Yeast Culture Is Beneficial for Improving the Rumen Fermentation and Promoting the Growth Performance of Goats in Summer
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsComments to the Author
Zhang et al. present a study of the “Antioxidant yeast culture is beneficial for improving rumen fermentation and promoting the growth performance of goats in summer”. These effects were tested by comparison of antioxidant capacity, fermentation and growth performance in in-vivo and by means of in-vitro fermentation and digestibility. The description of the statistics needs significant improvement including adding a descriptive model. No experimental design, replication through time not explained (where there different periods, cross over design, Latin square design, etc.). Pre-planned contrasts do not seem to have been evaluated (essential in a dose response study). Differences across treatments are not appropriately described to aid in the discussion that addresses best treatment among yeast doses. Consider a procedure of multiple comparisons when contrasts are significant. The presentation of data and writing in the manuscript is simplistic and does not address the effects of increased DMI affecting fermentation. There are major clarification points, edits, and some questions regarding calculation and values presented in the tables, methods, presentation and interpretation that preclude this manuscript from being accepted in the present form.
Abstract:
“P” needs to be in cursive through the abstract.
Ln 11: Clarify there was an in-vitro and an in-vivo experiment and justify the reason why the 0.6%DM dose was not included in the in-vivo experiment.
Introduction:
To make a point suggest only using 3 references max.
Ln 50: Use “fibrolytic” instead of “fibrinolytic”
Ln 50-62: Previous studies is used excessively. Improve flow and elaborate further being concise to build an in depth justification for your hypothesis.
Ln 68: Remove “Previous studies”
Ln 70: Improved grammar is needed
Ln 72: There is no such thing as “antioxidant yeast culture” should only be yeast culture. Is this a live or dead culture product?
Ln 69-74: These are biased few sentences; you are already anticipating the conclusion. Revise.
Elaborate on how your experiment will test your hypothesis!
Materials and Methods :
Ln: 77: Describe experimental design
Ln 77-83: Define abbreviations: i.e. THI, NHS HS
Ln 107: Seems to be out of place “These six goats as the donor of rumen fluid.”
Ln 108: Describe pressure suction device or proved commercial info
Ln 110: remove antioxidant
Ln 111-112: Report CFU or composition and justification of these doses
Ln 114: Do not start with a number after a period. Cite the methodology used for the in-vitro incubation
Ln 120: Add enough information in the table title to allow table to be stand alone
Ln: 122: Describe experimental design
Ln 124: Describe the crossbred origin of the goats and their mature body weight
Ln 126: 3 or 4 groups? Remove antioxidant, this is a marketing statement from the company (you can mention the intention of adding glutathione but not label it as such).
Ln 130: Justify the feeding times. Were the animals kept outdoors or indoors?
L 133: Replace “levels” with concentrations “throughout”
Ln 134: add a reference for this procedure
Table 2: Format is cumbersome and lacks detail (i.e. Content %DM?, add other important nutritional composition items of the diet such as DM, OM, NDF, ADF, NFC/NFC, starch, EE
Ln 140-141: Above, you mention that periods lasted for 30 d and here you are saying that fecal samples were collected on days 87 to 90 and blood samples were collected on day 90. Where periods repeated? In what manner? Revise
Ln 146-150: Assay product numbers needed for each index
Ln 161: Details of these methods is needed
Ln 162: Was feed provided as TMR? If so, was there a particle size analysis conducted to determine sorting? Justify
Ln 165: Briefly describe these procedures, specifying the differences on how DM were obtained for the invitro experiment
State your statistical model used, it is not clear from your stats description. What was your random effect? Was there a block effect? Did you have repeated measures!
Results and Discussion:
Remove antioxidant throughout
Ln: 178 Describe why you are doing this. This is important for the reader to un understand the logic behind these analysis. Again describe NHS and HS abreviations completely in table and at the beginning of the manuscript
Table 3
Clarify how you are using asterisks to separate means. Your asterisk model is confusing throughout.
pH : Based on your SEM, these all should be not significant. Also pH when sampling using suction devices there is significant saliva contamination that needs to be mentioned and often contaminate fermentation parameters
Enzymes : Based on your SEM, these all should be not significant
Table 5
Avicelase : Based on your SEM, these all should be not significant
Table 6
Specify the title to include digestibility and be specific when you refer to DM, NDF and ADF that you are referring to apparent digestibility coefficients.
DMI/ADG : Based on your SEM, these all should be not significant
Ln 140: sheep sheds?
Ln 239-261: This is redundant to what you presented in the introduction, delete. For the discussion, present your observed (not found) results, justify, and compare these to other studies and draw a concluding statement. This needs to be presented throughout your discussion.
Ln 274-291: Same as above. Elaborate more about the effects of yeast in Ph and the reason you did not see an effect on the in-vitro study with the 0.6% addition of yeast
Ln 293-305: Your apparent digestibility coefficients are quite high relative to the control. This needs to be justified citing other papers where these types of differences having been observed (from 10 to 15% differences)
Poor description of experimental design and statistical description/utilization of the methods makes this study impossible to replicate and the way presented the data might be completely random (No period replication). Without these the results presented here can be flawed and confounded.
Comments on the Quality of English Language
All presented above
Author Response
Response to Reviewer 1
Dear Reviewers,
We would like to express our sincere gratitude to you for the constructive comments and suggestions, which have greatly helped us improve the quality of the manuscript. Below, we provide point-by-point responses to each of the concerns raised. We hope that our revisions and clarifications adequately address the issues highlighted.
