Crude Glycerol Increases Neutral Detergent Fiber Degradability and Modulates Rumen Fermentative Dynamics of Kikuyu Grass in Non-Lactating Holstein Cows Raised in Tropical Conditions

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear authors, 

Please find in the attached doc all my suggestions and comments. I hope you can find those useful.

Thanks

Stefania

Comments for author File: Comments.pdf

Author Response

Answer: Dear Reviewer, we appreciate your valuable comments and suggestions to improve our manuscript.

General concept comments Article: The biggest area of weakness is the statistically analysis and the discussion section together with the conclusion. There is no hypothesis, and the lack of a clear hypothesis since the beginning does not allow a clear interpretation of the results. Table 3 and all those results of NDF degradability need to be reviewed. I was not able to understand why to different statistical designs were used in order to analyze the information, degradability using Orskov and Mcdonald Model can be analyze under a latin square design, and material incubated is not actually a treatment. Material and methods section is really confusing a need a deeper revision. In general the conclusion is not actually concluding the impacts or benefits that CG can have, and how this by product can be used in dairy cows. Need to connect more the results, discuss in greater depth and create a whole new conclusion.

Answer: The authors have addressed each of your comments about M&M, statistical analysis and discussion. In addition, we have included a hypothesis in the introduction. We also edited the conclusion to attending your suggestions. Thank you. Below we detail how we have addressed each of your comments:

Review:

Evaluating two harvesting times suggest that harvesting time of this type of grass has an effect on its degradability and it would be important to know how CG is going to improve ruminal degradability or not.

Answer: Thank you for your insightful comments and suggestions. We have revised the discussion section to address your concerns about the effect of harvesting time on the degradability of kikuyu grass and how CG supplementation interacts with this factor.

In the introduction it was mentioned that an improvement in NDF degradability could impact on DMI and also milk production, however in this work four non lactating cows were used, making difficult to make an extrapolation of the data. Even though non lactating cows were used, was any other measured taken to conclude if there was a greater performance achieved?

Answer: thank you for your question. Yes, we carried over a performance trial with lactating dairy cows and observed that supplementing grazing dairy cows with 1,500 g of CGL/cow/day did not induce any substitute effect on grass intake, but enabled higher total dry matter and energy intake, resulting in an increase in milk yield and protein milk content and no changes in the FA profile of milk. https://dx.doi.org/10.17268/sci.agropecu.2021.053. Thanks to this comment we realized that we did not consider this in our discussion. Thus, we included this trial to a better discussion.

There is a clear need to clearly connect the results in a general discussion. It seems to be implicit sometimes that the response in NDF degradability and ammonia concentrations and VFA are connected in certain way but it is not clear in the way it is presented in the manuscript. I suggest making an effort to connect all results to achieve a clear conclusion and recommendation.

Answer: You are right, we have failed to connect our discussion. Thank you for letting us know. We have edited our discussion to improve it.

Specific comments:

 

TITLE

Suggestion: Crude glycerol increases neutral detergent fiber degradability of kikuyu grass and modulates rumen fermentative dynamics in grazing Holstein cows in the tropics

Answer: We agree. The tittle was edited according to suggestions of you and reviewer 3. Both recommended included “grazing or raised”. 

ABSTRACT

Line 19: Holstein cows were used in a 4*4 latin square design

Answer: We agree. The edition was included in the abstract section.

Line 20: I am not sure if the best term is inclusion of CG, or if it is better to say infusion of CG

Answer: The authors agree with your suggestion and the edition was included in the abstract section.

Line 21: And two kikuyu …. regrowth were incubated in the rumen for …hs

Answer: The authors appreciate your suggestion. We included it in the abstract section.

Line 22: same than line 20

Answer: Edited.

Line 28 29: I suggest adding that the greater EDNDF was achieved for both harvesting times

Answer: Thank you for the comment, we added it at line 29.

INTRODUCTION

Line 33 38: suggest revision of how is written

Answer: We have revised the corresponding paragraph in the introduction to improve the writing.

