Effect of Feeding Lucerne and a Mixed Diet of Oats and Berseem Clover as a Source of Fresh Forage on Ruminal Characteristics and Nitrogen Use Efficiency in Dairy Cows during the Winter Period

Round 1

Reviewer 1 Report

General points about the manuscript: The manuscript brings interesting information about the effect of a mixed pasture of berseem clover/oats or alfalfa as a source of fibre and protein in a total mixed ration. This topic is relevant to comprehend the effects of these sources on the ruminal parameters, degradability of nutrients and also the efficiency of N utilization of dairy cows. In my opinion the manuscript has a good aim and the manuscript is well written.

My biggest concern about this experimental design is the number of experimental units. The experiment was conducted with only 3 cows, which converges to only 3 replicates per treatment. Animals, as biological unit, have great variation among them. This large variation can be overcome when great number of replicates is used, however in this case, there is nothing else to be done, as the experiment with only 3 cows was already conducted. Authors should present a strong explanation that justifies low replication number per treatment. Additionally, another concern is regarding the Period effect in the tables. Tables have Period effect, few parameters were statistically significant for effect of Period, however anything was said about it in the text. Authors need to rearrange these results in the Table in case they really want to present this P-values.

 

Specific considerations:

This manuscript is a mixture of British (BT) and American (AM) English, please be consistent and chose only one style. Maybe the one the Journal follows the most. Examples: Analysed (BT) vs. Analyzed (AM); fibre (BT); corn & alfalfa (AM), if it would be BT spelling, then it would be maize and lucerne, respectively.

Abstract: Please, include one sentence before the objective with the background of this study, for instance a question that addresses in a broad context the idea of conducting this experiment.

L21: TMR should be defined (spelled out) first time mentioned.

L27: Missing a period.

L64: This abbreviation was not used again. No need to be spelled out.

L87: Delete space between L and period.

L95: Please replace ‘improving’ by ‘improve’

L100: de is misspelled (dte)

L105: My biggest concern about this experimental design is the number of experimental units. The experiment was conducted with only 3 cows, which converges to only 3 replicates per treatment. Animals, as biological unit, have great variation among them. This large variation can be overcome when great number of replicates is used, however in this case, there is nothing else to be done, as the experiment with only 3 cows was already conducted. Authors should present a strong explanation that justifies low replication number per treatment.

L111: Please use: concentrate feed

L121: Something went wrong here, please fix this part.

L160: Would it be USA, instead of EE.UU?

L187: If 4 hours before feeding, then it should have been done at 5am. Please indicate correctly if 3 or 4h before feeding. The same for after feeding, if 4h, then it supposed to be at 13h.

L198: Missing the closing parenthesis.

L209: Do mean trend when P between 0.05 and 0.10? Or 0.10, instead of 0.01.

L211: 3.1, instead of 1.1. Please fix those next as well.

Table 2: NDIN and ADIN are not similarly mentioned in M&M section. Please check and fix.

Is the unit ‘%/kg’ correct?

Please include ‘NS’ in the footnote. Or, why not to include the real P-value even if non-significant? I would kindly suggest including the P-value, instead of ‘NS’. Same for other tables.

Include also in footnote the meaning of Tukey superscript letters. Same for other tables when this is needed.

Table 2 has Period effect, few parameters were statistically significant, however anything was said about it in the text. Authors need to rearrange these results in the Table in case they really want to present this P-value. The same for other tables.

These 3 paragraphs regarding results of Table 2 are long and boring to read. As a reader, I would kindly ask the authors to rearrange the presentation of results in a more fluent way.

L244: Delete one parenthesis.

The abbreviations (AA, TA, AV) in this manuscript are correlated to their scientific names. However, in text and tables, the names are expressed as common names (berseem clover, oat, …). It can be a bit hard to the reader all the time to correlate the abbreviation of the scientific name with the common name (e.g., TA is Berseem Clover). I would kindly suggest the authors to correct the abbreviations in a more comprehensive way (e.g., Berseem Clover, BC; and so on).