Best wishes,
Shuang Zhang
Zhang et al. present a study of the “Antioxidant yeast culture is beneficial for improving rumen fermentation and promoting the growth performance of goats in summer”. These effects were tested by comparison of antioxidant capacity, fermentation and growth performance in in-vivo and by means of in-vitro fermentation and digestibility. The description of the statistics needs significant improvement including adding a descriptive model. No experimental design, replication through time not explained (where there different periods, cross over design, Latin square design, etc.). Pre-planned contrasts do not seem to have been evaluated (essential in a dose response study). Differences across treatments are not appropriately described to aid in the discussion that addresses best treatment among yeast doses. Consider a procedure of multiple comparisons when contrasts are significant. The presentation of data and writing in the manuscript is simplistic and does not address the effects of increased DMI affecting fermentation. There are major clarification points, edits, and some questions regarding calculation and values presented in the tables, methods, presentation and interpretation that preclude this manuscript from being accepted in the present form.
Abstract:
Comment 1: “P” needs to be in cursive through the abstract.
Response 1: Thank you for pointing out the incorrect font of “p,” which has been replaced by “p” in lines 14 and 21.
Comment 2: Ln 11: Clarify there was an in-vitro and an in-vivo experiment and justify the reason why the 0.6%DM dose was not included in the in-vivo experiment.
Response 2: In vitro fermentation methods are firstly used for feed evaluation because in vitro evaluations are inexpensive and laborious savings. Next, a feeding experiment (in-vivo experiment) in heat-stressed goats can be conducted based on the results of this in vitro study. In the in vitro fermentation test, 0.6%DM did not improve the rumen fermentation of heat-stressed goats, so this concentration group was not set in further feeding tests.
Introduction:
Comment 3: To make a point suggest only using 3 references max.
Response 3: Thanks for your suggestion. There are few studies on improving rumen fermentation in heat-stress ruminants by feeding microecological agents, and few ruminants are involved in such studies. More than three citations are used at some point in this paper to include as many ruminant species as possible. Therefore, we retain the original number of references.
Comment 4: Ln 50: Use “fibrolytic” instead of “fibrinolytic”
Response 4: Thanks for pointing out the wrong words. "fibrinolytic" has been replaced by "fibrinolytic" in line 49.
Comment 5: Ln 50-62: Previous studies is used excessively. Improve flow and elaborate further being concise to build an in depth justification for your hypothesis.
Response 5: Thanks for your suggestion. Because there are very few studies on the effects of heat stress on goats, it is necessary for us to explain the rationality and significance of this study by referring to the previous studies. Moreover, after reviewing the previous studies, we conclude the paragraph by suggesting the necessity of finding an effective method to alleviate the adverse effects of heat stress on ruminants. Namely, "Therefore, we can determine that heat stress has a systemic adverse effect on ruminants. n this case, seeking an efficient and practical method to alleviate these adverse effects is necessary.” In lines 61-63.
Comment 6: Ln 68: Remove “Previous studies”
Response 6: Previous studies have been removed.
Comment 7：Ln 70: Improved grammar is needed
Response 7: The original sentence has been replaced by “So far, few studies have been on the application of yeast cultures in heat-stressed goats.” in line 71.
Comment 8：Comment: Ln 72: There is no such thing as "antioxidant yeast culture" should only be yeast culture. Is this a live or dead culture product?
Response 8: Thanks for your suggestion “antioxidant yeast culture” has been changed to “yeast culture” throughout the manuscript.
Comment 9: Ln 69-74: These are biased few sentences; you are already anticipating the conclusion. Revise.
Elaborate on how your experiment will test your hypothesis!
Response 9: Thank you for pointing out the inadequacy of this expression. We have revised the original expression in lines 73-74 according to your suggestion.
Materials and Methods :
Comment 10: Ln: 77: Describe experimental design
Response 10: We appreciate your insightful suggestion. Experimental design has been clearly described in lines 79-127
Comment 11: Ln 77-83: Define abbreviations: i.e. THI, NHS HS
Response 11: The full names of these abbreviations have been added in lines 80 and 86.
Comment 12: Ln 107: Seems to be out of place “These six goats as the donor of rumen fluid.”
Response 12: According to Cai et al., 2021 and Xue et al., 2022, through heat stress modeling, six goats were used as rumen fluid donors in vitro, which is reasonable.
1. Xue, L.G.; Zhou, S.Y.; Wang, D.; Zhang, F.Y.; Li, J.F.; Cai L.Y. The low dose of Saccharomyces cerevisiae is beneficial for rumen fermentation (both in vivo and in vitro) and the growth performance of heat-stressed goats. Microorganisms. 2022, 10: 1877.
2. Cai, L.Y.; Hartanto, R.; Zhang, J.; Qi, D.S. Clostridium Butyricum improves rumen fermentation and growth performance of heat-stressed goats in vitro and in vivo. Animals 2021, 11, 3261.
Comment 13: Ln 108: Describe pressure suction device or proved commercial info
Response 13: The device is briefly described on line 113. This non-invasive rumen collection device is patent-pending and will be described in detail in our subsequent article.
Comment 14: Ln 110: remove antioxidant
Response 14: Thanks for suggesting “antioxidant” before “yeast culture” has been removed throughout the manuscript.
Comment 15: Ln 111-112: Report CFU or composition and justification of these doses
Response 15: The live yeast cell in this yeast culture is 1.0 × 106 CFU/g. According to the existing studies, a concentration of 1.0 × 108 CFU/g of live cells of saccharomyces cerevisiae could improve rumen fermentation and growth performance of heat-stressed goats. Therefore, this study aimed to investigate the effects of yeast cultures (instead of live yeast cells) on antioxidant capacity, rumen fermentation, and growth performance of goats in summer.
Comment 16: Ln 114: Do not start with a number after a period. Cite the methodology used for the in-vitro incubation
Response 16: Thanks for your suggestion. The point you mentioned has been revised, and the methodology used for the in-vitro incubation has been cited as [5] in line 127.
Comment 17: Ln 120: Add enough information in the table title to allow table to be stand alone
Response 17: We appreciate your insightful suggestion. The title of Table 1 has been changed to “The primer sequences of heat-stress-related genes.” in line 128.