Line 43: ruminal degradable protein

Answer: Thank you for the comment, we edited it at line 43.

Line 44: excess of sugar alcohols in the rumen could cause a descend of ruminal pH affecting fiber degradability

Answer: We are very grateful for helping us improve our manuscript, the edition was added.

Line 46 47: producing propionate as the main fermentation product

Answer: The edition was added, thank you for helping us improve our manuscript.

Line 47: Additionally, part of the glycerol can be directly absorbed by the ruminal wall and…

Answer: The edition was added, thank you again for helping us improve our manuscript.

Line 53: NDF degradability as the degradability of other nutrient fractions …

Answer: it was edited at line 53.

Line 55: is still inconclusive. Literature reports have shown a lack of response ….

Answer: The authors are very grateful for your suggestions, and the edition was added.

Line 61: review this line “could be a sui feeding strategy”

Answer: We apologize, it was a typing error. Thanks for catching it.

Line63 64: ruminal degradability

Answer: it was edited at line 64.

MATERIALS AND METHODS

Line 75 77: Four non lactating Holstein cows (8 years old, 600±33 kgBW) provided with a ruminal fistula (No. 1 C 4", Bar Diamond, Inc) were used in this study in a 4*4 Latin square design.

Answer: The edition was added. Thank you.

Line 77 79: All cows had as libitum access to water and feed, and were grazing a Kikuyu pasture that had undergone 40 days of regrowth

Answer: We agree and edited this section. Thank you.

Table 1: IVDMD is a results obtained in this experiment?

Answer: No, it is not. This IVDMD was carried over in a daisy system, this is described in M&M at lines 147. In addition, we state it in the table footnote. Thank you for catching.

Line 86: Cows received each one of the four treatments in 4*4 Latin square design without replicate.

Answer: Thank you. The edition was added.

Line 87: I would make the same suggestion here than in the abstract portion. Is inclusion of infusion?

Answer: the authors agree with the suggestion and modified it.

Line 89: In addition, kikuyu forage harvested at 35 (DR35) and 45 (DR45) days of regrowth was incubated in the rumen … NEED to specify how.

Answer: We modified the sentence to a better description considering your suggestions. Thanks.

Table 2: IVDMD is a result of this experiment?

Answer: No, it is not. This IVDMD was carried over in a daisy system, this is described in M&M at lines 147. In addition, we state it in the table footnotes.

Line 95: Remove “the purchased”

Answer: We removed it.

Line 96: in this paragraph is described as CG infusion. I would suggest specifying the way that CG was infused or offered to the animal and use the same word throughout the article to make it clear. As it is described now it seems a liquid infusion directly into the rumen, however is with a syringe? Is a bolus?

Answer: The authors thank you for the comment, we have modified the term throughout the manuscript (using infusion). The CG was infused through the rumen cannula manually, the liquid was contained in a test tube, and through manual movements of the rumen content it was slowly incorporated in approximately 2-3 minutes, through movements that sought to homogenize the CG in the ruminal mat. For greater clarity, we have added a brief description in the M&M section.

Line 97 99: I suggest to move this paragraph above (line 86), when the design was described

Answer: We agree and moved this paragraph. Thank you for the recommendation.

 

Line 102: Hand Plucking is the hand clipping methodology?

Answer: Thank you for your inquiry. Yes, it is a manual collection methodology commonly used in grazing trials.

Line 103: Are those 4g expressed in DM? A bag size of 10x5 cm equals to 100cm2 of bag surface. In general literature recommends incubating 15 20 mg/cm2 of bag, In this case with 4 grams of DM in that bags is 40mg/cm2. I would appreciate if authors can justify that amount, also why grounding forage? Was it incubated dried? In grazing conditions when the animal is actually grazing the material we want to evaluate it forage will be incubated fresh and cut simulating mastication. Another suggestion is to say material was incubated x mg/cm2 of the bag and not putting approx 4 g.

