Effect of Astragalus membranaceus Root on the Serum Metabolome of Preweaning Dairy Calves

Round 1

Reviewer 1 Report

All comments and suggestions are inserted in attached pdf file.

Comments for author File: Comments.pdf

Author Response

We gratefully thank you for your constructive remarks and useful suggestions, which has exactly enabled us to improve the manuscript. Each suggested revision and comment  was accurately incorporated and considered. Below the comments are response point by point and the revisions are indicated.

1.Comment: Line 86 it is necessary to present if there are some antioxidants presented in feed composition.

Reply: Thanks for your suggestion. The concentrate supplement is a commercial product, which is mainly used to provide protein and energy to calves and without additional antioxidants. In addition, all calves were fed the same basic diet during the experiment, so we believe that it will not affect our experimental results.

2.Comment: Line 87 please note if there is a single dose or not.

Reply: Thanks for your advice. We have revised it at line 87-88 of the revised manuscript.

3.Comment: Line 88 details about AMR properties or composition could be provided? at least total antioxidant capacity?

Reply: Thanks for your advice. We have provided the properties and composition of the superfine powder of AMR at line 89-90 of the revised manuscript.

4. Comment: Line 97 what reference materials and control serum have been used for quality control of the results?

Reply: Thanks for your comment. The quality control of diagnostic assays used the Human Assayed Multi-sera-Level 3 (purchased from Randox Laboratories Ltd. CAT.NO. HE1532, LOT NO. 1079UE) and 10 stability tests were conducted before the formal test.

5. Comment: Line 99 provide the exact name of the kits.

Reply: Thanks for your advice. We have provided it at line 102 of the revised manuscript.

6. Comment: Line106-108 how this quality control sample have been exactly used?

Reply: Thanks for your comment. The quality control samples were used to determine system stability over the entire experiment before testing. We have explained it at line 156-159 of the revised manuscript.

7. Comment: Line 126 what is the version of data based used?

Reply: Thanks for your comment. There is no exact version information of the database Shanghai Applied Protein Technology Co. Ltd used, but as far as we know, it based on the online databases, such as METLIN (https://metlin.scripps.edu/), Bovine Metabolome Database (https://bovinedb.ca/) and MassBank (http://www.massbank.jp/). We have revised it at line 126-127 of the revised manuscript.

8. Comment: Line145 misspell word.

Reply: Thank you so much for your careful check. We have corrected the “deceasing” to “decreasing” at line 146 of the revised manuscript, we feel sorry for our carelessness.

9. Line 275-276

Reply: Thanks for your comment. We are sorry to say we can’t address this comment in the review report.

10. Comment: Line280-281 provide the references.

Reply: Thanks for your advice. We have added references in the corresponding place (at line 287) of the revised manuscript.

11. Comment: Line 285-287 This statement is described for plants. Provide references for animal metabolic pathway.

Reply: We appreciate for your comment. We have added reference for animal metabolic pathway at line 293-296 of the revised manuscript.

12. Comment: Line 318-324 a reformulation is needed.

Reply: Thanks for your valuable suggestion. We have reformulated it at line 327-331 of the revised manuscript.

13. Comment: Line 343-345 There are not enough parameters studied in order to be able to have this conclusion. In discussion is not mentioned at all AMR influence on SOD. Overall antioxidant capacity is a generic status given by a lot of factors. To evaluate it, at least total antioxidant capacity of serum should be performed.

Reply: Thanks for your comment. We have revised it at line 355-357 of the revised manuscript and discussion of SOD was supplemented at line 331-336 of the revised manuscript.

14. Comment: Line 356 Considering all above observations, the conclusion should be reformulated regarding influence on antioxidant capacity.

Reply: Thanks for your valuable advice. We have reformulated the conclusion at line 369-381 of the revised manuscript.

Thanks again for your kind suggestions and comments, which helped a lot in improving the manuscript.

