Nutritional Characteristics and Digestibility of Woody and Herbaceous Native Plants from Tropical Flooded Savannas Ecosystems

Round 1

Reviewer 1 Report

 

Improve the methodology especially sample collection

Improve the discussion with scientific explanations for your findings

Comments for author File: Comments.pdf

Author Response

Dear reviewer

The authors appreciate the insightful comments.

We attach the corrections and answers

First reviewer’s responses

 Comment

Introduction

Line 42- Increase - improve grammar

Response: corrected

Line 44- Improve English grammar and sentence flow

 

Response: your suggestion was taken into account modifying the paragraph as follows:

Productive intensification implies an efficient resources use to guarantee a balance nutrition, health and welfare of animals, allowing an increase in productivity per unit area

 Line 49- Improve the flow of sentences and grammar

Response: your suggestion was taken into account modifying the paragraph as follows:

The implementation of the intensify livestock models (eg. Silvopastoral systems) are viable productive alternatives to be implemented under flooded savannahs conditions, however is limit by the scarce knowledge…….

Comment

Methods

Selection of the plant material:  The suggestion of livestock producers

Give references where these were suggested- line 86

Response: We do not have a specific reference because we only participated in some meetings held with livestock farmers associated with the Livestock Committee of the region. However, this information was included in the document.

 Plant material and sampling

Not clear if the collection was done once at a point in time or if they were repeated sample

collection to avoid results by chance line 95

Response: The observation was clarified within the document according to the following paragraph:

At each observation point, leaves of plants selected for the study were triplicate sampling (approximately 500g) in a 100 m radius. According to the species availability, all the samples were taken within the same observation point or were complemented of other traveled points. Different specimens of the same species were sample to guarantee representativeness.

Replications not indicated. How many times in that same season was the samplking done?

Response: in the previous comment, it is specified that triplicate leaf samples were taken (approximately 500g per sample = 1,5 Kg  per specie) within the same or different observation point depending on the plants presence among the travelled routes. The most common was that samples were complemented with species from different observation points

Any reason or explanation why sample collection was carried out during the transition period from dry to the rainy season? Results of which cannot be attributed to either dry or wet season.

The seasonal nutritional composition/ value of these forage species is of relevance especially when it comes to inclusion in diets line 90

Response: the sampling was carried out in the transition period from dry to rainy season because it was expected to identify plants that showed nutritional potential and that would withstand the dry period in the area, which is prolonged (6 months) and with adverse environmental conditions. Likewise, with the beginning of the rains, it was expected to increase the diversity of species to be evaluated. These studies, in addition to contemplating the evaluation of the plants during the dry and rainy seasons, must also be carried out in transition times to know the vegetation dynamics.

Following your suggestion this information was included in the text.

Line 96- grammar

Response: corrected

Describe the actual plant material that was considered for nutritional analysis. Leaves, flowers, stems, and twigs or all. Please indicate for all the categories that were considered as this affects the nutritional composition and digestibility results.

The nature of the plant material used determines the nutritional quality

Now that we are considering a cut and carry system as opposed to free grazing/browsing, it’s important for you to be clear on the plant material used In nature, it is difficult for the animals to pick out only leaves when it is freely grazing/browsing. Even in cut and carry, it is difficulot to only cut outr leaves. Analysis samples for trees, shrubs, climbing plants, and herbs, please specify the sample collection- what samples did you take to the lab?

Response: the information was specified in the text according with the following paragraph

“At the each observation points, leaves, midribs, rachis (compound plants) and petioles of selected species for the study were triplicate sampling (approximately 500g per sample) in a 100 m radius”.

Improve the methodology section.

Response: the suggestion was taken into account

Comment

Results

Be consistent in write up. In the table, you use trees, while in the writing up you use arboreal species 25.3.1

Response: the suggestion was taken into account

These results are interesting protein sources- more research should be conducted to study how the protein content varies across the season These will present important protein supplements to ruminants, especially CP is the limiting material Interesting finding CP Arbored /trees- 8-25.3 Shrubs – 2.5-25.8 Climbing – 8.8- 19 Herbs- 17.5-27.3

NDF, ADF, AND LIGNIN

What about maturation

Could also be attributed to the samples taken to the lab

Could have included twigs and some branches, thus directly affecting the results of the

structural component

Response: we specified that samples were constitute of leaves, midribs, rachis (in compound plants) and petioles.