L254: Why is the DM not shown in the Table? Because it wasn’t significative? As well as the pH was also not significant. Please include DM intake in the Table.

L262: Please indicate that this is according to Tukey test.

L266: Include one extra digit in P-value. Also, L271. Please check this throughout the manuscript (at least 2 digits for P-value).

L270: Why is ‘DM soluble’ in italic?

L280: The description of treatments here (CON, MIX and LEG) in this footnote is not the same as in other tables. Please be consistent.

Indicate the real P-value, instead of <0.05 or NS. Equally to what was done in Table 6. Please indicate P-value in the same way as in Table 6 for all other tables.

Table 6: Remove horizontal lines in the middle of the table.

Write a more informative title for Table 6. Follow the same idea of title across the titles of the Tables, of course just changing the description of parameters in different tables.

L320: There is not NS in this table.

L364: lead us to accept the second… OR lead us to the acceptance of second.

L386: Please spell out NNP.

L390: Do you mean “preserved forage”?

L414-415: Is this the way of including citation? Please check throughout the manuscript. Follow the Journal’s guideline and be consistent.

L429: Please format this paragraph in the same way as others.

L441: I would not even say ‘minor effect’, because actually there was no effect at all, especially in pH and the major VFAs. Please rearrange this sentence in a way that conclusion matches better the results.

L451: Should you include the initials of authors still in the filiation in order to include them here? Similarly to this previous published paper: https://doi.org/10.3390/ruminants1020011

L456: This sentence is repeated twice, delete one of them. Also delete the quotation mark in the end of the sentence.

L466: Was the approval by the ethics committee done after completion of the experiment? If so, this is not ideal. Approval should be given before the experiment with animals start.

Mention DOI link always in the same way (with https, or without), be consistent and follow Journal’s guideline.

L490: Should each word of the title start with capital letter? Check throughout according to Journal’s guideline.

L511: Delete spaces in half-sib.

L527 – Here scientific name is in italic; L532 – Here it’s not in italic. Please check throughout according to Journal’s guideline and be consistent.

L576: Delete 1 after Growth.

L578: Should the journal’s name be abbreviated? Please check throughout according to Journal’s guideline.

L599: Delete 1,2.

 

Best regards.

Author Response

Comments and Suggestions for Authors

Reviewer 1

General comments

General points about the manuscript: The manuscript brings interesting information about the effect of a mixed pasture of berseem clover/oats or alfalfa as a source of fibre and protein in a total mixed ration. This topic is relevant to comprehend the effects of these sources on the ruminal parameters, degradability of nutrients and also the efficiency of N utilization of dairy cows. In my opinion the manuscript has a good aim and the manuscript is well written.

My biggest concern about this experimental design is the number of experimental units. The experiment was conducted with only 3 cows, which converges to only 3 replicates per treatment. Animals, as biological unit, have great variation among them. This large variation can be overcome when great number of replicates is used, however in this case, there is nothing else to be done, as the experiment with only 3 cows was already conducted. Authors should present a strong explanation that justifies low replication number per treatment. Additionally, another concern is regarding the Period effect in the tables. Tables have Period effect, few parameters were statistically significant for effect of Period, however anything was said about it in the text. Authors need to rearrange these results in the Table in case they really want to present this P-values.

Authors’ response: We agree with the reviewer that n=3 is quite low and would have been ideal to have more cows to replicate the treatments. However, considering that 1) rumen fistulated cow surgery and maintenance is quite expensive, and 2) the maintenance of such animals has been criticized by the society due to their animal welfare side-effects, even that they proportionate important information regarding rumen function, their usage and numbers has been reduced quite drastically. However, the usage of 3x3 Latin Square design when assessing rumen fermentation and other metabolic parameters is still common as you can see from previous publications from some of the co-authors of this study (Vargas-Bello-Perez et al., 2016, Animal or Vargas-Bello-Perez et al., 2015 J Dairy Sci ) and some other groups as well (e.g. Phakachoed et al 2012, LIvest Sci; Wang et al., 2014, Animal Prod Sci; Wanapat et al., 2013, Afr J Agric Res). Moreover, as commented in section 2.4, in order to reduce the within animal variation, we used three set of bags in each fermentation so the ruminal conditions variation was taken into account.