Reviewer 2 Report

The work demonstrates the effect of AMR supplementation on the serum metabolite profiles of preweaning dairy claves. Advanced metabolomics analysis in combination with chemometric methods were applied for data evaluation and interpretation. The manuscript is well prepared and clearly written with appropriate English language level. In my humble opinion, however, several minor points as indicated below should be additionally clarified.

• Line 85-87: The number of experimental calves used in this study seemed to be very limited. A recommendation should be given in order to validate the positive effects of AMR supplementation found in this research.
• Line 87: How did the authors decide the suitable quantity (20 g/d) and feeding period (14 d) of AMR supplementation in this work? What would be the hypothesis for the application of these AMR dose and duration?
• Line 300/320/352 : Could the authors speculate these interesting health positive effects of AMR supplement on the mature cows?

Author Response

We gratefully thank you for your constructive remarks and useful suggestions, which has exactly enabled us to improve the manuscript. Each suggested revision and comment was accurately incorporated and considered. Below the comments are response point by point. 

1.Comment: Line 85-87 The number of experimental calves used in this study seemed to be very limited. A recommendation should be given in order to validate the positive effects of AMR supplementation found in this research.

Reply: Thanks for your comment. The number of experimental calves designed in this work was according to several published articles. For example, a study explored the effect of Astragalus root extract on the average daily gain, immunity, antioxidant status and ruminal microbiota of early weaned yak calves, the authors selected six weaned yak calves per group [1]. In another article authors selected eight weaned lambs per group to investigate the effects of dietary Astragalus membranaceus root supplementation on growth performance, blood metabolites, rumen fermentation, immune responses and antioxidant status [2]. One study used six male Tibetan sheep per group to examine the effect of Astragalus membranaceus root supplementation on DM intake, growth performance, rumen fermentation and immunity [3]. Based on these researches, we think eight calves is enough to validate the positive effects of AMR supplementation found in this research. We also intend to carry out an experiment which involved more calves for further research.

[1] Wei, H.; Ding, L.; Wang, X.; Yan, Q.; Jiang, C.; Hu, C.; Wang, G.; Zhou, Y.; Henkin, Z.; Degen, A.A. Astragalus root extract improved average daily gain, immunity, antioxidant status and ruminal microbiota of early weaned yak calves. J. Sci. Food. Agric. 2021, 101, 82-90. https://doi.org/10.1002/jsfa.10617.

[2] Zhong, R.Z.; Yu, M.; Liu, H.W.; Sun, H.X.; Cao, Y.; Zhou, D.W. Effects of dietary Astragalus polysaccharide and Astragalus membranaceus root supplementation on growth performance, rumen fermentation, immune responses, and antioxidant status of lambs. Anim. Feed Sci. Technol. 2012, 174, 60-67. https://doi.org/10.1016/j.anifeedsci.2012.02.013.

[3] Wang, X.J.; Ding, L.M.; Wei, H.Y.; Jiang, C.X.; Yan, Q.; Hu, C.S.; Jia, G.X.; Zhou, Y.Q.; Henkin, Z.; Degen, A.A. Astragalus membranaceus root supplementation improves average daily gain, rumen fermentation, serum immunity and antioxidant indices of Tibetan sheep. Animal 2021, 15, 100061. https://doi.org/10.1016/j.animal.2020.100061.

2. Comment: Line 87 How did the authors decide the suitable quantity (20 g/d) and feeding period (14 d) of AMR supplementation in this work? What would be the hypothesis for the application of these AMR dose and duration?