Line 254-258, seems redundant- we are explaining nutrient composition not intake at this moment. This is a misplaced statement and reference (31) Please give a more detailed discussion of your results. Give extra scientific meaning and explanation of your results

Response: the paragraph was restructured to give greater clarity about the idea that is mentioned

Line 274: Explain this finding “ Although high fibre levels werte found in some of the evaluated species, digestibility was not affected in many of them”. Give a more scientific explanation to this occurance. The explanation given (8)n isn not satisfactory

• How about the methodology bused? Could it have a hand in this outcome? Well I am

just thinking loud – but explain this further.

It is known fact that as fibre increases, digestibility reduces, however in thisn study, it is the opposite – please explain why?

Response: we appreciate the suggestion, which led us to review the trends and it was possible to observe that the assertion was being made for a particular case and not the general trend. In this way, the paragraph was restructured to provide greater clarity about what is intended to be mentioned.

More explanation needed about RFV and what is the implication of your finding to livestock nutrition

Response: The explanation of the RFV meaning, the way to estimate and interpret it, was  explained both in the materials and methods section, and in the presentation of results and discussion. Similarly, to explain the implication of the RFV results, the following paragraph was included in the results and discussion section:

The RFV is an index that do not consider the protein content of the forages, how-ever it is usefulness for the comparison of two or more similar forages for energy in-take potential. RFV values of 100 represent a forage with a 53 % and 41 % of NDF and ADF respectively. In this way, values higher than 100 are accepted, while in dairy cows nutrition, values greater than 150 are always desirable [15]. According to Table 3, 28.6 % of the studied plants presented an acceptable RFV (equal or higher than 100). Only the 14.3 % of the plants meet the suggested requirements for dairy cattle (RFV≥150), while 21.4 % of the species presented RFV values equal or greater than 125, which can be used to feed animals with lower nutritional requirements such as those used for breeding and fattening under flooded savannah conditions. The low RFV index values observed among the plants can be attributed to the fact that this index is affected by the fiber levels (NDF and ADF). Among the studied plants, 54.8 % and 23.8 % attained FDN and FDA contents, respectively, higher than the base values used to construct an acceptable RFV index. The observed fiber variability between plants is an expect behavior in species growing during a transition period from dry to rainy seasons. Some plants have endured a prolonged drought period trying to adapt to the tropical conditions, while others begin their growth process encouraged by the first rains (44)

English corrected

Author Response File: Author Response.pdf

Reviewer 2 Report

Manuscript number Agriculture-1898272

Title Nutritional Characteristics and Digestibility of Woody and Herbaceous Native Plants from Tropical Flooded Savannas Ecosystems

 

Overall comments,

Researchers paid close attention to a study that evaluated the chemical components and digestibility of native woody and herbaceous plants. Although this work is intriguing, after a comprehensive reading, just some issues need to be revised.

 

Abstract

General

            -

Introduction

General

-There are many several previous studies on alternative forages which similar your manuscript. Therefore, what is the research gap from this review obtained?

-What new knowledge from this review did you obtain?

-Add more experimental hypotheses.

Materials and methods

General

            -

Specific

-

Results and Discussion

General

            -

Specific

-L333-336: It should be explained in more detail mechanically.

-L341-342: It should be explained in more detail mechanically.

-L381-383: Add more detail to the mechanism.

-L381-404: Add more previous studies.

-L417-418: How can it reduce methane?

-L417-418: How can it reduce ammonia?

-417-422: Can you explain more about how did it happen?

Conclusion

General

-

Reference

 

-Modify format according to Journal style

Author Response

Dear reviewer

The authors appreciate the insightful comments.

We attach the corrections and answers

Second reviewer’s responses

 Comment

 Introduction

General

-There are many several previous studies on alternative forages which similar your manuscript. Therefore, what is the research gap from this review obtained?

Response: Based on your suggestion, the following paragraph was added in the introduction:

Livestock activity under flooded savannah conditions requires a change towards more sustainable systems, however, to achieve this goal it is required to strengthen the knowledge about the nutritional properties that native species can provide, which, due to their adaptive capacities, constitute the main option for the establishment of sus-tainable livestock models in the region.

-What new knowledge from this review did you obtain?