Comment 18: Ln: 122: Describe experimental design
Response 18: Thanks for your insightful suggestion. The experimental design has been well described in lines 130–142.
Comment 19: Ln 124: Describe the crossbred origin of the goats and their mature body weight
Response 19: The crossbred goat in this study was Boer (♂) × Macheng Black (♀) crossbred goat, which is the first filial generation of male Boer goat and female Macheng Black. Such crossbred goats are widely raised in the Jianghuai region of China. The mature (12 months) is 28.60 ± 4.83 kg (Zhang, N.; Suo, X.J.; Chen, M.X.; Li, X.F. Study on quality traits of F1 generation cross between Boer Goat and Macheng Black Goat. J. Anhui Agri. Sci. 2008, 36, 1344–11345.).
Comment 20: Ln 126: 3 or 4 groups? Remove antioxidant, this is a marketing statement from the company (you can mention the intention of adding glutathione but not label it as such).
Response 20: Thank you for pointing out the error in this group; it should be three groups; it has been revised in line 133. “antioxidant” before “yeast culture” has been removed throughout the manuscript. The glutathione content was specifically presented to indicate the differentiation of this yeast culture. The homogeneity of such studies can be avoided by using this special yeast culture.
Comment 21: Ln 130: Justify the feeding times. Were the animals kept outdoors or indoors?
Response 21: Thank the reviewer for the question. Goats were kept in a natural ventilation house equipped with slatted floors, manure scraper systems, and individual feeding pens. The feeding time in the original text needs to be corrected; it should be between 8:00 a.m. and 17:00 p.m. We have modified it in line 137.
Comment 22: Ln 133: Replace “levels” with concentrations “throughout”
Response 22: Thanks for your constructive comment. “levels” have been changed to “concentrations” in lines 121, 133, 140, and 143.
Comment 23: Ln 134: add a reference for this procedure
Response 23: A reference [5] has been added for this procedure in line 142.
Comment 24: Table 2: Format is cumbersome and lacks detail (i.e. Content % DM?, add other important nutritional composition items of the diet such as DM, OM, NDF, ADF, NFC/NFC, starch, EE
Response 24: Thank the reviewer for the question. “Content % (DM basis) has been added in Table 2. For feed preparation, refer to Xue et al., (2002). We have focused on the listed items but not on the other indicators you mentioned. In future research, we will try our best to improve the formula table.
Xue, L.G.; Zhou, S.Y.; Wang, D.; Zhang, F.Y.; Li, J.F.; Cai L.Y. The low dose of Saccharomyces cerevisiae is beneficial for rumen fermentation (both in vivo and in vitro) and the growth performance of heat-stressed goats. Microorganisms 2022, 10, 1877.
Comment 25: Ln 140-141: Above, you mention that periods lasted for 30 d and here you are saying that fecal samples were collected on days 87 to 90 and blood samples were collected on day 90. Where periods repeated? In what manner? Revise
Response 25: We apologize for the confusion caused by a clerical error in the collection time of fecal samples. “87 to 89” has been changed to “27 to 29” in line 141, and “87 to 89” has been changed to “28 to 30” in line 148.
Comment 26: Ln 146-150: Assay product numbers needed for each index
Response 26: We ignored this assay product numbers when we did the corresponding testing. Since the number of each batch of products is different, we cannot provide it now. I'm sorry! We should pay attention to this problem in the future study.
Comment 27: Ln 161: Details of these methods is needed
Response 27: Thanks for your suggestion. In the original text, the testing steps have been briefly but emphatically described and cited in the literature. Therefore, there is no need to go into the details of test of these short-chain fatty acids.
Comment 28: Ln 162: Was feed provided as TMR? If so, was there a particle size analysis conducted to determine sorting? Justify
Response 28: Thanks for your question. This feed is TMR; its particle size is shown in the figure below:
Figure 1. TMR Feed particle model
Comment 29: Ln 165: Briefly describe these procedures, specifying the differences on how DM were obtained for the in vitro experiment
Response 29: The description of the detection method will take up a lot of space and make the article appear cumbersome. Here we show the testing process as follows:
Van Soest fiber analysis
• Principle and Scope
• Reagents:
1. Neutral Detergent Fiber
2. Acid Detergent Fiber
• Procedure
1. Neutral Detergent Fiber
2. Acid Detergent Fiber
Principle and Scope ________________________________________
The concept behind the detergent fiber analysis is that plant cells can be divided into less digestible cell walls (contains hemicellulose, cellulose and lignin) and mostly digestible cell contents (contains starch and sugars). Van Soest separated these two components successfully by use of two detergents: a neutral detergent (Na-lauryl sulfate, EDTA, pH =7.0) and an acid detergent (cetyl trimethyl ammonium bromide in 1 N H2SO4).
Hemicellulose, cellulose and lignin are indigestible in non-ruminants, while Hemicellulose and Cellulose are partially digestible in ruminants. NDF = Hemicellulose + Cellulose + Lignin ADF = Cellulose + Lignin
Neutral Detergent Fiber is a good indicator of "bulk" and thus feed intake. Acid detergent fiber is a good indicator of digestibility and thus energy intake
Figure: Principle of detergent partitioning of the fiber fraction by Van Soest
Although aquaculture does not deal with ruminants at all, it may be useful to be aware of these analysis. For instance dealing with plant material as imortant feed ingredient or dealing with herbivorous fish.
