Effects of Fermented Kefir as a Functional Feed Additive in Litopenaeus vannamei Farming
Round 1
Reviewer 1 Report
This MS aims to investigate whether fermented kefir could serve as a functional feed additive in Litopenaeus vannamei farming. By using RT-PCR, this study confirmed that six immune related genes of shrimp (i.e. beta-glucan binding protein, prophenoloxidase, crustin, peaneidin, lysozyme, and superoxide dismutase) were up-regulated after feeding with kefir. Furthermore, the treatment with kefir not only slightly increased the size/weight of farming shrimps, but also improved the survival rate of shrimps under WSSV challenge. Taken together, this is a straightforward MS and the provided information may be useful for shrimp farming. Specific comments are as the following:
- (Abstract, lines 20-21) The authors mentioned that the treatment of kefir could upregulate “six of 10 genes” crucial for innate immunity of L. vannamei. However, this reviewer only sees 6 genes in this MS. What are the other four genes? If there are any related results, they should also be included in Figure 2.
- (Lines 85-86) This should be Table 2. Further, the title of this table is missing.
- (Figure 2B) I doubt the accuracy of the quantitative results of this figure. For instance, based on 0.4% kefir treated shrimps in Figure 2A, in the four groups of experimental shrimps, only one lysozyme gene was significantly up-regulated, while the other three showed almost no signal. After averaging, the standard deviation should be much larger than the one shown in Figure 2B. Although it may not affect the conclusion, the results should be presented more accurately.
Author Response
Response to Reviewer 1 Comments
First of all, I appreciate your kindly comments and fixed my manuscript according to reviewer comments. Please find revised manuscript uploaded.
Point 1: (Abstract, lines 20-21) The authors mentioned that the treatment of kefir could upregulate “six of 10 genes” crucial for innate immunity of L. vannamei. However, this reviewer only sees 6 genes in this MS. What are the other four genes? If there are any related results, they should also be included in Figure 2.

Response 1: I fixed “six of 10 genes to “six of 7 genes” and added new Figure 2 according to reviewer 1 comment in revised manuscript.
Point 2: This should be Table 2. Further, the title of this table is missing.
Response 2: I fixed “Table 1” to “Table 2” and added the title of this table in revised manuscript.
Point 3: I doubt the accuracy of the quantitative results of this figure. For instance, based on 0.4% kefir treated shrimps in Figure 2A, in the four groups of experimental shrimps, only one lysozyme gene was significantly up-regulated, while the other three showed almost no signal. After averaging, the standard deviation should be much larger than the one shown in Figure 2B. Although it may not affect the conclusion, the results should be presented more accurately.

Response 3: I re-analysed the expression level of lysozyme gene and hemacyanin. And, I fixed the result and added new Figure 2 according to reviewer comment.
Reviewer 2 Report
In this manuscript, the authors describe the effects of the use of fermented kefir as functional feed additive in the shrimp species Litopenaeus vannamei. They monitored the effects of this treatment on the weight of the tested individuals, on the expression level of several classical genes involved in immune response (crustin, proPO, SOD, etc.) and after a controlled infection with the WSSV (White Spot Syndrome Virus).
This is an interesting and valuable study to improve our knowledge on possible feed additive (economically cheap) to improve both immune system and productivity of L. vannameil in shrimp farming, as an alternative of the use of antibiotics. The manuscript is generally clear and well organized, however I think that the significance of the results should be even more highlighted, and I do have some minor concerns about methodological approaches used here. I do not see any major methodological reason that could justify a refusal to publish this study.
Introduction (global comment): I was a bit frustrated at the end of the end of the reading of this introduction. To me, it does not explain enough the potential impact of your results (which are really interesting!). I have noted some potential points for an improvement in this direction, which are listed below.
Introduction:
Line 32/33: The authors said “whiteleg shrimp, is an important aquaculture species in the Asia-Pacific region [1]”. Please specify why/how important (notably economically), to highlight the importance of the studied model (and thus, the impact of your study).