Response: Based on your suggestion, the following paragraph was added in the conclusion:

These results are promising since constitute the first report evaluating the individual and join nutritional contribution of flooded savanna flora diversity as a productive strategy in search of nutritional alternatives for livestock activity in the region.

-Add more experimental hypotheses.

Response: Based on your suggestion, the following paragraph was added in the conclusion:

We test the hypothesis that native flora biodiversity from flooded savannahs present optimal nutritional characteristics to be used individually or in conjunction as nutri-tional alternatives to improve the livestock systems sustainability in the region. 

Comment

Results and Discussion

General

 Specific

-L333-336: It should be explained in more detail mechanically.

The idea was explained in greater depth: Inside the cluster 2, shrubs and trees were the most representative species (78.6 %). This kind of plants are recognized by the presence of secondary metabolites that in sufficient quantities can present antimicrobial properties affecting ruminal microbiota population responsible for substrate degradation which is reflect in a reduce in the feed utilization efficiency [46]. Since this study did not evaluate the presence of sec-ondary metabolites, this hypothesis requires further research.

-L341-342: It should be explained in more detail mechanically.

The idea was explained in greater depth: Inside this group, proper CP levels was found which constitute a substrate for ruminal populations that can be degraded quickly contributing to the higher IVDMD [42]. Digestibility was also promote by the low fiber NDF fraction observed, indicating that partial digestible (cellulose and hemicellulose) and undigestible (lignin) fractions were in low concentrations, allowing ruminal microorganism to easily access the non-fibrous substrate and the cellulose and hemi-cellulose component of the cell wall for their degradation [15,35].

-L381-383: Add more detail to the mechanism.

The idea was explained in greater depth: This observation agree with literature reports indicating that higher fiber fraction limit the access of ruminal microorganisms to the substrate, which reduces its degradation rate, while high CP and ash contents are easily degradaded contribuiting to the digestibility[29,45].

-L417-418: How can it reduce methane?

The idea was explained in greater depth: Some positive effects included the reduction of ammonia production and methane emissions through the selective control of ruminal microorganisms in charge of its production the reduction of ruminal ammonia production and methane emissions [37,46].

-L417-418: How can it reduce ammonia?

The idea was explained in greater depth: Some positive effects included the reduction of ammonia production and methane emissions through the selective control of ruminal microorganisms in charge of its production the reduction of ruminal ammonia production and methane emissions [37,46].

-417-422: Can you explain more about how did it happen?      

The idea was explained in greater depth: Some positive effects included the reduction of ammonia production and methane emissions through the selective control of ruminal microorganisms in charge of its production the reduction of ruminal ammonia production and methane emissions [37,46].

Comment

Reference 

-Modify format according to Journal style

Response: The document was adjusted according to the journal format

Author Response File: Author Response.pdf

Reviewer 3 Report

Nutritional Characteristics and Digestibility of Woody and  Herbaceous Native Plants from Tropical Flooded Savannas  Ecosystems

 

The present work provides useful information (chemical composition and potential nutritional values) of many tropical plants.

However, the work lacks of a clear objective or hypothesis, hence, it seems the authors have made a large collection of samples and put the results through as many statistical procedures as they could.

The information per se is valuable and it seems to be of little use to perform a multiple comparison among so many different species. Why do so?

Probably the creation of a cluster or PCA could be useful if there was a hypothesis or clear objective supporting this analysis.

I encourage authors to provide a clear objective or hypothesis and perform the analysis needed to test the hypothesis or accomplish the objective.

An additional problem with the present approach is that authors are attempting to explain every single chemical component and its range of result values. Given the large data set, this is difficult to achieve and prone to yield over generalization or contradictions.

From my point of view, there is nothing wrong and it is equally valuable, to provide just tabular result of lesser know plants without the need to compare all of them in multiple comparison test.

some additional comments are in the file, however, the manuscirpt itself could have large modifications if the suggestions (approach) is modified.

