Seasonal Variation in Chemical Composition, Ruminal Fermentation, and Biological Characteristics of Paulownia shan tong: In Vitro Potential Use by Sheep and Goats

Round 1

Reviewer 1 Report

This manuscript cannot be published in this form in the Fermentation journal. The experimental design is not clear, as your objective was to determine the nutritive values of P. shan tong during two seasons and with two animals inocula (sheep and goat), but in the methods part you mentioned only the sheep as ruminal fluid donors!! also in all incubations you have to incubate the samples collected in winter and spring using the two inoculate sources, also in all tables, it is not clear which samples were incubated in goats or sheep inocula. another issue, If you would like to use it as an alternative feed resource, comparisons with traditional feed have to be made. Where is your control in the treatments? Moreover, it is not clear if the plants were planted or naturally cultivated? in the studies where you have to compare two seasons you must obtain results from two years at least, or the evaluation will be biased and unreal!

The hypothesis of this work is not clear, why you measured the nutritive value in two seasons? may you have to mention the feed security during these two seasons, and why you selected these two seasons specifically?    

what is the novelty of this study? leaves of P. shan tong are already considered a forage source for ruminants.

Please insert more information about the plant collection, what was the life stage each season? plant sample size? how many trees were collected for each plant sample? how many plots are for each plant sample? how many true statistical and analytical repetitions were made for all the obtained results?

The description of the methods part of the in vitro assay is poor, please insert more information for preparing the ruminal fluid, the number of syringes that were done for each plant, and how many GP runs.

Here are some other minor issues:

Line 38, please write the full name of all abbreviations in their first mentions (e.g, CE, GAE, ..), an check this issue through the manuscript.

Line 39, what do you mean by asymptotic?

In vitro is italic throughout the manuscript.

In lines 31 to 32, you have to present the treatments in place of  DM, OM, NDF, ADF,...

Line 46, the conclusion has to be changed, you don't have any comparisons with any traditional source to insert such a conclusion.

In tables, you have to insert the data of the incubation of both seasons in each ruminal source to get the interaction effect as you tested.

Line 111, fiberteck ??

For the statistical analyses, did you use the one-way ANOVA for all the experimental parameters!!  please insert the models that you used in the chemical composition and for the in vitro assay, what was your experimental unit for all of your obtained results?  which significance degree did you use?  correlation!! where are the tables of the correlations that you did?

Line 215, which variables?

In the manuscript, you used " Leaves obtained from the 1st harvest", what do you mean by the 1st harvest? where these data, no mentions for this either in the methods or the results part!!  Also, what was the 2nd harvest?

Conclusions part is very poor.

 

 

 

Author Response

1. This manuscript cannot be published in this form in the Fermentation journal. The experimental design is not clear, as your objective was to determine the nutritive values of  shan tongduring two seasons and with two animals inocula (sheep and goat), but in the methods part you mentioned only the sheep as ruminal fluid donors!! also in all incubations you have to incubate the samples collected in winter and spring using the two inoculate sources, also in all tables, it is not clear which samples were incubated in goats or sheep inocula. another issue, If you would like to use it as an alternative feed resource, comparisons with traditional feed have to be made. Where is your control in the treatments? Moreover, it is not clear if the plants were planted or naturally cultivated? in the studies where you have to compare two seasons you must obtain results from two years at least, or the evaluation will be biased and unreal!

AU Response 1-: We have expanded the materials and methods section to include a more detailed description of the design. We clarified the fact that both sheep and goats were used as source of ruminal fluid. We have also clarified that plants were cultivated and described with greater detail how and when they were cultivated and sampled. We will also take into consideration in future studies the need to collect two years of data. We have also mentioned the limitations of our design in the conclusions section.

2. The hypothesis of this work is not clear, why you measured the nutritive value in two seasons? may you have to mention the feed security during these two seasons, and why you selected these two seasons specifically?    