Reagents ________________________________________
Reagents for NDF determination (Neutral detergent fiber)
1. Neutral detergent solution:
o Sodium borate decahydrate (Na2B4O7 - 10 H2O) 6.81 g
o Disodium ethylenediaminetetraacetate (EDTA, C10H14N2Na2O8)
o 18.61 g Sodium lauryl sulfate neutral (C12H25NaO4S)
o 30 g 2-ethoxyethanol (Ethylene glycol monoethyl ether, Cellosolve, C4H10O2)
o 10 ml Disodium phosphate anhydrous (Na2HPO4) 4.56 g
o Distilled water 1000 ml. Pour Sodium borate and disodium EDTA in a beaker and dissolve with a part of distilled water while heating. Add lauryl sulfate and 2-ethoxyethanol. Separately dissolve disodium phosphate in part of distilled water while heating until complete solution is obtained. Mix the two solutions and the remaining distilled water and control the pH which must be between 6.9 and 7.1
2. n-octanol (C8H18O) octilic alcohol
3. Sodium sulfite anhydrous (Na2SO3)
4. Acetone
Reagents for ADF determination (Acid detergent fiber)
1. Acid detergent solution:
o Cetyltrimethylammonium bromide technical grade (C19H42BrN) 20 g
o Sulfuric acid 1 N (H2SO4, 49.04 g/l) 1 l
o Dissolve tensioactive into acid while stirring to promote dissolution.
2. n-octanol (C8H18O) octilic alcohol.
3. Acetone.
Procedure ________________________________________
Procedure for NDF determination (Neutral detergent fiber)
1. Grind the air dried sample to pass 1 mm screen.
2. Weigh in a crucible 1 g of grinded sample with 1 mg approximation.
3. Add 100 ml of neutral detergent solution at room temperature into crucible with 0.5 g of sodium sulfite and some drops of n-octanol.
4. Heat to boiling and reflux 60 minutes from onset of boiling.
5. Filter and wash 3 times with boiling water, then twice with cold acetone.
6. Dry 8 hours at 105 °C and let cool in a desiccator.
7. Weigh.
8. Calculate neutral detergent fiber: NDF % = (weight of crucible + weight of residue) - weight of crucible / weight of sample x 100. Neutral detergent solubles: NDS % = 100 - NDF %.
9. Ash in a muffle at 550 °C 2 hours and let cool in a desiccator.
10. Weigh.
11. Calculate ash insoluble in neutral detergent: loss on ashing / weight of sample x 100.
Procedure for ADF determination (Acid detergent fiber)
1. Grind the air dried sample to pass 1 mm screen.
2. Weigh in a crucible 1 g of grinded sample with 1 mg approximation.
3. Add 100 ml of acid detergent solution at room temperature and some drops of n-octanol.
4. Heat to boiling and reflux 60 minutes from onset of boiling.
5. Filter and wash 3 times with boiling water, then twice with cold acetone.
6. Dry 8 hours at 105 °C and let cool in a desiccator.
7. Weigh.
8. Calculate acid detergent fiber: ADF % = (weight of crucible + weight of residue) - weight of crucible / weight of sample x 100.
9. Ash in a muffle at 550 °C 2 hours and let cool in a desiccator.
10. Weigh.
11. Calculate ash insoluble in acid detergent: loss on ashing / weight of sample x 100.
However, we still retained the original way of writing in the manuscript, which is also the expression of many similar articles. The feed, fermented feed substrates, and fecal samples were placed in a 105 ± 2°C oven and dried at atmospheric pressure until the weight was constant and the mass lost was water. The remainder is dry matter mass. The digestibility of DM, was calculated as follows: (DM content in feed-DM content in feces or fermentation broth)/DM content in feedstuff ×100 = DM digestibility (%).
Comment 30: State your statistical model used, it is not clear from your stats description. What was your random effect? Was there a block effect? Did you have repeated measures!
Response 30: We are very sorry for the error in our statistics. We reorganized the original data and used the R language for statistics again. The details are described in the “Statistical Analysis” section in lines
Results and Discussion:
Comment 31: Remove antioxidant throughout
Response 31: Thanks for suggesting “antioxidant” before “yeast culture” has been removed throughout the manuscript.
Comment 32: Ln: 178 Describe why you are doing this. This is important for the reader to un understand the logic behind these analysis. Again describe NHS and HS abreviations completely in table and at the beginning of the manuscript
Response 32: Thank the reviewer for the question. Physiological parameters, temperature and humidity index, heat stress-related genes, and serum cortisol concentration determined the occurrence of heat stress. The goats can meet the donor criteria of rumen fluid in vitro fermentation. It is instructive for the feeding test of heat-stressed goats in subsequent trials. The full names of the nouns are given when they first appear throughout this manuscript.
Comment 33: Table 3
Clarify how you are using asterisks to separate means. Your asterisk model is confusing throughout.
Response 33: We apologize for the confusion caused by the expression in our description of the difference between groups in the same row. The original statement that confused you has been changed to “The data are expressed as the means and SEM. In the same row, values with “*” are significantly different (p < 0.05).” in lines 228-229. The original Table 3 has been changed to the current Table 4.
Comment 34: pH : Based on your SEM, these all should be not significant. Also pH when sampling using suction devices there is significant saliva contamination that needs to be mentioned and often contaminate fermentation parameters
Response 34: Thank the reviewer for the question. We are very sorry for the error in our statistics. We reorganized the original data and used the R language for statistics again. The correct statistical result of pH value will be redisplayed in table 4 (The original Table 3 has been changed to the current Table 4). In order to avoid contamination of rumen contents by saliva, the initial 20 ml of rumen contents were discarded when rumen contents were collected.
Comment 35: Enzymes : Based on your SEM, these all should be not significant
Response 35: We are very sorry for the error in our statistics. We reorganized the original data and used the R language for statistics again. The correct statistical result of Enzyme activity will be redisplayed in the Table 4 (The original Table 3 has been changed to the current Table 4).
Comment 36: Table 5
Avicelase : Based on your SEM, these all should be not significant
Response 36: We are very sorry for the error in our statistics. The original data were reorganized and analyzed using the R language for statistics again. The correct statistical result of SCFAs and enzymes will be redisplayed in Table 6 (The original Table 5 has been changed to the current Table 6.)