 

regards

Comments for author File: Comments.pdf

Author Response

Third reviewer’s responses
Comment
The information per se is valuable and it seems to be of little use to perform a multiple comparison
among so many different species. Why do so?
Response: The objective of the multiple comparison was to try to identify within each group of
plants the ones that presented the best behavior in statistical terms. The above allowed to sift the
evaluated species for the presentation of the results.
Probably the creation of a cluster or PCA could be useful if there was a hypothesis or clear
objective supporting this analysis.
Response: Based on your suggestion, the following paragraph was added in the introduction:
We test the hypothesis that native flora biodiversity from flooded savannahs present optimal
nutritional characteristics to be used individually or in conjunction as nutritional alternatives to
improve the livestock systems sustainability in the region. The objective of the present study was
to evaluate the individual and join chemical compo-sition and digestibility of native plants from
flooded savannas ecosystems as a nutritional alternative for grazing ruminants.
I encourage authors to provide a clear objective or hypothesis and perform the analysis needed to
test the hypothesis or accomplish the objective.
Response: your comment was taken into account
An additional problem with the present approach is that authors are attempting to explain every
single chemical component and its range of result values. Given the large data set, this is difficult
to achieve and prone to yield over generalization or contradictions.
Response: since the study aims to characterize the nutritional composition, we consider that it is
necessary to make an individual description of each component. Subsequently with this
information we identified those species with the most desirable nutritional variables from the
livestock point of view.
From my point of view, there is nothing wrong and it is equally valuable, to provide just tabular
result of lesser know plants without the need to compare all of them in multiple comparison test.
Response: we appreciate your suggestion, however we consider that the way the results are
presented allows a quick identification of the plants with the most desirable characteristics in
terms of animal nutrition.
Comment
some additional comments are in the file, however, the manuscirpt itself could have large
modifications if the suggestions (approach) is modified.
Response to comments that were in the text
Reviewer: a better description of the objetive or an hypothesis must be presented in agreement
with the methodology employed.
Response: The objetive was reestructured according with the presented data.
Reviewer: why plants which can not be used in animal nutrition were evaluated?th

Response: the study focused on plant species with nutritional potential and that can be consumed
by grazing animals, without affecting their welfare
Reviewer: it is important to describe the nature of the sample.
Response: the highlighted paragraph indicates the sample collection methodology
Reviewer: see coments regarding objetive/hypothesis
Response: hypothesis and objetive were modified according with the obtained results.
Reviewere: Unless the author have data for the same plants in a different season, there is no strong
support for this statements
Response: The authors pretend to explain the increase in NDF with a valid argument that is
typically observed in tropical plants. Climatic variables in warm conditions accelerate the fiber
content accumulation at a higher rate than tin temperate grasses. For this reason the paragraph
mentions "could be attributed". Also the argument is accompanied by a reference.
With these supports, we consider that the idea is valid to support the results
Reviewer: this will contradict the explanation for NDF and lignin as younger leaves will not have
increased content of fiber
Response: the idea was reestructured for a better understanding
English corrected

Author Response File: Author Response.pdf

Reviewer 4 Report

The manuscript presents an evaluation of chemical composition and digestibility of 42 plants from flooded savannas, in order to be used as fodder for ruminants. The topic is very interesting and with a high scientific soundness. However, the material and methods, and results and discussion should be improved. The material and methods should be developed. The collection and the analyses methods should be more explained and described, especially for the digestibility method. The results and discussion should be divided into two sections. The discussion is poor, it needs to be improved and the results needs to be more discussed. I suggest to read articles of Chebli et al., to inspire you.

 

There are some comments below:

 

The manuscript study chemical composition more than nutritional characteristics. Digestibility is one of the nutritional characteristics. It is better to mention chemical composition, or just nutritional characteristics without digestibility.

Line 6 & 49: “limited”

Line 64-66: the use of native plants as a supplementation could induce a degradation of the vegetation and the ecosystem.

Section 2.1: It will be better if authors could add a map

Line 69: which animal species exactly.

Line 84-85: the authors have chosen plants according to production, morphology, etc. Could authors explain more the minimum level of production and the characteristics of morphology.

The material and method section should be developed, especially the collection method and samples analyses. Authors did not present the number of samples, and the number by category of plants. Also, the digestibility should be more explained. The used liquor and the animal species, and their provenance should be mentioned.

Based on the digestibility and chemical composition, authors could calculate the Metabolizable energy

It is more convenient if the results and discussion section could be divided into 2 sections separately, to more describe results and discuss them.

The authors should report DM of each plant, because it is an important parameter. Also, component contents were presented in %. This is % of dry matter?? Please precise.

Table 2: the table contains too much information. The “,” in values should be changed by “.”. The letters of means comparison should be added in the footnotes. SEM should be calculated for parameters and by category, not by species.