Line 34/36: You did not specified the impact of Vibrio sp. on the economy based on L. vannamei farming. Still in the same way of highlighting your study, it might be interesting to describe (briefly) the impact of this pathogen on shrimp farming.
Line 42/46: The authors exposed briefly some effects of a kefir treatment on “animals and humans [15]”. Could you develop a bit more than you did? It would be useful to the reader to understand why kefir might be such useful in your case. Do you have any example of an effect on invertebrate species to illustrate your point?
Here are just a few thoughts to improve the introduction and make it more punchy (especially since this introduction is extremely short), still with the idea of highlighting your study.
Materials & Methods:
Line 61: The authors said “viable cells were counted“. What do you mean by “viable”? How did you check the viability of those cells? How did you count the cells? (KOVA slide, automated cell counter, etc.)
Line 83: The authors said “using ImageJ software”. Please add the reference of the software. I am not really familiar with the use of the intensity of the amplified cDNA to “quantify” the amount of mRNA (and thus the expression of a gene). Did not you have the possibility to use a RT-qPCR approach instead? Furthermore, did you have the occasion to check the stability of the expression of your housekeeping gene? Because if the expression of this housekeeping gene is not statistically constant, it might be problematic for your interpretations. For example, I know that geNorm software, from Vandesompele et al. (2002), is used for this kind of checking for RT-qPCR analysis, may be an equivalent is available for ImageJ?
Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Paepe, A., Speleman, F., 2002. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3, research0034.1–research0034.11.
Line 85: Concerning the Table 1, the author gave no explanation concerning the choice of the studied genes… Please just add a sentence noticing that those genes are quite representative of the global immune response in crustacean for example (and I do not question the choice of these genes at all).
Furthermore (and to be picky), you checked the proPO gene activity and the Hemocyanin activity (known to play also a role in the proPO cascade), but do you know if the mRNA is actually transcribed (and thus, if the proPO cascade is more effective)? Using a proteic approach with L-DOPA substrate for example, which will inform you directly about the PO activity and effectiveness… If you still have some haemolymph from the individuals you used, it is clearly possible to check that (with the same kind of protocol as Söderhäll & Häll (1984).
Söderhäll, K., Häll, L., 1984. Lipopolysaccharide-induced activation of prophenoloxidase activating system in crayfish haemocyte lysate. Biochim. Biophys. Acta 797 (1), 99–104.
Line 108: why do not you use a Kaplan-Meier survival test to estimate the survival rate? Classily, a log-rank test is applied after Kaplan-Meier test to compare the survival rates between conditions (instead of t test). I think it is better to use Kaplan-Meier approach here (even if the results will not change, to my opinion). You can easily perform it using R software, with the “survival” (Therneau & Grambsch, 2000) coupled with the package “KMsurv” (Klein & Moeschberger, 1997).
Therneau, T.M., Grambsch, P.M., 2000. Modeling Survival Data: Extending the Cox Model. Springer, New York.
Klein, J.P., Moeschberger, M.L., 1997. Survival Analysis Techniques for Censored andTruncated Date. Springer, New York.
Results:
Line 143: Table 1 > It is Table 2 and the legend said “Tables should be placed in the main text near to the first time they are cited.” Please revise it.
Line 150: The author said “7.65 ´107/mL on an average », please also specify the standard deviation. Also, please add the statistical test to prove the non-significance of the evolution of the hemocyte density. Furthermore, what about the viability of the haemocyte? Did you check also this? (Using a blue tripan coloration for example: blue cells are dead cells, and white cells are living cells)
Line 189: About the Figure 4. To me, it is difficult to see the scale below the body of the shrimps, due to the poor quality of the picture (poor definition, dark contrasts). If these images were of good quality, I would have no problem keeping them as illustrations, however it is not the case, and the histogram below (Figure 4 E, F and G) is the most important data here.
Discussion:
Line 199: The author said “Litopenaeus vannameiis one of the popular species in shrimp farming in Asia and America [16]” which is quite redundant with the first sentence of the introduction, and no more informative…
Line 201: “Penaeus monodon” is not in italic, please correct.