We agree with the reviewer’s second concern regarding the exclusion of the Period effect from the tables and therefore we have removed this effect from the manuscript.

Specific considerations:

This manuscript is a mixture of British (BT) and American (AM) English, please be consistent and chose only one style. Maybe the one the Journal follows the most. Examples: Analysed (BT) vs. Analyzed (AM); fibre (BT); corn & alfalfa (AM), if it would be BT spelling, then it would be maize and lucerne, respectively.

Authors’ response: Suggestion accepted. The British English was used along the entire manuscript

Abstract: Please, include one sentence before the objective with the background of this study, for instance a question that addresses in a broad context the idea of conducting this experiment.

Authors’ response: Suggestion accepted.

L21: TMR should be defined (spelled out) first time mentioned.

Authors’ response: Suggestion accepted

L27: Missing a period.

Authors’ response: Suggestion accepted

L64: This abbreviation was not used again. No need to be spelled out.

Authors’ response: Suggestion accepted. The abbreviation was removed

L87: Delete space between L and period.

Authors’ response: Suggestion accepted

L95: Please replace ‘improving’ by ‘improve’

Authors’ response: Suggestion accepted

L100: de is misspelled (dte)

Authors’ response: Suggestion accepted

L105: My biggest concern about this experimental design is the number of experimental units. The experiment was conducted with only 3 cows, which converges to only 3 replicates per treatment. Animals, as biological unit, have great variation among them. This large variation can be overcome when great number of replicates is used, however in this case, there is nothing else to be done, as the experiment with only 3 cows was already conducted. Authors should present a strong explanation that justifies low replication number per treatment.

Authors’ response: Please see our response to the general comments.

L111: Please use: concentrate feed

Authors’ response: Suggestion accepted.

L121: Something went wrong here, please fix this part.

Authors’ response: Suggestion accepted. The paragraph was formatted accordingly.

L160: Would it be USA, instead of EE.UU?

Authors’ response: Suggestion accepted.

L187: If 4 hours before feeding, then it should have been done at 5am. Please indicate correctly if 3 or 4h before feeding. The same for after feeding, if 4h, then it supposed to be at 13h.

Authors’ response: Since feeding occurred at approximately 10:00h we would like to maintain the phrase as it is written to better reflect the sampling and feeding intervals

L198: Missing the closing parenthesis.

Authors’ response: Suggestion accepted.

L209: Do mean trend when P between 0.05 and 0.10? Or 0.10, instead of 0.01.

Authors’ response: Suggestion accepted. Values were fixed accordingly.

L211: 3.1, instead of 1.1. Please fix those next as well.

Authors’ response: Suggestion accepted. Numbers were fixed accordingly.

Table 2: NDIN and ADIN are not similarly mentioned in M&M section. Please check and fix.

Authors’ response: NDIN and ADIN were used in the M&M as well.

Is the unit ‘%/kg’ correct?

Authors’ response: Unit and values were fixed.

Please include ‘NS’ in the footnote. Or, why not to include the real P-value even if non-significant? I would kindly suggest including the P-value, instead of ‘NS’. Same for other tables.

Authors’ response: The NS values have been replaced for the actual P values in the tables.

Include also in footnote the meaning of Tukey superscript letters. Same for other tables when this is needed.

Authors’ response: Suggestion accepted

Table 2 has Period effect, few parameters were statistically significant, however anything was said about it in the text. Authors need to rearrange these results in the Table in case they really want to present this P-value. The same for other tables.

Authors’ response: Suggestion accepted. As already mentioned, we removed the effect of Period form the tables of the manuscript.

These 3 paragraphs regarding results of Table 2 are long and boring to read. As a reader, I would kindly ask the authors to rearrange the presentation of results in a more fluent way.

Authors’ response: Suggestion accepted. We have made corrections to improve this section.

L244: Delete one parenthesis.

