscript.
Conclusions
The authors no replied part of the objective these work. What the influence of substituting fish meal protein with insect meal protein for jade perch? Answer in the conclusions.
Author Response
Dear Reviewer
Thanks very much for taking your time to review this manuscript. I really appreciate all your comments and suggestions! Please find my itemized responses in below and my revisions/corrections in the re-submitted files. Thanks again!
Review 2
General comments
Discussion
Line 429: “Black soldier fly is a sustainable feed ingredient option rich in micronutrients” Why the use Black soldier fly is a sustainable? Explain in the manuscript.
A: The sentence of “In insect protein, black soldier fly has several major advantages over other insect species. The species is polyphagous that use well-defined substrates, producing insects of a defined quality of macro- and micronutrients.” Was added in Introduction of manuscript.
Line 443-456 Can the Black soldier fly have antinutrients? Can the quitine cause prejudice in the absorption of nutrients? Explain in the manuscript.
A: The sentence of “Furthermore, antinutritional factors are compounds that reduce the absorption of nutrients, such as phytic acid in soybeans. Therefore, in this study, black soldier fly maybe contains antinutrients, which is likely caused by the different chemical compositions of soybean meal on which the insect feed.” was added in Discussion of manuscript.
There are no references for black soldier fly containing quitine.
Lines 466-468 “Ref. [38] noted that a black soldier fly meal content higher than 5.3% in feed caused damage to fish intestinal villi, created vacuoles in the hepatopancreas, and reduced lipid accumulation in tissues”. Why? Explain in the manuscript.
A: The sentence of “Ref. [38] noted that a black soldier fly meal protein content higher than 5.3% in feed caused damage to fish intestinal villi, created vacuoles in the hepatopancreas, and reduced lipid accumulation in tissues. This may result from the chitin exist in black soldier fly meal. Chitin is widely distributed in insects, and chitin polysaccharides account for approximately 20% of the dry weight of insect shells [39]. Ref. [40] indicated that chitin and its derivatives were reported to play a significant role on decreasing fatty acids synthesis and decreased the digestibility of crude protein.” was added in Discussion of manuscript.
Lines 493-511 It is unclear how butyric acid promotes an increase in villous height. "Ref. [44], namely that butyric acid promoted nutrient digestion and absorption by increasing the number of epithelial cells and reducing the pH level in the fish intestine". How does it promote cell growth? Could the better health of the intestinal epithelium be due to the antioxidant action of butyric acid? Explain in the manuscript.
A: The sentences of “Ref. [44] indicated that butyric acid can inhibit harmful bacteria and promote the reproduction of beneficial bacteria after entering bacterial cells. Butyrate can also induce the synthesis of r intestinal epithelial mRNA and protein, thus maintaining intestinal health, accelerate the proliferation of intestinal villus, and promoted nutrient absorption. “ and “The nutrients composition of diet will affect the construction of intestinal villi, especially protein sources and its amino acids composition. Many histological studies have been investigated the impact of various dietary protein sources and unbalance amino acids composition in fish including rainbow trout [51], gilthead sea bream [52] and common carp [53]. Partial amino acid deficiency in black soldier fly meal protein-based diets (BM and BMB) adversely affects fish intestinal epithelium. Therefore, the histological effects on intestine observed in this study maybe partly explained by amino acids unbalance in black soldier fly meal. The experimental results of this study revealed that amino acids composition balance in diet, adding butyric acid significantly increased the length of the anterior intestinal villi and thus benefited the intestinal health and nutrient absorption of jade perch fingerlings.” were added in Discussion of manuscript.
Lines 537-538 "Ref. [52] added 2 g/kg of butyric acid to the feed of largemouth bass (Micropterus salmoide) with an initial weight of 12.3 g and noted increased ALT and AST activities in the liver". The increase in transaminases (ALT and AST) in the liver may be due to increased protein synthesis and not due to liver damage. The increase in transaminases in the blood, indicates liver damage. Check the information in the manuscript.
A: The sentence of “Ref. [52] added 2 g/kg of butyric acid to the feed of largemouth bass (Micropterus salmoide) with an initial weight of 12.3 g and noted increased ALT and AST activities in the liver, which was consistent with the histopathological changes.” was revise.
Lines 555-556 “Butyric acid is an effective immunity booster that removes harmful reactive oxygen species (ROS) from the cellular environment, thereby reducing oxidative stress [55,56]” How? The butyric acid is eletrons donator? It is unclear from the manuscript discussion how butyric acid acts to enhance antioxidant response. Explain in the manuscript.
A: The sentence of “Sodium butyrate administration increased antioxidant genes expression, such as SOD, CAT, GPx, GST and GR gene, and consequently these enzyme activities in intestine of fish, which led to lower ROS formation in the intestinal tissues, thereby reducing oxidative stress [55,56].” was revise.
Conclusions
The authors no replied part of the objective these work. What the influence of substituting fish meal protein with insect meal protein for jade perch? Answer in the conclusions.
A: The sentence of “Complete substitution of FM with black soldier fly meal derived from flies fed with soybean meal considerably reduced the growth performance of jade perch fingerlings and changed their muscle protein contents and lipid contents. Black soldier fly meal supplemented with butyric acid may effectively partially substitute FM in the diet of the jade perch fingerlings without adversely affecting their growth performance.” was added in conclusions.
Author Response File: Author Response.pdf