Line 206: “Vibrio parahaemolyticus" is not in italic, please correct.
Line 206/207: You indicated that the “high-density farming of this shrimp results in the outbreak of infection by viruses and pathogenic bacteria [4]”. Do you have any quantitative information on the impact of these pathogens? (Concerning the amount of shrimp in tons, or the size/weight of the shrimps, etc.) It might be useful to add these kind of information in the discussion, still to strengthen the impact pf your results.
In the same way, do you have any idea of the estimated (probably low) cost of using kefir complement to feed shrimp in a farming place? (In order to be both efficient and not too impactful economically)
Author Response
Response to Reviewer 2 Comments
First of all, I appreciate your kindly comments and fixed my manuscript according to reviewer comments. Please find out revised manuscript uploaed.
Point 1: Line 32/33: The authors said “whiteleg shrimp, is an important aquaculture species in the Asia-Pacific region [1]”. Please specify why/how important (notably economically), to highlight the importance of the studied model (and thus, the impact of your study)

Response 1: I fixed this sentence to “the Asia-Pacific region because this region is the largest producer contributing to nearly 80% of the value and volume of the global shrimp market.”
Point 2: Line 34/36: You did not specified the impact of Vibrio sp. on the economy based on L. vannamei farming. Still in the same way of highlighting your study, it might be interesting to describe (briefly) the impact of this pathogen on shrimp farming.
Response 2: I fixed this sentence to “Vibrio spp. that cause tail necrosis, shell disease, red disease, loose shell syndrome (LSS), and white gut disease (WGD)”
Point 3: Line 42/46: The authors exposed briefly some effects of a kefir treatment on “animals and humans [15]”. Could you develop a bit more than you did? It would be useful to the reader to understand why kefir might be such useful in your case. Do you have any example of an effect on invertebrate species to illustrate your point?.

Response 3: According to reviewer comment, I added one example studied the effect of kefir on trout in revised manuscript. And, our study is first case to investigate the effect of kefir on shrimp as one of invertebrate species.
Point 4: Line 61: The authors said “viable cells were counted“. What do you mean by “viable”? How did you check the viability of those cells? How did you count the cells? (KOVA slide, automated cell counter, etc.)
Response 3: I fixed “viable cells” to “viable colonies”.
Point 4: Line 83: The authors said “using ImageJ software”. Please add the reference of the software. I am not really familiar with the use of the intensity of the amplified cDNA to “quantify” the amount of mRNA (and thus the expression of a gene). Did not you have the possibility to use a RT-qPCR approach instead? Furthermore, did you have the occasion to check the stability of the expression of your housekeeping gene? Because if the expression of this housekeeping gene is not statistically constant, it might be problematic for your interpretations. For example, I know that geNorm software, from Vandesompele et al. (2002), is used for this kind of checking for RT-qPCR analysis, may be an equivalent is available for ImageJ?
Response 4: Firstly, I agreed with reviewer comment. We carried out RT-PCR instead of RT-qPCR because we did not have the machine operating RT-qPCR. And this method in my manuscript was already reported in Fish & Shellfish Immunology (2007). So, I fixed some parts of method in revised manuscript with adding one reference.
Point 5: Line 85: Concerning the Table 1, the author gave no explanation concerning the choice of the studied genes… Please just add a sentence noticing that those genes are quite representative of the global immune response in crustacean for example (and I do not question the choice of these genes at all).
Furthermore (and to be picky), you checked the proPO gene activity and the Hemocyanin activity (known to play also a role in the proPO cascade), but do you know if the mRNA is actually transcribed (and thus, if the proPO cascade is more effective)? Using a proteic approach with L-DOPA substrate for example, which will inform you directly about the PO activity and effectiveness… If you still have some haemolymph from the individuals you used, it is clearly possible to check that (with the same kind of protocol as Söderhäll & Häll (1984).