AU Response 2: We explained the reason behind selecting these two specific seasons. They were selected because they corresponded to seasons with significant environmental characteristics. Season 1 is the pruning season (Winter) and season 2 is the beginning of a new vegetative cycle (Spring).

3. What is the novelty of this study? leaves of P. shan tong are already considered a forage source for ruminants.

AU Response3: P. shan tong has recently been introduced to Tunisia. Also, there is a scarcity of data regarding the nutritive values of the plant while using both sheep and goats’ ruminal fluids. Different ruminant species have different ability to digest forages due to differences in their microbiota. We also believe that contrasting the nutritive value of the leaves during two separate growth and environmental conditions add value to this study.

4. Please insert more information about the plant collection, what was the life stage each season? plant sample size? how many trees were collected for each plant sample? how many plots are for each plant sample? how many true statistical and analytical repetitions were made for all the obtained results?

AU Response 4: Materials and Methods were expanded to include the requested information. `

5. The description of the methods part of the in vitroassay is poor, please insert more information for preparing the ruminal fluid, the number of syringes that were done for each plant, and how many GP runs.

AU Response 5: Materials and Methods were expanded to include the requested information.

6. Here are some other minor issues:

6.1. Line 38, please write the full name of all abbreviations in their first mentions (e.g, CE, GAE, ..), an check this issue through the manuscript.

AU Response6.1: Done

6.2. Line 39, what do you mean by asymptotic?

AU Response 6.2: The model used to fit gas production data (equation (7)) is an exponential function, monotonically increasing up to an upper asymptote. The value of this asymptote is represented by parameter “A”, commonly defined by people using this invitro gas production technique as the asymptotic gas production. It is the amount of fermentation gas that can be potentially produced if the sample was incubated for an infinite time.

6.3. In vitro is italic throughout the manuscript.

AU Response 6.3: Done

6.4. In lines 31 to 32, you have to present the treatments in place of  DM, OM, NDF, ADF,...

AU Response 6.4: Addressed.

6.5. Line 46, the conclusion has to be changed, you don't have any comparisons with any traditional source to insert such a conclusion.

AU Response 6.5: the conclusions have been modified and comparison to other forages has been removed

6.6. In tables, you have to insert the data of the incubation of both seasons in each ruminal source to get the interaction effect as you tested.

AU Response 6.6: The interaction was significant only in few cases, and the most important was the comparisons for the main effects.

6.7. Line 111, fiberteck ??

AU Response 6.7: Corrected in the revised manuscript

6.8. For the statistical analyses, did you use the one-way ANOVA for all the experimental parameters!!  please insert the models that you used in the chemical composition and for the in vitro assay, what was your experimental unit for all of your obtained results?  which significance degree did you use?  correlation!! where are the tables of the correlations that you did?

AU Response 6.8: there was a wrong description of statistical methods. The statistical analyses actually made have been described in the revised manuscript.

6.9 Line 215, which variables?

AU Response 6.9: corrected in the revised manuscript

6.10. In the manuscript, you used " Leaves obtained from the 1st harvest", what do you mean by the 1st harvest? where these data, no mentions for this either in the methods or the results part!!  Also, what was the 2nd harvest?

AU Response 6.10: Corrected to clarify that the 1^st harvest is the winter harvest and the 2^nd harvest is the Spring harvest

6.11. Conclusions part is very poor.

AU Response 6.11: The conclusions have been modified

Author Response File: Author Response.docx

Reviewer 2 Report

Dear Authors,

This study aimed to determine the seasonal variation (two seasonal harvests during Winter and Spring) of some chemical and biochemical characteristics of Paulownia shan tong leaves.

The  manuscript presents an interesting topic. It also has a  scientific value. However, some mistakes must be fixed before the publication.

My main objection to this work is lack of a  short subsection, where the Authors could  summarise the obtained results and show mutual relationships. It requires applying the more advanced statistical method such as Cluster Analysis or PCA method. It needs to be improved. 

 I have  also the following other suggestions, for Authors to further improve their manuscript:

- line 99: is „40°C” the room temperature?