Answer: Thank you for your inquiry regarding in situ procedures. Yes, the 4 g is expressed in DM. We have included it in M&M for better understanding.  The in situ bag method is widely because the measurements are performed in the rumen, where the degradation process is presumed to be more reliable than in vitro. There are many variants for the in situ incubation technique (Tedeschi and Fox, 2018, page 148). But, in general, the in situ method recommends to use forage be dried and ground before putting it into nylon bags (recommendation for in situ procedures of feed evaluation by NRC 2001). The particle size is important for the ruminal microorganisms to adhere to the feed particles after entering bags through pores in the nylon mesh and compensate for the lack of rumination of forages (Nocek, 1988). The utilization of particles ground to pass a 2-mm screen sieve seems to favor an equilibrium point between controlling the loss of particles and an adequate specific surface for microbial degradation (Krizans et al. 2015). Nocek (1988) also explains that the exact amount of sample to be incubated and bag size relation will depend on the potential extent of ruminal digestion of a given ingredient or forage in relation to extended ruminal incubation times and the number of chemical analyses that one desired to be conducted on the residue. In this sense, we choice to incubated 4 g of Kikuyo, by the relatively higher values of  the in vitro dry matter digestibility (IVDMD) observed at 48 h (712.0    and 601.0 g/kg DM), considering that our last incubation time was 72h. Looking to ensure that there will be sufficient sample residue available at the end of incubation for NDF analysis. Other studies also have used Ankom bags of 5 × 10 cm and 50 μg micrometer porosity, weighed, filled with 5 g of ground sample (to pass a 2-mm screen), and sealed (Vanzant et al., 1998; Cagle et al., 2020; Despal et al., 2022).

• National Research Council. 2001. Nutrient requirements of dairy cattle. 7th ed. Washington, DC. 381 pp.
• Nocek J. E. In situ and other methods to estimate ruminal protein and energy digestibility: a review J. Dairy Sci. 1988 71 2051 -2069
• Krizsan S. J., Rinne M., Nyholm L., and Huhtanen P. New recommendations for the ruminal in situ determination of indigestible neutral detergent fibre Anim. Feed Sci. Technol. 2015 205 31 -41
• Tedeschi, L. O., and D. G. Fox. 2018. The Ruminant Nutrition System:An Applied Model for Predicting Nutrient Requirements and Feed Utilization in Ruminants. 2nd ed. XanEdu, Acton, MA.
• Vanzant, E. S., R. C. Cochran, and E. C. Titgemeyer. 1998. Standardization of in situ techniques for ruminant feedstuff evaluation. Anim. Sci. 76:2717–2729. https: / / doi .org/ 10 .2527/ 1998 .76102717x.
• Cagle, C.M.; Fonseca, M.A.; Callaway, T.R.; Runyan, C.A.; Cravey, M.D.; Tedeschi, L.O. Evaluation of the effects of live yeast on rumen parameters and in situ digestibility of dry matter and neutral detergent fiber in beef cattle fed growing and finishing diets. Applied Animal Science, v.36, Issue 1, 2020, Pages 36-47
• Despal D, Alifianty OF, Pratama AP, Febrianti F, Evvyernie D, Wijayanti I, Nuraina N, Agustiyani I, and Rosmalia A (2022) In situ degradation of dairy cattle feedstuffs using reusable local nylon fabric bags, Veterinary World, 15(9): 2234–2243.

Line 106: were incubated in duplicate … (can remove line 107)

Answer: We are grateful for your suggestion. It was edited.

Line 107: were actually incubated at T0?

Answer: Yes, we incubated in the rumen bags at time 0, considering approximately 20 min. We modified the sentence to include this information in M&M.

Line 108: at room temperature?

Answer: Yes, it is. We added a statement about it to better understand.

Line 110: were the bags incubated at the same time and then removed? Or removed all together?

Answer: We appreciate the question. The bags were incubated at the same time and then removed. We have edited the sentence to more clarify.

Line 107 and line 110 are describing different things

Answer: You are right, we apologize for the mistake. We have edited this sentence to avoid confusion.

Line 115 116: it is not necessary to specify why you are measuring NDF content on the residues.