Reply: Thanks for your rigorous consideration. The feeding amount of the superfine powder of AMR was according to our previous study and the study of Wu et al. We found that adding 10-50 g/d Astragalus Polysaccharides (APS) could significantly improve the antioxidant capacity of lactating dairy cows [1]. Also, Wu et al found added 20g/d APS modulated the physiological responses and alleviated the performance losses of transported heifers caused by long-distance transportation [2]. The AMR used in this work accounts for more than 20% APS. Therefore, combing with the above researches, the content of APS in AMR and considering the difference of body weight among lactating dairy cow(600-800 kg), heifers(315±6 kg) and preweaning calves in our experiment(56±4 kg), we believe that the feeding amount of 20 g/d is moderate. For the feeding period, the main purpose of this work was to determine whether the short-period supplementation of AMR was helpful to improve preweaning calves‘ health and provide reference for future research, so we think 14 d feeding period is suitable.

[1] Shen, Y.J.; Zhou, J.W.; Wang, B.; Li, J.S.; Shen, L.H.; Yu, S.M.; Cao, S.Z. Effect of Astragalus polysaccharides on the Activity of Antioxidant Enzyme in Lactating Holstein cows. Natural Product Research and Development 2014, 26, 244-247, https://doi.org/10.16333/j.1001-6880.2014.02.007. (in Chinese)

[2] Wu, X.; Cao, N.; Zhou, Z.; Beck, P.A.; Wu, H.; Meng, Q. Effect of anti-inflammatory compounds or antibiotic administration on receiving performance and physiological responses of transported heifers. J. Anim. Sci. 2020, 98, skaa036. https://doi.org/10.1093/jas/skaa036.

3. Comment: Line 300/320/352 Could the authors speculate these interesting health positive effects of AMR supplement on the mature cows?

Reply: Thanks for your comment. As a traditional Chinese medicine, Astragalus membranaceus has broad bioactivities and was used to treat diabetes, colitis and other disease in recent years. In livestocks, varieties of studies have found Astragalus membranaceus was benefcial to improve growth performance, antioxidant capacity and immunity. In this work, we have proved AMR was able to enhance calves’ antioxidant and anti-inflammatory capacity from the perspective of serum metabolites. For mature cows, studies have found dairy cows in transition period experienced huge transition from late pregnancy to early lactation, the physiological conditions, nutritional status, and metabolic responses of cows undergo marked changes, the energy demand during this period is higher than the intake, thus resulting in a negative energy balance [1]. The development of negative energy balance will further exacerbate postpartum oxidative stress response and inflammation response [2,3]. Our previous study also found transition dairy cows experienced drastic metabolic changes and such changes reflected the cows were under metabolic stress [4]. Based on these researches we speculate that AMR has the potential to be used in the transition dairy cows and plays a role in alleviating such stress status via its antioxidant and anti-inflammatory capacity, which may be helpful to realize a healthy transition for perinatal cows.

[1] Esposito, G.; Irons, P.C.; Webb, E.C.; Chapwanya, A. Interactions between negative energy balance, metabolic diseases, uterine health and immune response in transition dairy cows. Anim. Reprod. Sci. 2014, 144, 60-71. https://doi.org/ 10.1016/j.anireprosci.2013.11.007.

[2] Bell, A.W. Regulation of organic nutrient metabolism during transition from late pregnancy to early lactation. J. Anim. Sci. 1995, 73, 2804-2819. https://doi.org/10.2527/1995.7392804x.

[3] Overton, T.R.; McArt, J.A.A.; Nydam, D.V. A 100-Year Review: Metabolic health indicators and management of dairy cattle. J. Dairy Sci. 2017, 100, 10398-10417.

[4] Luo, Z.Z.; Shen, L.H.; Jiang, J.; Huang, Y.X.; Bai, L.P.; Yu, S.M.; Yao, X.P.; Ren, Z.H.; Yang, Y.X.; Cao, S.Z. Plasma metabolite changes in dairy cows during parturition identified using untargeted metabolomics. J. Dairy Sci. 2019, 102, 4639-4650. https://doi.org/10.3168/jds.2018-15601.

Thanks again for your kind suggestions and comments, which helped a lot in improving the manuscript.

 

 

Round 2

Reviewer 1 Report

After evaluation of revised manuscript I recommend it for publishing.

Author Response

Thanks again for your valuable comments and suggestions.