- line 102: please give the temperature,

- Table 1: in the articles written in English, we use dots instead of commas when entering numerical values. Please correct it, 

- the tables do not indicate statistically significant differences between the investigated samples,

- under the individual tabels is a lack of the names of used statistical tests

- reference no. 14 – please complete the names of used methods, which are listed in lines: 104-105,

- how many repetitions were done for each experiment?

- Line 155: the source reference for this method is:

Brand-Williams, W.; Cuvelier, M.E.; Berset, C. L.W.T. Use of a free radical method to evaluate antioxidant activity. LWT Food Sci. Technol. 1995, 28, 25–30. http://dx.doi.org/10.1016/S0023-6438(95)80008-5.

However, it can be  used with some modifications.

- the reference no 2, isn’t available online,

- the reference no. 1: please, give more details, 

- more than 30% (19 references among total 54 presented ) of the cited literature is older than 15 years. 

From my standpoint, this manuscript could be considered for publication in Journal – Fermentation,  after major revision given the above aspects.

Author Response

Reviwer comments

1. This study aimed to determine the seasonal variation (two seasonal harvests during Winter and Spring) of some chemical and biochemical characteristics of Paulownia shan tong leaves.

The manuscript presents an interesting topic. It also has a scientific value. However, some mistakes must be fixed before the publication.

1. My main objection to this work is lack of a short subsection, where the Authors could  summarise the obtained results and show mutual relationships. It requires applying the more advanced statistical method such as Cluster Analysis or PCA method. It needs to be improved. 

AU Response 1: As we have only compared two seasons (for chemical composition) and its in vitro fermentability in two species (sheep and goat), we believe that this sort of multivariate methods will not provide any additional information to that obtained by the anova for each variable

2.  I have also the following other suggestions, for Authors to further improve their manuscript:

2.1. - line 99: is „40°C” the room temperature?

AU Response 2.1.: Yes, the room temperature was 40 ± 2 °C

 2.2. line 102: please give the temperature

AU Response 2.2: Done

2.3. Table 1: in the articles written in English, we use dots instead of commas when entering numerical values. Please correct it, 

AU Response 2.3: Done

2.4. the tables do not indicate statistically significant differences between the investigated samples,

AU Response 2.4: this has been shown in the revised manuscript, although it would not be necessary, when there are only two levels of a factor (winter vs spring), if the P values is less than 0.055 there is a significant difference between both means.

2.5. under the individual tables is a lack of the names of used statistical tests

AU Response 2.5: the statistical tests used are described in the Material and methods section of the revised manuscript

2.6. reference no. 14 – please complete the names of used methods, which are listed in lines: 104-105,

AU Response 2.6: Done

2.7. how many repetitions were done for each experiment?

AU Response 2.7: there were three replicates per season

2.8. Line 155: the source reference for this method is:

AU Response 2.8: Done

2.9.Brand-Williams, W.; Cuvelier, M.E.; Berset, C. L.W.T. Use of a free radical method to evaluate antioxidant activity. LWT Food Sci. Technol. 1995, 28, 25–30. http://dx.doi.org/10.1016/S0023-6438(95)80008-5.

              However, it can be  used with some modifications.

AU Response 2.9: The reference has been replaced.

2.10. the reference no 2, isn’t available online,

AU Response 2.10: Now, it is available at:  https://doi.org/10.3390/agriculture12081199.

2.11. the reference no. 1: please, give more details, 

AU Response2.11: The reference has been replaced with another one.

2.12. more than 30% (19 references among total 54 presented ) of the cited literature is older than 15 years. 

AU Response 2.12.: Many old references, except those of methods, have been replaced with more recent ones.

2.13. From my stand point, this manuscript could be considered for publication in Journal – Fermentation,  after major revision given the above aspects.

AU Response 2.13: all aspects were addressed in the new version

 

Author Response File: Author Response.docx

Reviewer 3 Report

This work is good, well designed, analysed, written and discussed. I just have just found some typos errors and formatting errors to be corrected. please refer to the attached Manuscript for yellow highlighted words and sentences to be corrected.