Comment 37: Table 6
Specify the title to include digestibility and be specific when you refer to DM, NDF and ADF that you are referring to apparent digestibility coefficients.
Response 37: Thanks for your suggestion. The original title of Table 7 (The original Table 6 has been changed to the current Table 7) has been changed to “The DMI, ADG, AND the apparent digestibility of DM, NDF, and ADF of goats with yeast culture supplementation in the summer.” in lines 259-260.
Comment 38: DMI/ADG : Based on your SEM, these all should be not significant
Response 38: We are very sorry for the error in our statistics. The original data were reorganized and analyzed using the R language for statistics again. The correct statistical result of DMI and ADG will be redisplayed in Table 7 (The original Table 6 has been changed to the current Table 7.)
Comment 39: Ln 140: sheep sheds?
Response 39: Thank the reviewer for the question. “sheep sheds” has been changed to “goat houses” in line 266.
Comment 40: Ln 239-261: This is redundant to what you presented in the introduction, delete. For the discussion, present your observed (not found) results, justify, and compare these to other studies and draw a concluding statement. This needs to be presented throughout your discussion.
Response 40: We appreciate your constructive suggestions. The “Discussion” section has been rewritten according to your suggestion. In addition, since we added the antioxidant capacity and rumen fermentation parameters of goats in the non-heat stress group and the heat stress group in the results, we added the discussion on this result in the first paragraph of the discussion.
Comment 41: Ln 274-291: Same as above. Elaborate more about the effects of yeast in Ph and the reason you did not see an effect on the in-vitro study with the 0.6% addition of yeast
Response 41: Thank the reviewer for the question. In this paper, we explain the reason why pH can be increased from the perspective of yeast culture promoting rumen microbial activity is because yeast culture mainly promotes the growth of lactic acid utilization bacteria by changing the structure of microbial flora in the rumen so that lactic acid produced in the rumen can be utilized and rumen pH can be increased to promote rumen fermentation. The yeast culture needs to reach a certain concentration to promote the activities of rumen microorganisms. We speculated that 0.6% of yeast culture could not promote the utilization of its substrate by lactic acid-utilizing bacteria, so it had no effect on the improvement of pH. This hypothesis needs to be confirmed in our subsequent studies on the effects of yeast cultures on rumen microorganisms.
Comment 42: Ln 293-305: Your apparent digestibility coefficients are quite high relative to the control. This needs to be justified citing other papers where these types of differences having been observed (from 10 to 15% differences)
Response 42: Thank the reviewer for the question. The use of microecological preparations under heat stress can significantly improve the digestibility of DM, NDF, and ADF, which has been reported in previous literature. Please refer to the following literature:
1. Cai, L.Y.; Li, M.; Zhou, S.Y.; Xu, Q.B. The mixture of Saccharomyces cerevisiae and Clostridium butyricum could promote rumen fermentation and improve the growth performance of goats in hot summer. Metabolites 2023, 13, 104.
2. Xue, L.G.; Wang, D.; Zhang, F.Y.; Cai, L.Y. Prophylactic feeding of Clostridium butyricum and Saccharomyces cerevisiae were advantageous in resisting the adverse effects of heat stress on rumen fermentation and growth performance of goats. Animals 2022, 12, 2455.
3. Xue, L.G.; Zhou, S.Y.; Wang, D.; Zhang, F.Y.; Li, J.F.; Cai L.Y. The low dose of Saccharomyces cerevisiae is beneficial for rumen fermentation (both in vivo and in vitro) and the growth performance of heat-stressed goats. Microorganisms 2022, 10,1877.
4. Cai, L.Y.; Hartanto, R.; Xu, Q.B.; Zhang, J.; Qi, D.S. Saccharomyces cerevisiae and Clostridium butyricum could improve B-vitamin production in the rumen and growth performance of heat-stressed goats. Metabolites 2022, 12, 766.
5. Cai, L.Y.; Hartanto, R.; Zhang, J.; Qi, D.S. Clostridium Butyricum improves rumen fermentation and growth performance of heat-stressed goats in vitro and in vivo. Animals 2021, 11, 3261.
5. Cai, L.Y.; Yu, J.K.; Hartanto, R.; Qi, D.S. Dietary supplementation with Saccharomyces cerevisiae, Clostridium butyricum and their combination ameliorate rumen fermentation and growth performance of heat-stressed goats. Animals 2021, 11, 2116.
The main reason for the improvement of rumen digestibility of heat-stressed goats by yeast culture was that it promoted the activity of fibrinolytic bacteria and greatly increased the utilization rate of fiber in feed. In subsequent studies, we will analyze the effects of yeast cultures on rumen microbial composition and function of heat-stressed goats.
Comment 43: Poor description of experimental design and statistical description/utilization of the methods makes this study impossible to replicate and the way presented the data might be completely random (No period replication). Without these the results presented here can be flawed and confounded.
Comment 43: We are very sorry for the poor description of the experimental design and statistical description. We have redescribed the experimental design in the Materials and Methods sections to ensure that readers can clearly understand our experimental process. Moreover, we also re-analyzed and statistically analyzed the original data of the experiment to ensure the accuracy of the data. Thank you again for your valuable comments and suggestions on our articles. Under your guidance, the quality of our articles has been greatly improved.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for Authors
The authors examined the effects of a commercial antioxidant yeast strain at 3 different feeding levels, on both ruminal fermentation (in vitro and in vivo) and animal performance in young goats subjected to heat stress. The data convincingly show that the yeast preparation enhances VFA production, animal growth, and serum antioxidant markers. Some of the authors’ explanations for the effects are a little weak and are not fully backed up by literature citations. In particular, the yeast’s effects on the rumen microbial community remain rather opaque, as no microbial population assessments were carried out. Regardless, there is some useful information here, in a livestock system (goats) that has not received much research attention.