Was DM measured too?

Answer: The authors agree and deleted the sentence. We included the DM in this line to a better understanding of.

Line 119: why 4 hours after?

Answer: dear reviewer, thank you for your question. Considering the fermentation profile of crude glycerol previously reported by other works, we choose to collect 4h after infusion to check the possible “pick” of volatile fatty acid production and its effects on rumen pH drop.

Line 122: potentiometer or phmeter?

Answer: We checked it and is pHmeter, thanks for catching it. It was edited.

Line 126: the subsample for VFA was acidified too for preservation?

Answer: No, is not. The VFA subsample was immediately stored at -4 °C.  

Line 139: nitrogen contents are referring to CP?

Answer: We determined the N content, and then the Crude protein was calculated as N*6.25. We added a statement about it in this section.

Line 168: why is Diet a plot? Was not the same diet for the 4 animals (grazing+supplement)? Why is not analyzed as a latin square as all the other variables??

Answer: Thank you for your question and the opportunity to clarify our experimental design. We checked and this is a typing error. In this section, we mistakenly refer to forage harvested on different days of regrowth as “diet”. We apologize for the mistake and edit it. 

RESULTS

Line 176 177: the kikuyu grass incubates is not a different diet, is a different material incubated while the animal is receiving the same diet and a treatment (CG). Is the same situation than when is incubated forages, a RTM, or any other feedstuff, but this does not mean that the material incubated is making a difference if all the cows are receiving same diet, and all treatments (CG) over time. I firmly suggest reviewing this, and justifying the decisions taken forward. Table 3 and all those results of NDF degradability need to be reviewed.

Answer: You are totally right. We mistakenly refer to forage harvested on different days of regrowth as “diet”. We edited table 3 and all results about these results to improve it.

DISCUSSION

Line 217: Here it starts with CG infusion, same suggestion than before to unify the terms used through the manuscript

Answer: the authors agree with the suggestion. We edited the manuscript according it.

First part of discussion (NDF deg) it is written properly, but cannot review it right now before stats are reviewed

Answer: thank you so much for your collaboration. We have answer your question about statistical section. Above.

Line 255: was expected a different response?

Answer: this is a good question. We expected a change, mainly when compared to control with a high level of CG infusion. But, as discussed in this paragraph, the literature is still inconsistent.

Line 255 267: it needs to clarify how was CG administrated in this cow. Is it possible to replicate it in commercial conditions?

Answer: Thank you for your comment, we have included the CG infusion in M&M section. In addition, we have included a practical recommendation for CG use in farm or commercial conditions in the discussion section.

Line 269: that aligns with …. (Instead of conforms)

Answer: thank you for the recommendation.

Line 279 281: make a connection between VFA response and NDF degradability. Can this impact on animal performance? Milk production? Body condition score?.

Answer: The authors agree and include an additional statement in the discussion.

CONCLUSIONS

Line 299: again appear inclusion

Answer: thank you for the comment. We edited it.  

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors,

Congratulations on your accomplished research. Methods for improving dairy cow productivity are always welcome and add significant value to the bovine livestock sector. The introduction is well-written and structured; however, it might be beneficial to include an additional paragraph highlighting that other authors have also demonstrated the positive effects of various orally administered substances. For example, here is an article that demonstrates the positive effect of zeolite: "The effect of oral administration of zeolite on the energy metabolism and reproductive health of Romanian spotted breed in advanced gestation and postpartum period." Consider citing this work. Additionally, the last sentence should ideally begin with "The aim of the study was."

L 48 "by the rumen wall", maybe "by the rumen mucosa" sounds better

L 61 could be a sui feeding? what this means? maybe you rewrite this

L64 are not is

L 71 nonlactating Holstein cows, were they in the dry period?The materials and methods section is well-written and allows for the replication of the study.

The materials and methods section is well-written and allows for the replication of the study.

The results, discussion, and conclusions are well-written and easily understandable for the reader. The study clearly demonstrates the beneficial effect of glycerol on the digestion of kikuyu grass in Holstein cattle.