Authors’ response: Suggestion accepted

The abbreviations (AA, TA, AV) in this manuscript are correlated to their scientific names. However, in text and tables, the names are expressed as common names (berseem clover, oat, …). It can be a bit hard to the reader all the time to correlate the abbreviation of the scientific name with the common name (e.g., TA is Berseem Clover). I would kindly suggest the authors to correct the abbreviations in a more comprehensive way (e.g., Berseem Clover, BC; and so on).

Authors’ response: Suggestion accepted, we have changed the use of AA, TA and AV to LUC, BC and OAT throughout the entire manuscript.

L254: Why is the DM not shown in the Table? Because it wasn’t significative? As well as the pH was also not significant. Please include DM intake in the Table.

Authors’ response: Suggestion accepted.

L262: Please indicate that this is according to Tukey test.

Authors’ response: Suggestion accepted. We have included this information in al tables.

L266: Include one extra digit in P-value. Also, L271. Please check this throughout the manuscript (at least 2 digits for P-value).

Authors’ response: Suggestion accepted.

L270: Why is ‘DM soluble’ in italic?

Authors’ response: We have removed the italic font.

L280: The description of treatments here (CON, MIX and LEG) in this footnote is not the same as in other tables. Please be consistent.

Authors’ response: Suggestion accepted. We have amended the treatments description to maintain consistency.

Indicate the real P-value, instead of <0.05 or NS. Equally to what was done in Table 6. Please indicate P-value in the same way as in Table 6 for all other tables.

Table 6: Remove horizontal lines in the middle of the table.

Write a more informative title for Table 6. Follow the same idea of title across the titles of the Tables, of course just changing the description of parameters in different tables.

Authors’ response: We have made all the changes suggested.

L320: There is not NS in this table.

Authors’ response: NS has been removed from the table

L364: lead us to accept the second… OR lead us to the acceptance of second.

Authors’ response: Phrase has been amended accordingly

L386: Please spell out NNP.

Authors’ response: Suggestion accepted.

L390: Do you mean “preserved forage”?

Authors’ response: Phrase has been amended accordingly

L414-415: Is this the way of including citation? Please check throughout the manuscript. Follow the Journal’s guideline and be consistent.

Authors’ response: We have amended this citation according to the Journal’s guideline

L429: Please format this paragraph in the same way as others.

Authors’ response: Suggestion accepted.

L441: I would not even say ‘minor effect’, because actually there was no effect at all, especially in pH and the major VFAs. Please rearrange this sentence in a way that conclusion matches better the results.

Authors’ response: We have amended the conclusion including “almost no effects” rather than “no effects’ alone as suggested by the reviewer since we consider that there were some changes observed in some VFA proportions.

L451: Should you include the initials of authors still in the filiation in order to include them here? Similarly to this previous published paper: https://doi.org/10.3390/ruminants1020011

Authors’ response: We prefer to maintain this format to easily identify the authors contribution

L456: This sentence is repeated twice, delete one of them. Also delete the quotation mark in the end of the sentence.

Authors’ response: The sentence has been removed.

L466: Was the approval by the ethics committee done after completion of the experiment? If so, this is not ideal. Approval should be given before the experiment with animals start.

Authors’ response: The details of the approval committee protocol were wrong; we have amended to the correct details. The protocol was approved before the experimental procedures occurred.

Mention DOI link always in the same way (with https, or without), be consistent and follow Journal’s guideline.

Authors’ response: Suggestion accepted.

L490: Should each word of the title start with capital letter? Check throughout according to Journal’s guideline.

Authors’ response: Suggestion accepted. References’ words of the titles now start with capital letter

L511: Delete spaces in half-sib.

Authors’ response: Suggestion accepted.

L527 – Here scientific name is in italic; L532 – Here it’s not in italic. Please check throughout according to Journal’s guideline and be consistent.

Authors’ response: Suggestion accepted.

L576: Delete 1 after Growth.

Authors’ response: Suggestion accepted.

L578: Should the journal’s name be abbreviated? Please check throughout according to Journal’s guideline.

Authors’ response: Suggestion accepted. All references have been checked

L599: Delete 1,2.

Authors’ response: Suggestion accepted.