Specific comments:
L13: Change “will” to “can?
L82-83: Presumably HS and NHS refer to heat-stressed and non-heat stressed, respectively? This should be stated explicitly here.
L103: Here and throughout the manuscript, please express centrifugation as relative centrifugal force (x g) rather than rotor speed (rpm), as different rotor diameters and geometries will yield different RCF at the same rotor speed.
L114: Provide literature reference for McDougall buffer.
L113-115: Was a chemical reducing agent added to the buffer to ensure anaerobic conditions?
L129-133: Were goats fed ad libitum, or was the dietary amount restricted?
Table 2: Are the amounts listed on a dry matter basis?
L158: Rather than the “mixed liquor”, do the authors mean that the supernatant from the centrifugation was injected into the GC?
Table 3: “Total SCFA” is a misnomer, because the stated values are the sum of acetic, propionic and butyric acids only, and do not include valeric or the branched-chain (isobutyric, isovaleric and 2-methylbutyric) acids. Thus, the row should be labeled “Sum A+P+B). It would be more useful to substitute molar percentages of end products for the mM concentrations in the Table.
Table 4: Why are there no data for test group 1?
Table 5: Why are there no data for test group 3?
Table 6: Indicate in the heading or by footnote that the values for DM, ADF and NDF are digestibilities. Why are the units for DM digestibility listed as “U/mL”?
L257-264: The paragraph is slightly confusing. It only makes sense if the study (ref. [40]) compared only the effects of heat stress, and did not include a study of yeast supplementation. Is that the case?
L274-288: In both the in vitro (Table 3) and in rumine (Table 4) experiments, pH values upon yeast supplementation were higher than in the controls, despite the fact that VFA concentrations were higher. This is the opposite of what one might expect. The authors are speculating that the yeast are acting by stimulating lactic acid utilizing bacteria. But they are relying on other studies for their interpretation, as they did not quantify microbial community composition, nor did they quantify lactate in the in vitro or in vivo samples. Is it also possible that the yeast preparations have additional buffering capacity?
L295: Do the authors mean DMI here, rather than DM?
L301-307: The reviewer is a bit skeptical here. Firstly, yeast are not known to produce either cellulases or hemicellulases, Can the authors provide citations supporting production of either type of enzyme by this particular commercial yeast strain? Secondly, there is a substantial body of literature indicating that cellulose degradation in the rumen occurs primarily via cell-bound enzymes, and degradation is limited by the availability (accessible surface area) of the cellulose, rather than by the concentration or activity of cellulolytic microbes or enzymes. Consequently, enhancement of cellulose degradation by the treatment is unlikely to be due to more enzyme. The authors should at least mention this.
L307-308: This statement needs a supporting literature citation.
L331: Indicate here that this was the optimum addition amount for the preparation studied (i.e., will vary with strain and with density of the yeast in the preparation.
Minor edits:
L11-13: Please rewrite as complete sentence.
L50: Change “fibrinolytic” to “fibrolytic”.
L107: Incomplete sentence. Please rewrite as, “The above six goats were used as donors of rumen fluid”.
L108: Insert “by stomach tubing” after “collected”.
L113: Do not begin sentences with numerals. Change to “Feed substrate (400 mg),….”
L115: Change “flack” to “flask”.
L201: Here, and also in the footnotes for Tables 4, 5, and 6, change “with no, the same asterisk” to “while no asterisk”.
L208: Insert “the two” after “between”.
Table 5: Change “CMCaes” to “CMCase”.
L232-234: Move this last sentence to the beginning of the paragraph.
Comments on the Quality of English Language
English is generally adequate.
Author Response
Response to Reviewer 2
Dear Reviewers,
We would like to express our sincere gratitude to you for the constructive comments and suggestions, which have greatly helped us improve the quality of the manuscript. Below, we provide point-by-point responses to each of the concerns raised. We hope that our revisions and clarifications adequately address the issues highlighted.
Best wishes,
Shuang Zhang
The authors examined the effects of a commercial antioxidant yeast strain at 3 different feeding levels, on both ruminal fermentation (in vitro and in vivo) and animal performance in young goats subjected to heat stress. The data convincingly show that the yeast preparation enhances VFA production, animal growth, and serum antioxidant markers. Some of the authors' explanations for the effects are a little weak and are not fully backed up by literature citations. In particular, the yeast's effects on the rumen microbial community remain rather opaque, as no microbial population assessments were carried out. Regardless, there is some useful information here, in a livestock system (goats) that has not received much research attention.
Specific comments:
Comment 1: L13: Change “will” to “can?
Response 1: Thanks for your suggestion. Since we think “will" is better than "can," therefore "will" has been retained in the original place.
Comment 2: L82-83: Presumably HS and NHS refer to heat-stressed and non-heat stressed, respectively? This should be stated explicitly here.
Response 2: Thank you for your question. You are correct that NHS and HS refer to non-heat stress and heat-stressed and heat-stressed, respectively. The full names of NHS, HS, and THI abbreviations have been added in lines 80 and 86.
Comment 3: L103: Here and throughout the manuscript, please express centrifugation as relative centrifugal force (x g) rather than rotor speed (rpm), as different rotor diameters and geometries will yield different RCF at the same rotor speed.
Response 3: Thanks for your suggestion. The rpm has been replaced by x g in lines 109, 124, and 151.
Comment 4: L114: Provide literature reference for McDougall buffer.
Response 4: Thank for your reminding. The literature reference has been provided as [30] in line 118.
Comment 5: L113-115: Was a chemical reducing agent added to the buffer to ensure anaerobic conditions?
Response 5: Thank you for your question. No chemical-reducing agent has been added to the buffer to ensure anaerobic conditions.
Comment 6: L129-133: Were goats fed ad libitum, or was the dietary amount restricted?