Comments for author File: Comments.pdf

Author Response

Reviewer commens: 3: Comments in reviewer’s PDF addressed.

1. This work is good, well designed, analysed, written and discussed. I just have just found some typos errors and formatting errors to be corrected. 
2. Please refer to the attached Manuscript for yellow highlighted words and sentences to be corrected.

AU Response: Authors thank the reviewer for his/her valuable comments. All issues raised by the reviewer have been addressed. Hope the present revision is acceptable for publishing in Fermentation.

 

Round 2

Reviewer 1 Report

However, the authors responded in the response letter that they clarified the hypothesis and why they selected these two seasons but no modifications were done in the introduction regarding these issues.

Please insert the importance of the plant secondary metabolites of this plant and why you have to measure them in the introduction part.

 In the in vitro assay, you did three runs in three different weeks, so for each ruminal inoculum (sheep or goat)  how many animals you got used? you said that you used four sheep and four goats how you used these animals as inoculum donors?

Line 206, the pH inoculum was maintained at 6.8 to 6.9, for what, which animal source? and this was which run? please clarify,  if it is an average you have to insert the value ±SE.

In line 212, you said that  "All treatments were incubated with 3 replicates per run" , and in line 222, you said that "(one substrate/harvest time/run) with 2 syringes", please insert the true analytical repetitions you did for each run/ sample.

 

Write the full name of the SEM under each table. 

Author Response

1/However, the authors responded in the response letter that they clarified the hypothesis and why they selected these two seasons but no modifications were done in the introduction regarding these issues.

AU: The introduction was modified to better explain the importance of the two seasons selected.
2/Please insert the importance of the plant secondary metabolites of this plant and why you have to measure them in the introduction part.
Au: Done
 3/In the in vitro assay, you did three runs in three different weeks, so for each ruminal inoculum (sheep or goat)  how many animals you got used? you said that you used four sheep and four goats how you used these animals as inoculum donors?
AU: The description was rather confusing in the previous version. The actual incubation scheme followed is described in the revised manuscript.
4/Line 206, the pH inoculum was maintained at 6.8 to 6.9, for what, which animal source? and this was which run? please clarify,  if it is an average you have to insert the value ±SE.
AU: The description was a bit confusing in the previous version. The buffering capacity of the medium is very strong and maintains pH within a narrow range. pH is measured not to detect differences but just to check that the incubations start at the intended pH level.
5/In line 212, you said that  "All treatments were incubated with 3 replicates per run", and in line 222, you said that "(one substrate/harvest time/run) with 2 syringes", please insert the true analytical repetitions you did for each run/ sample.
AU: The description was rather confusing in the previous version. The actual incubation scheme followed is described in the revised manuscript.
6/Write the full name of the SEM under each table.
AU: defined in each table footnote

 

Reviewer 2 Report

Dear Authors,

Thank you very much for the responses to the review.

The revised version of  manuscript has not been corrected taking into account the presentation of the results. Unfortunately, the  main objection to this work is still lack of summarization of  the obtained results and showing mutual relationships by applying the more advanced statistical methods.

From my standpoint, this manuscript  isn’t appropriate for publication in Journal – Fermentation,  given the above aspects.

Author Response

Dear reviwer, tha,ks for your comments. We tried to take them intoaccount in this current version. Hope is in accordance with your requests

1/The revised version of  manuscript has not been corrected taking into account the presentation of the results. Unfortunately, the  main objection to this work is still lack of summarization of  the obtained results and showing mutual relationships by applying the more advanced statistical methods.

AU: To address this issue, multivariate analyses (clustering UPGMA) have been performed. The results are presented in the revised manuscript, showing the significant discrimination between harvest seasons and between sheep and goats, based on the mutual relationships among all the variables recorded. This should not be a reason for rejection the manuscript considering also the assessments by the other reviewers.