 

 

Author Response File: Author Response.docx

Reviewer 2 Report

General comments

The manuscript evaluated the effect of feeding alfalfa and a mixed diet of oats and berseem clover as a source of fresh forage to dairy cows. Overall, the manuscript is within the scope of the journal, the experiment is well designed, and the findings are novel.

A limitation of this study is that it included 3 cows per treatment. Perhaps this small number of replicates was the reason for relatively small number of significant findings throughout the manuscript. Moreover, the authors have correctly pointed out that values were averaged as one value per cow per period. However, it is not clear how the authors have dealt with outliers. If one datapoint (cow) was considered an outlier and was removed, only two datapoints would remain to create a mean for that treatment. The authors can address this issue by including references that support this design and number of animals and/or discuss this limitation.

It should be noted that the title mentions dairy cows and the introduction mentions lactating dairy cows (L41) but the experiment was actually conducted in dry cattle.

Specific comments

L38 perhaps mention other countries with similar conditions to show that this research is applicable on a global scale, which it is.

L42 Consider saying that total mixed rations based on concentrates and preserved forages are offered.

L42 What about partial mixed rations? Are they offered too in these climates?

L44 During the forage conservation process

L58 should this be et al?

L86 of dairy cattle

L100 de Chile

L122 Were any measures taken to avoid spoilage of the green matter during the day?

L146 rumen fluid samples

L174 Consider including a reference in this paragraph to support this methodology

L187 after (12:00h) feeding, remove the

L248 Consider replacing the word participation or reformulation this sentence

L254 DMI

L255 DMI is not shown in table 4 or in any table

L255 Consider reformulating this sentence. It isn’t the diet that had similar ruminal N-NH4 content but rather the ruminal fluid of cows fed these two diets. Please consider this comment for other similar sentences in the results section and discussion sections.

L414 et al?

L439 Consider reformulating the conclusion to avoid treatment abbreviations. This way the conclusion can be read independently from the main text. Just a suggestion.

Author Response

Comments and Suggestions for Authors

Reviewer 2

General comments

The manuscript evaluated the effect of feeding alfalfa and a mixed diet of oats and berseem clover as a source of fresh forage to dairy cows. Overall, the manuscript is within the scope of the journal, the experiment is well designed, and the findings are novel.

A limitation of this study is that it included 3 cows per treatment. Perhaps this small number of replicates was the reason for relatively small number of significant findings throughout the manuscript. Moreover, the authors have correctly pointed out that values were averaged as one value per cow per period. However, it is not clear how the authors have dealt with outliers. If one datapoint (cow) was considered an outlier and was removed, only two datapoints would remain to create a mean for that treatment. The authors can address this issue by including references that support this design and number of animals and/or discuss this limitation.

Author: We agree with the reviewer’s comments that the low number of replications is one of the major limitations of our study and this may be somewhat related to the lack of differences among treatments that we found, especially regarding the rumen fermentation parameters. Therefore, as suggested by the reviewer, we have highlighted this limitation in the first paragraph of the discussion. Please also see our response to reviewer 1 for further explanations.

Moreover, we agree with the reviewer that n=3 is quite low and would have been ideal to have more cows to replicate the treatments. However, considering that 1) rumen fistulated cow surgery and maintenance is quite expensive, and 2) the maintenance of such animals has been criticized by the society due to their animal welfare side-effects, even that they proportionate important information regarding rumen function, their usage and numbers has been reduced quite drastically. However, the usage of 3x3 Latin Square design when assessing rumen fermentation and other metabolic parameters is still common as you can see from previous publications from some of the co-authors of this study (Vargas-Bello-Perez et al., 2016, Animal or Vargas-Bello-Perez et al., 2015 J Dairy Sci ) and some other groups as well (e.g. Phakachoed et al 2012, LIvest Sci; Wang et al., 2014, Animal Prod Sci; Wanapat et al., 2013, Afr J Agric Res).

It should be noted that the title mentions dairy cows and the introduction mentions lactating dairy cows (L41) but the experiment was actually conducted in dry cattle.