Response 6: Thank you for your question. The goats were fed twice daily (8:00 h; 17:00 h) with a 1.20 kg/day maintenance diet and had free access to water. Goats were fed in restricted feeding mode.
Comment 7: Table 2: Are the amounts listed on a dry matter basis?
Response 7: Thank you for your question. The amounts have been listed on a dry matter basis, and “Content % (DM basis) has been added in Table 2.
Comment 8: L158: Rather than the “mixed liquor”, do the authors mean that the supernatant from the centrifugation was injected into the GC?
Response 8: Thank you for your question. The answer is “yes!”. The mixed liquor was injected into a Chrompack CP-Wax 52 fused silica column (30 m × 0.53 mm × 1.00 µm) of gas chromatography equipment.
Comment 9: Table 3: “Total SCFA” is a misnomer, because the stated values are the sum of acetic, propionic and butyric acids only, and do not include valeric or the branched-chain (isobutyric, isovaleric and 2-methylbutyric) acids. Thus, the row should be labeled “Sum A+P+B). It would be more useful to substitute molar percentages of end products for the mM concentrations in the Table.
Response 9: Thanks for your question and suggestion. Total SCFA is the sum of acetic propionic acid, a butyric acid. Moreover, it is also feasible and instructive to use units “mmol/L”. Previous studies have provided us with scientific references, and they are listed here:
- Cai, L.Y.; Li, M.; Zhou, S.Y.; Xu, Q.B. The mixture of Saccharomyces cerevisiae and Clostridium butyricum could promote rumen fermentation and improve the growth performance of goats in hot summer. Metabolites 2023, 13, 104.
- Xue, L.G.; Wang, D.; Zhang, F.Y.; Cai, L.Y. Prophylactic feeding of Clostridium butyricum and Saccharomyces cerevisiae were advantageous in resisting the adverse effects of heat stress on rumen fermentation and growth performance of goats. Animals 2022, 12, 2455.
- Xue, L. G., Zhou, S. Y., Wang, D., Zhang, F. Y., Li, J. F., Cai L. Y. * The low dose of Saccharomyces cerevisiae is beneficial for rumen fermentation (both in vivo and in vitro) and the growth performance of heat-stressed goats. Microorganisms 2022, 10: 1877.
- Cai, L.Y.; Hartanto, R.; Xu, Q. B.; Zhang, J.; Qi, D.S. Saccharomyces cerevisiae and Clostridium butyricum could improve B-vitamin production in the rumen and growth performance of heat-stressed goats. Metabolites 2022, 12, 766.
- Cai, L. Y., Hartanto, R., Zhang, J., Qi, D. S*. Clostridium Butyricum improves rumen fermentation and growth performance of heat-stressed goats in vitro and in vivo. Animals 2021, 11, 3261.
- Cai, L.Y.; Yu, J.K.; Hartanto, R.; Qi, D.S. Dietary supplementation with Saccharomyces cerevisiae, Clostridium butyricum and their combination ameliorate rumen fermentation and growth performance of heat-stressed goats. Animals 2021, 11, 2116.
Therefore, original expressions in the text have been maintained.
Comment 10: Table 4: Why are there no data for test group 1?
Response 10: Thanks for your question. We apologize for the confusion caused by a clerical error in our description of test groups. The “test 2 and test 3” in Table 5 have been changed to “test 1 and test 2”. Due to the addition of experimental data, the original Table 4 becomes Table 5.
Comment 11: Table 5: Why are there no data for test group 3?
Response 11: Thanks for your question. There was no test group 3.
Comment 12: Table 6: Indicate in the heading or by footnote that the values for DM, ADF and NDF are digestibilities. Why are the units for DM digestibility listed as “U/mL”?
Response 12: Thanks for your suggestion. In the title of Table 6, that the values for DM, ADF and NDF are digestibilities has been emphasized. We apologize for the confusion caused by a clerical error in the units for DM digestibility and “U/mL”
has been changed to % in Table 7. Due to the addition of experimental data, the original Table 6 becomes Table 7
Comment 13: L257-264: The paragraph is slightly confusing. It only makes sense if the study (ref. [40]) compared only the effects of heat stress, and did not include a study of yeast supplementation. Is that the case?
Response 13: Thanks for your question. Supplementing yeast culture under non-heat stress conditions also has a good antioxidant stress effect, but the antioxidant effect of yeast culture under heat stress was only discussed in this study.
Comment 14: L274-288: In both the in vitro (Table 3) and in rumine (Table 4) experiments, pH values upon yeast supplementation were higher than in the controls, despite the fact that VFA concentrations were higher. This is the opposite of what one might expect. The authors are speculating that the yeast are acting by stimulating lactic acid utilizing bacteria. But they are relying on other studies for their interpretation, as they did not quantify microbial community composition, nor did they quantify lactate in the in vitro or in vivo samples. Is it also possible that the yeast preparations have additional buffering capacity?
Response 14: Thank you for your questions. Since we did not study the effect of yeast culture on rumen microorganisms of heat-stressed goats, we could not investigate
the abundance and function of rumen lactic acid utilizing bacteria and lactic acid-producing bacteria. Therefore, speculative explanations are being made with the help of existing literature. To explain this mechanism of action, rumen microbiome, and rumen metabolome studies must be conducted, which is also the work we will carry out in the future. According to the literature, yeast has no additional buffering capacity. Due to the addition of experimental data, the original Table 3 and 4 becomes Table 4 and 5.
Comment 15: L295: Do the authors mean DMI here, rather than DM?
Response 15: I apologize for confusing you by using abbreviations directly.
DMI is the abbreviation for dry matter intake, and the full name has been added to line 170. DM is the abbreviation for dry matter.