 

Comments on the Quality of English Language

Minor editing of English language required

Author Response

Dear authors,

Congratulations on your accomplished research. Methods for improving dairy cow productivity are always welcome and add significant value to the bovine livestock sector. The introduction is well-written and structured; however, it might be beneficial to include an additional paragraph highlighting that other authors have also demonstrated the positive effects of various orally administered substances. For example, here is an article that demonstrates the positive effect of zeolite: "The effect of oral administration of zeolite on the energy metabolism and reproductive health of Romanian spotted breed in advanced gestation and postpartum period." Consider citing this work. Additionally, the last sentence should ideally begin with "The aim of the study was."

Answer: Dear reviewer, we appreciate your valuable comments and suggestions to improve our manuscript. We have edited the objective sentence according to your recommendation. And including the work recommended in our introduction section.  In addition, below we detail how we have addressed each of your comments

 

L 48 "by the rumen wall", maybe "by the rumen mucosa" sounds better

Answer: The authors appreciate your suggestion and modified it.

L 61 could be a sui feeding? what this means? maybe you rewrite this

Answer: We apologize, it was a typing error. Thanks for catching it.

L64 are not is

Answer: The authors appreciate your suggestion and modified it.

L 71 nonlactating Holstein cows, were they in the dry period?

Answer: Yes, the cows were during the dry period.

The materials and methods section is well-written and allows for the replication of the study.

Answer: Thank you for your comment. You will see some editions in this section to address attending the recommendations of other reviewers.

The results, discussion, and conclusions are well-written and easily understandable for the reader. The study clearly demonstrates the beneficial effect of glycerol on the digestion of kikuyu grass in Holstein cattle.

Answer: The authors are very grateful for your comments.  You will see some editions in these section to address attending the recommendations of other reviewers.

Reviewer 3 Report

Comments and Suggestions for Authors

Manuscript ID dairy-3101193 

Brief Abstract: In the paper entitled “Crude glycerol increases neutral detergent fiber degradability and modulates rumen fermentative dynamics of kikuyu grass in Holstein cows in the tropics”, the authors investigated the effects the effects of increased crude glycerol (CG) inclusion (derived from biodiesel production) on ruminal neutral detergent fiber (NDF) degradability in situ and on fermentation parameters in 4 non-lactating Holstein cows fed a diet containing predominantly containing Pennisetum clandestinum Hochst as forage essence. The authors used 4 non-lactating cows (fitted with rubber rumen cannulas), randomized in a 4 × 4 Latin square design. Treatments consisted of rumen inclusion of CG at the following levels: 0, 500, 1,000, and 1,500 g/animal/day. The authors evaluated intraruminal incubation of two forages (Pennisetum clandestinum Hoch.) harvested at 35 and 45 days of regrowth. Results showed that CG inclusion in the rumen increased effective degradability of neutral detergent fiber in both incubated forages. Total volatile fatty acids (VFA) and pH values ​​in rumen fluid were not affected by CG inclusion, even if, the molar proportions of propionate and butyrate increased at the expense of acetate at all levels of CG inclusion. The authors conclude that CG supplementation increases the degradability of ruminal neutral detergent fiber (NDF), improving rumen fermentation dynamics in cows grazing on Pennisetum clandestinum Hochst forage under tropical conditions.

Although the paper is potentially interesting and in line with the aims of the journal, it has limitations that prevent an objective assessment, which I list below:

- The authors use (I think I am interpreting correctly) a pasture with forage essences of Pennisetum clandestinum Hochst (sown? spontaneously grown?) which, in the case of natural meadows, are mixed with other forage essences. It would be necessary to know the "specific contribution" of the other forage essences, especially if they belong to the family of Fabaceae and Poaceae. 

- Although the experiment is potentially interesting, I wonder what the practical implications might be for normally reared animals, particularly lactating cows. My question is, how can the experimental data obtained be reproduced in daily practice?

By cannulating all animals in a herd

By treating the harvested feed (Pennisetum clandestinum Hochst) with GC

Something else?