Author: We acknowledge the reviewer’s comments regarding this aspect. However, and despite that we used dry dairy cows in this experiment we used both, animals with dairy cows genetics and to evaluate diets used for lactating dairy cows; therefore, we would like to maintain the mention to ‘dairy cows’ in the tittle and the introduction of the manuscript.

Specific comments

L38 perhaps mention other countries with similar conditions to show that this research is applicable on a global scale, which it is.

Author: Suggestion accepted. We included some other Mediterranean climate regions.

L42 Consider saying that total mixed rations based on concentrates and preserved forages are offered.

Author: Suggestion accepted.

L42 What about partial mixed rations? Are they offered too in these climates?

Author: Even that partial mixed rations are used as well they are less common, and, to the best of our knowledge, there is no clear documentation about their usage. However, we have included a comment to better reflect this aspect.

L44 During the forage conservation process

Author: Suggestion accepted.

L58 should this be et al?

Author: Suggestion accepted.

L86 of dairy cattle

Author: Suggestion accepted.

L100 de Chile

Author: Suggestion accepted.

L122 Were any measures taken to avoid spoilage of the green matter during the day?

Author: No other measure than those commented in the following paragraph were used: ‘Green matter not used in the morning feed was stored indoors protected from rain and direct sunshine exposure until the afternoon feed.’ However, comments were included to beter represent the conservation we carried out.

L146 rumen fluid samples

Author: Suggestion accepted.

L174 Consider including a reference in this paragraph to support this methodology

Author: Suggestion accepted. We included a reference for the methodology used for ruminal incubation.

L187 after (12:00h) feeding, remove the

Author: Suggestion accepted.

L248 Consider replacing the word participation or reformulation this sentence

Author: Suggestion accepted.

L254 DMI

Author: Suggestion accepted.

L255 DMI is not shown in table 4 or in any table

Author: Suggestion accepted. DMI has been included in the table

L255 Consider reformulating this sentence. It isn’t the diet that had similar ruminal N-NH4 content but rather the ruminal fluid of cows fed these two diets. Please consider this comment for other similar sentences in the results section and discussion sections.

Author: Suggestion accepted. We have reviewed these aspects in all the sections of the manuscript.

L414 et al?

Author: Suggestion accepted.

L439 Consider reformulating the conclusion to avoid treatment abbreviations. This way the conclusion can be read independently from the main text. Just a suggestion.

Author: Suggestion accepted.

 

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Authors have attended all required suggestions and comments. The manuscript has gone through intense revision and has improved since the submission. At this stage I don’t have any additional comment. I’m in favour of the acceptance and publication of this manuscript, if also in line with the Editorial decision.

Kind regards.

Author Response

We appreciate the reviewer's acceptance of the manuscript for publication.

Kind regards,

Daniel Enriquez-Hidalgo

Reviewer 2 Report

The authors have addressed all of my comments and suggestions.

My recommendation is that the paper should be accepted for publication.

I have made minor suggestions that could be considered by the authors.

 

L4 should the 4 be in subscript?

L96 in situ should be in italics

L239 of the LEG diet

L241 as above

L267 VFA is already volatile fatty acids, no need to write VFAs

L281 here you have a space between P < 0.05, elsewhere there is no space

L294 as above

L300 as above

In all tables, the title for the last column should be P value instead of diet

Author Response

L4 should the 4 be in subscript?

Authors’ response:

L96 in situ should be in italics

Authors’ response: Suggestion accepted.

L239 of the LEG diet

Authors’ response: Suggestion accepted.

L241 as above

Authors’ response: Suggestion accepted.

L267 VFA is already volatile fatty acids, no need to write VFAs

Authors’ response: Suggestion accepted.

L281 here you have a space between P < 0.05, elsewhere there is no space

Authors’ response: Suggestion accepted. I have replaced and formatted all the P values according to the Journal’s format (e.g. p < 0.05)

L294 as above

Authors’ response: Suggestion accepted.

L300 as above

Authors’ response: Suggestion accepted.

In all tables, the title for the last column should be P value instead of diet

Authors’ response: Suggestion accepted.