Response 5: I added “antimicrobial peptides and the immune defense system against major pathogens” as a phrase according to reviewer comment. And I agreed reviewer comment regarding mRNA. Now, we cannot carried out the experiment because we do not have any haemolymph.
Point 6: Line 108: why do not you use a Kaplan-Meier survival test to estimate the survival rate? Classily, a log-rank test is applied after Kaplan-Meier test to compare the survival rates between conditions (instead of t test). I think it is better to use Kaplan-Meier approach here (even if the results will not change, to my opinion). You can easily perform it using R software, with the “survival” (Therneau & Grambsch, 2000) coupled with the package “KMsurv” (Klein & Moeschberger, 1997).
Response 6: According to reviewer comment, I re-analysed my data by Kaplan-Meier Survival analysis and fixed to “Survival rate by kefir treatment was calculated by Kaplan-Meier survival analysis provided by GraphPad Prism 8 (GraphPad Software, CA, US)” in revised manuscript. Also, I added new Figure 3 analysed by Kaplan-Meier survival analysis in revised manuscript.
Point 7: Line 143: Table 1 > It is Table 2 and the legend said “Tables should be placed in the main text near to the first time they are cited.” Please revise it.
Response 7: According to reviewer comment, I fixed the legend of Table 2 to “Table 2. The optimized medium condition for kefir fermentation”
Point 8: Line 150: The author said “7.65´107/mL on an average », please also specify the standard deviation. Also, please add the statistical test to prove the non-significance of the evolution of the hemocyte density. Furthermore, what about the viability of the haemocyte? Did you check also this? (Using a blue tripan coloration for example: blue cells are dead cells, and white cells are living cells)
Response 8: According to reviewer comment, I added the Table regarding the number of total hemocyte. Through trypan blue staining, we already checked that the viability of hemocyte in each group were almost lived.
Point 9: About the Figure 4. To me, it is difficult to see the scale below the body of the shrimps, due to the poor quality of the picture (poor definition, dark contrasts). If these images were of good quality, I would have no problem keeping them as illustrations, however it is not the case, and the histogram below (Figure 4 E, F and G) is the most important data here.
Response 9: I added modified pictures and black arrow to see the differences more clear.
Point 10: Line 199: The author said “Litopenaeus vannameiis one of the popular species in shrimp farming in Asia and America [16]” which is quite redundant with the first sentence of the introduction, and no more informative…
Response 10: According to reviewer comment, I omitted this sentence.
Point 11: Line 201: “Penaeus monodon” is not in italic, please correct.
Response 11: I fixed “Penaeus monodon” to “Penaeus monodon” in revised manuscript.
Point 12: Line 206: “Vibrio parahaemolyticus" is not in italic, please correct.
Response 12: I fixed “Vibrio parahaemolyticus” to “Vibrio parahaemolyticus” in revised manuscript.
Point 13: Line 206/207: You indicated that the “high-density farming of this shrimp results in the outbreak of infection by viruses and pathogenic bacteria [4]”. Do you have any quantitative information on the impact of these pathogens? (Concerning the amount of shrimp in tons, or the size/weight of the shrimps, etc.) It might be useful to add these kind of information in the discussion, still to strengthen the impact pf your results.
In the same way, do you have any idea of the estimated (probably low) cost of using kefir complement to feed shrimp in a farming place? (In order to be both efficient and not too impactful economically)
Response 13: I rewrote this sentence to “Moreover, high-density farming of this shrimp that can produce yields of 20,000 to 100,000 kg/ha/year results in the outbreak of infection by viruses and pathogenic bacteria because of eutrophication by unconsumed feeds and lots of shrimp feces” in revised manuscript. And the estimation of cost of using kefir complement to feed is quite hard because each farming use different feed additives individually. However, if the farm need a functional feed additive containing multiple bacteria beneficial to shrimp, kefir fermentation is more advantageous economically because multiple bacteria can be obtained through kefir fermentation at once, compared to other products that mix bacteria after fermentation of each bacteria individually.