Line 245: reference

Line 258: maintance ?

Table 3 : check the comma in the values.

Line 417-421: please check these sentences.

Line 456-460: Should be deleted.

Author Response

Dear reviewer

The authors appreciate the insightful comments.

We attach the corrections and answers

Fourth reviewer’s responses

Comment

There are some comments below:

 The manuscript study chemical composition more than nutritional characteristics. Digestibility is one of the nutritional characteristics. It is better to mention chemical composition, or just nutritional characteristics without digestibility.

Response: your suggestion was taken into account

Line 16 & 49: “limited”

Response: corrected

Line 64-66: the use of native plants as a supplementation could induce a degradation of the vegetation and the ecosystem.

Response: Some of the studied plants are already used for feeding grazing ruminants in the area. The objective is to increment the knowledge about their nutritional properties so that they can be used in agroforestry systems in the region, such as silvopastoral systems. Under this regenerative livestock approach, the opposite is expected to degrade the ecosystem. These production schemes have shown to improve soil characteristics, welfare of grazing animals and wildlife, in addition to other ecosystem services like carbon fixation trough the constant growth dynamics of the vegetation.

Section 2.1: It will be better if authors could add a map

Response: your suggestion was considered in the document

Line 69: which animal species exactly.

Response: corrected

Line 84-85: the authors have chosen plants according to production, morphology, etc. Could authors explain more the minimum level of production and the characteristics of morphology.

Response: the selection criteria of the plants were specified in the document through the following paragraph: “ the degree of plants availability and greenness during the sampling season, the ab-sence of morphological structures  like spines or the presences of  toxic com-pounds that may  compromise the animals welfare, the suggestion of  livestock farmers associated with the Livestock Committee of the region  or other productive attributes (timber, medicinal, among others)”.

The material and method section should be developed, especially the collection method and samples analyses. Authors did not present the number of samples, and the number by category of plants. Also, the digestibility should be more explained. The used liquor and the animal species, and their provenance should be mentioned.

Response: the collection methodology and digestibility conditions were specified in more detail in the materials and methods section.

Based on the digestibility and chemical composition, authors could calculate the Metabolizable energy

Response: we appreciate the suggestion, however in this study we rather not to mention any estimation of energy content of the plants, because we are conducting other fermentation experiment with the aim of identify ruminal parameters with the most promising plants obtained in this first phase. We are planning to estimate gross energy directly in the laboratory using a calorimeter bomb, and these results include them together with the ruminal parameters possibly in another document that will be submitted for publication

It is more convenient if the results and discussion section could be divided into 2 sections separately, to more describe results and discuss them.

Response: we appreciate the suggestion, however we consider that the document is in accordance with the journal format. Also, the join sections presents a logical sequence of results discussion (we try to improve de discussion of some results according to your recommendation).  Finally, since such modifications could eliminate previous changes made to the document based on comments made by the other reviewers, we prefer to retain the current format.

The authors should report DM of each plant, because it is an important parameter. Also, component contents were presented in %. This is % of dry matter?? Please precise.

Response: According to your suggestion, dry matter was included in the analysis. The results were described and discussed. Similarly, in the tables it was specified that the reported percentages are with respect to dry matter.

Table 2: the table contains too much information. The “,” in values should be changed by “.”. The letters of means comparison should be added in the footnotes. SEM should be calculated for parameters and by category, not by species.

Response: Table 2 was divided to simplify the results presentation. The decimals were constructed with points, and the letters of mean comparison were set as superscripts. SEM were estimated by categories and included at the end of each category.

Line 245: reference

Response: Corrected

Line 258: maintance ?

Response: the idea was restructured to give greater clarity: “nevertheless, some reports show that a partial inclusion of these plants in the animals diet can improve their performance in terms of milk or meat production, increase forage biomass production and enrich the diet nutritional for grazing herbivores and provide shade, an important factor that guarantee animal welfare specially in grazing ruminants under tropical environment”.

Table 3 : check the comma in the values.

Response: Corrected

Line 417-421: please check these sentences.

Response: Corrected

Line 456-460: Should be deleted.

Response: Corrected

English corrected

 

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

The manuscript has been improved and recommended for publication.

Author Response

Dear reviewer

The authors appreciate the insightful comments.