- In addition, the title needs to be changed to “Crude glycerol increases neutral detergent fiber degradability and modulates rumen fermentative dynamics of kikuyu grass in non-lactating Holstein cows raised in the tropical conditions”.

 

Comments on the Quality of English Language

In my opinion, a moderate editing of the English language is required.

Author Response

Manuscript ID dairy-3101193

 

Brief Abstract: In the paper entitled “Crude glycerol increases neutral detergent fiber degradability and modulates rumen fermentative dynamics of kikuyu grass in Holstein cows in the tropics”, the authors investigated the effects the effects of increased crude glycerol (CG) inclusion (derived from biodiesel production) on ruminal neutral detergent fiber (NDF) degradability in situ and on fermentation parameters in 4 non-lactating Holstein cows fed a diet containing predominantly containing Pennisetum clandestinum Hochst as forage essence. The authors used 4 non-lactating cows (fitted with rubber rumen cannulas), randomized in a 4 × 4 Latin square design. Treatments consisted of rumen inclusion of CG at the following levels: 0, 500, 1,000, and 1,500 g/animal/day. The authors evaluated intraruminal incubation of two forages (Pennisetum clandestinum Hoch.) harvested at 35 and 45 days of regrowth. Results showed that CG inclusion in the rumen increased effective degradability of neutral detergent fiber in both incubated forages. Total volatile fatty acids (VFA) and pH values ​​in rumen fluid were not affected by CG inclusion, even if, the molar proportions of propionate and butyrate increased at the expense of acetate at all levels of CG inclusion. The authors conclude that CG supplementation increases the degradability of ruminal neutral detergent fiber (NDF), improving rumen fermentation dynamics in cows grazing on Pennisetum clandestinum Hochst forage under tropical conditions.

Answer: Dear reviewer, we are so grateful for your comments and contributions as reviewers of our manuscript.

Although the paper is potentially interesting and in line with the aims of the journal, it has limitations that prevent an objective assessment, which I list below:

Answer: Thank you. Below we detail how we have addressed each of your comments:

- The authors use (I think I am interpreting correctly) a pasture with forage essences of Pennisetum clandestinum Hochst (sown? spontaneously grown?) which, in the case of natural meadows, are mixed with other forage essences. It would be necessary to know the "specific contribution" of the other forage essences, especially if they belong to the family of Fabaceae and Poaceae.

Answer: thank you for your comments. Yes, cows were raised in Pennisetum clandestinum Hochst pastures, thus as recommended by you and reviewer 1, we edited the title, for more clarity. In addition, our experimental area (paddocks) was composed by almost 98% of forage Kikuyo. We understand your concern about forage-specific contribution, once in farm conditions, the normal find is a heterogenous pasture with a mix of several species, however, as this is an experimental area, the effort to maintain homogeneous pastures is high (manual and chemical controls). Of course, we observed some Poaceae such as Holcus lanatus and Trifolium repens, but these represented less than 2% of the total forage in our trial.

 - Although the experiment is potentially interesting, I wonder what the practical implications might be for normally reared animals, particularly lactating cows. My question is, how can the experimental data obtained be reproduced in daily practice?

Answer: we appreciate your question. This type of approach is useful to define the better level of inclusion of novel or alternative feedstuffs, determined firstly the effects on rumen fermentation and fiber degradation, for in a subsequent trial evaluated performance, reproductive or environmental implications, with a lower number of treatments. Putting the feedstuffs directly into the rumen is a good approach to guarantee the real dose of tested feedstuffs, for example avoiding errors such as animal selection or losses during the supplement intake. As included in our discussion, we development a subsequent performance trial with lactating dairy cows, and observed that reported that supplementing grazing dairy cows with 1,500 g of GL per day increased by 14.2% the milk yield. Thus, incorporating CG into supplements could be a suitable nutritional strategy to optimize the utilization of kikuyu forage and enhance the performance of dairy cows.  