Comment 16: L301-307: The reviewer is a bit skeptical here. Firstly, yeast is not known to produce either cellulases or hemicellulases. Can the authors provide citations supporting the production of either type of enzyme by this particular commercial yeast strain? Secondly, a substantial body of literature indicates cellulose degradation in the rumen occurs primarily via cell-bound enzymes, and degradation is limited by the availability (accessible surface area) of the cellulose rather than by the concentration or activity of cellulolytic microbes or enzymes. Consequently, the enhancement of cellulose degradation by the treatment is unlikely due to more enzymes. The authors should at least mention this.
Response 16: Thanks for your constructive suggestion. Firstly, yeast could produce cellulases! According to Zhang et al. (2016), Saccharomyces cerevisiae Y3 could produce Cellulose endoglucanase (3.27 U/mL), Cellulose exoglucanase (2.22 U/mL), xylanase (4.21 U/Ml). According to Somporn et al. (2013), Candida chanthaburiensis, a new yeast species (KKU-FW10), could produce cellulase (58.24 U/mL). According to Zhang et al. (2007), Aureobasidium pullulans could produce CMCase (4.51 U/mg). There are also a lot of yeast strains that could produce cellulase, which are not listed here. Secondly, our research on the mechanism of cellulase is not in-depth, and the mechanism and mode of action of fiber decomposition have not been confirmed by experiments but only demonstrated according to existing reports. Finally,
Since we did not study the effect of yeast culture on rumen microorganisms of heat-stressed goats, we could not determine the effect of yeast culture on cellulase or digestibility from the composition, function and changes of microorganisms, so we could only infer this. We will conduct further research based on the current findings to clarify this issue.
References:
Zhang, L.L.; Wei. XB; Ma, G.; Zhang, R.J. Screening and identification of cellulase producing strains. Feed Industry 2016, 14, 5. DOI：10.13302/j.cnki.fi.2016.14.010
Somporn, S.; Polson, M.; Sasitorn, J.; Kanit, V.; Sukanda, V.; Jutaporn, S. Candida konsanensis sp. nov. a new yeast species isolated from jasminum adenophyllum in thailand with potentially carboxymethyl cellulase-producing capability. World Journal of Microbiology & Biotechnology 2013, 29,1481-1486. DOI: 10.1007/s11274-013-1313-5.
Zhang, L.; Chi, Z.M. Screening, identification and optimization of fermentation conditions of a cellulase-producing Marine yeast. Journal of ocean university of China: Natural Science, 2007, S2, 8. DOI：CNKI:SUN:QDHY.0.2007-S2-019
Comment 17: L307-308: This statement needs a supporting literature citation.
Response 17: Thanks for your suggestion. This statement is based on our experimental results and is not described in other literature. Therefore original sentence has been revised to “In this study, the activities of cellulolytic enzymes were enhanced in the rumen by the supplementation of yeast culture, which is speculated to be the primary cause of the increased digestibilities of DM, NDF, and ADF.” In lines 310-312.
Comment 18: L331: Indicate here that this was the optimum addition amount for the preparation studied (i.e., will vary with strain and with density of the yeast in the preparation.
Response 18: Thanks for your constructive suggestion. “Therefore, given the problems existing in the feeding stage and specific growth stage of goats, the selection of excellent yeast strains and the optimal addition amount of their cultures based on the targeted test results has a positive guiding significance for the intensive goat culture.” Has been added in lines 353-356.
Minor edits:
Comment 19: L11-13: Please rewrite as complete sentence.
Response 19: Thanks for your constructive suggestion. The “Abstract” section has been rewritten.
Comment 20: L50: Change “fibrinolytic” to “fibrolytic”.
Response 20: Thank you for pointing out our typo. “fibrinolytic” has been changed to “fibrolytic” in line 49.
Comment 21: L107: Incomplete sentence. Please rewrite as, “The above six goats were used as donors of rumen fluid”.
Response 21: Thanks for your constructive suggestion. The original sentence has been revised to “On the day 14, rumen contents were collected from all the goats of HS." in line 112.
Comment 22: L108: Insert “by stomach tubing” after “collected”.
Response 22: Thanks for your suggestion. The original sentence has been revised to “The rumen contents were collected using a negative pressure suction device, which was made by ourselves and consists of two parts: a flexible stomach tube and a vacuum pump (Jin Teng GM-0.33A, Tianjin, China).” in lines 112-114.
Comment 23: L113: Do not begin sentences with numerals. Change to “Feed substrate (400 mg),….”
Response 23: Thanks for your constructive suggestion. The original sentence has been revised to “Next, 400 mg dry feed substrates, and 8.0 mL rumen fluid, 32.0 mL of McDougall’s buffer, and yeast culture were added to a 100 mL flask, and all flasks were pre-warmed using a water bath at 39 °C.” in lines 117-119.
Comment 24: L115: Change “flack” to “flask”.
Response 24: Sentences containing flack has been removed.
Comment 25: L201: Here, and also in the footnotes for Tables 4, 5, and 6, change “with no, the same asterisk” to “while no asterisk”.
Response 25: Thanks for your suggestion. The footnotes for Tables 3-7 have been changed to “The data are expressed as the means and SEM. In the same row, values with “*” are significantly different (p < 0.05).”
Comment 26: L208: Insert “the two” after “between”.
Response 26: Thanks for your suggestion. “the two” has been inserted after “between” in lines 216 and 257.
Comment 27: Table 5: Change “CMCaes” to “CMCase”.
Response 27: Thanks for your suggestion. In Table 6 (Originally it was Table 5), “CMCaes” has been changed to “CMCase."
Comment 28: L232-234: Move this last sentence to the beginning of the paragraph.
Response 28: We appreciate your constructive suggestions. The "Discussion" section has been rewritted according to your suggestion. In addition, since we added the antioxidant capacity and rumen fermentation parameters of goats in the non-heat stress group and the heat stress group in the results, we added the discussion on this result in the first paragraph of the discussion.
Author Response File: Author Response.pdf