Reviewer 3 Report

Nutritional Characteristics of Native Plants from Tropical 2 Flooded Savannas Ecosystem

In the previous review round, I suggested including a hypothesis to be tested or a clear objective.

The authors have included both. After reading the whole manuscript (revised version) I suggest keeping to the objective and deleting the hypothesis.

It is clear that this hypothesis was not tested, nor can be with current data. Authors are linking the nutritional characteristics of plants with an improvement in livestock systems sustainability in the region. There is no data to test this statement. In fact, the approach of the present manuscript is very well in line with the objective and the hypothesis was forgotten in the whole manuscript.

 

additional comments. Unless the journal allows the current formatting, In all tables, the full scientific names should be included. Authors should consider that tables must stand alone for any reader and currently they are not. Any reader would need to search in the main text to find the complete/full name.

 

Please double check the IVDMD result values for G. americana and P. hispidium
in the first case, it seems that even half the lignin was digested in 48h and in the second case, it is equivalent to obtaining just the lignin as residue in the bag after only 48 h.

Having said that, I would double-check all IVDMD values to make sure no calculation error was involved in the whole data set.

If the IVDMD values (any) are corrected, then the PCA and clusters analysis would need to be verified. if any result changes, the manuscript sections would need to be revised accordingly.

Mainly based on the above observation I do recommend a major revision as at this stage I can not properly assess the impact of double checking results.

A further aspect which need to be addressed is the following.

 Authors worked with 2 animals to obtain rumen liquor for the iVDMD. Likely several incubations procedures due to the number of samples. However, the Institutional Review Board Statement was not included in the manuscript

Author Response

Dear reviewer

The authors appreciate the insightful comments.

We attach the corrections and answers

Third reviewer’s responses (Round2)

Comment

• In the previous review round, I suggested including a hypothesis to be tested or a clear objective.

The authors have included both. After reading the whole manuscript (revised version) I suggest keeping to the objective and deleting the hypothesis.

It is clear that this hypothesis was not tested, nor can be with current data. Authors are linking the nutritional characteristics of plants with an improvement in livestock systems sustainability in the region. There is no data to test this statement. In fact, the approach of the present manuscript is very well in line with the objective and the hypothesis was forgotten in the whole manuscript.

 Response: the hypothesis was deleted.

Comment

additional comments. Unless the journal allows the current formatting, In all tables, the full scientific names should be included. Authors should consider that tables must stand alone for any reader and currently they are not. Any reader would need to search in the main text to find the complete/full name.

 Response: we tried to include the plants complete names within the tables, however in some cases the format was altered making them very large. In this way, it was preferred to put the full names at the end of each table

• Please double check the IVDMD result values for G. americana and P. hispidium
  in the first case, it seems that even half the lignin was digested in 48h and in the second case, it is equivalent to obtaining just the lignin as residue in the bag after only 48 h.

Having said that, I would double-check all IVDMD values to make sure no calculation error was involved in the whole data set.

If the IVDMD values (any) are corrected, then the PCA and clusters analysis would need to be verified. if any result changes, the manuscript sections would need to be revised accordingly.

Mainly based on the above observation I do recommend a major revision as at this stage I can not properly assess the impact of double checking results.

 Response: We appreciate your comment. We review the raw data to verify if there were calculations errors in %IVDMD. The data obtained were the following:

According to what was observed, the values obtained in each repetition are consistent.

Similarly, your comments led us to conduct a more detailed literature search to identify plants with similar results. We found that plants such as Croposma robusta, Griselinia littoralis and Pittosporum crassifolium have presented digestibilities between 78.4 and 78.8% with levels of DM (31.5 - 43%), PC (6.2 - 7.9%), NDF (32.1 - 37.4%), ADF (20.7 - 22.4%) and lignin (9.3 - 12%). In our case, the G. Americana and P. hispidium plants presented almost twice the protein content, to which the results obtained can be largely attributed.

 

We will continue to carry out studies on these plants to deepen these preliminary results.

• A further aspect which need to be addressed is the following.

 Authors worked with 2 animals to obtain rumen liquor for the iVDMD. Likely several incubations procedures due to the number of samples. However, the Institutional Review Board Statement was not included in the manuscript

 Response:  The ethical endorsement granted by the Institutional Committee for animals care and Use (CICUA) of the National University of Colombia, Medellin, was included in the document.

 

Author Response File: Author Response.pdf