By cannulating all animals in a herd

Answer: Thank you for your inquiry regarding cannulating cows. As previously explained, we choose the in situ bag method widely because the measurements are performed in the rumen, where the degradation process is presumed to be more reliable than in vitro. And the use of rumen cannulate cows was necessary for our experimental procedures, but in commercial herds, the recommendation is to include the CG into the supplement, as we carried over in our subsequent performance trial.

By treating the harvested feed (Pennisetum clandestinum Hochst) with GC, Something else?

Answer: we are grateful for your inquiry. However, we did not treat the harvested forage. The harvested forage at 35 (DR35) and 45 (DR45) days of regrowth was used to be incubated into the rumen of cows that received the ruminal infusion, looking to evaluate the possible effect of harvesting time of this type of grass has an effect on its degradability and know how CG is going to improve ruminal degradability or not.

- In addition, the title needs to be changed to “Crude glycerol increases neutral detergent fiber degradability and modulates rumen fermentative dynamics of kikuyu grass in non-lactating Holstein cows raised in the tropical conditions”.

Answer: The authors agree and edit the tittle according to your suggestions.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Authors, 

Thanks for including all the suggestions and edits. Manuscript has improved substantially. I added some minor comments. 

Thanks,

 

38 / 5.000  

Resultados de traducción

Resultado de traducción

 

Comments for author File: Comments.pdf

Author Response

Article: All the comments and suggestions were addressed properly. Now a clear Hypohesis is stated which is essential to understand the “why” of this research, and allowed a more clear conclusion.

Answer: Thank you for your thorough review and positive feedback on our manuscript. We appreciate your acknowledgment of the improvements made, particularly in clarifying the hypothesis and enhancing the conclusions. Your suggestions have been invaluable in strengthening the overall quality of our research.

Review:

Evaluating two harvesting times suggest that harvesting time of this type of grass has an effect on its degradability and it would be important to know how CG is going to improve ruminal degradability or not. This was now clearly addressed and a conclusion about it is stated.

Answer: We are pleased to hear that our revisions have adequately addressed this issue and provided a clear conclusion.

Appreciate the effort to add all the suggestions and connect the results obtained. If the authors consider it properly could make a reference or a comment that more research is needed to evaluate the responses in lactating cows and measure the response in milk production

 Answer: we agree and have included a statement in the conclusion section emphasizing the potential for future research in this area. Once again, thank you for your constructive comments and for helping us enhance our work.

Specific comments:

Some small specific comments are stated here:

ABSTRACT

Line 19: Holstein cows were used in a 4*4 latin square design (instead of latin square 4*4)

Answer: We agree and edited it.

INTRODUCTION

Line 62: starts with a 7, is this a reference? Or maybe a typo error

Answer: Yes, it is a typo error, thank you for catching it.

MATERIALS AND METHODS

Line 96: to estimate or to measure in situ ruminal NDF degradability.

Answer: We added the edition, thank you.

 

Line 97: The experimental period included four 16-day periods (13 days for adaptation to the diet and 3 days for measurements), for a total of 64 days.

Answer: The sentence was edited. We are very grateful for helping us improve our manuscript.

Line 119: into the rumen and removed at 0, 3, 6 …. Adding that can remove this phrase “After the designated incubation times were completed, the bags were taken out of the rumen,”

Answer: We added the edition, thank you.

Reviewer 3 Report

Comments and Suggestions for Authors

The paper has been considerably improved, also as a result of suggestions made during the review process. I think Tables 3 and 4 should be checked, especially the letters indicating significance level, should be superscripted.

Author Response

The paper has been considerably improved, also as a result of suggestions made during the review process. I think Tables 3 and 4 should be checked, especially the letters indicating significance level, should be superscripted.

Answer: Thank you for your positive feedback and for acknowledging the improvements made to the paper due to the review process. We appreciate your meticulous attention to detail, particularly your observation regarding the formatting of Tables 3 and 4. We have double-checked our tables, and ensure that the letters indicating significance levels are correctly superscripted in both tables. We are committed to making these adjustments and ensuring that the final version of the paper meets the highest standards. Thank you once again for your valuable input.