Effects of Dietary Folic Acid Supplementation on Growth Performance and Immune Parameters in Weanling Piglets

Round 1

Reviewer 1 Report

The work focuses on effects of dietary folic acid supplementation on growth performance and immune parameters in weanling piglets. In general, the manuscript is well-written. However, some of the following points must be assessed and corrected. Please take into account all of the suggestions provided below.

1.     Line 33: Write the recommendation and conclusion with more accuracy.

2.     The introduction should explain why the authors applied the current selected levels of FA in the trial and

3.     Why were the immunological state and growth performance of weanling piglets taken into consideration? in the introduction, please clarify them.

4.     Line 75: Please provide the initial body weight±SD of the piglets.

5.     Why do the FA levels not seem to be consistent? Why is 3.0 mg/kg suddenly higher than 0.33 mg/kg?, please clarify

6.     How were FA levels incorporated into the diets? Explain these, please

7.     What underlying processes result in the immunological and growth alterations in piglets fed FA levels?  The underlying causes of these changes must be thoroughly discussed in the discussion section.   

The work focuses on effects of dietary folic acid supplementation on growth performance and immune parameters in weanling piglets. In general, the manuscript is well-written. However, some of the following points must be assessed and corrected. Please take into account all of the suggestions provided below.

1.     Line 33: Write the recommendation and conclusion with more accuracy.

2.     The introduction should explain why the authors applied the current selected levels of FA in the trial and

3.     Why were the immunological state and growth performance of weanling piglets taken into consideration? in the introduction, please clarify them.

4.     Line 75: Please provide the initial body weight±SD of the piglets.

5.     Why do the FA levels not seem to be consistent? Why is 3.0 mg/kg suddenly higher than 0.33 mg/kg?, please clarify

6.     How were FA levels incorporated into the diets? Explain these

7.     What underlying processes result in the immunological and growth alterations in piglets fed FA levels?  The underlying causes of these changes must be thoroughly discussed in the discussion section.   

Author Response

Dear Sr.

   We appreciate your review and suggestions of our manuscripts. We take into account all of the suggestions you provided. I corrected and revised some points. I would be happy to reply to your suggestions one by one, as shown below.

Suggestion 1.     Line 33: Write the recommendation and conclusion with more accuracy.

We revised it to be: In conclusion, an adequate dietary FA level of 0.66 mg/kg or more is necessary to improve the immunity and growth performance of weaned piglets from 21 to 45 days of age. The suitable dietary FA level for feed efficiency was 5.83 mg/kg in weaned piglets weighing between 7 to 11 kg.

Suggestion 2.     The introduction should explain why the authors applied the current selected levels of FA in the trial and

We have added explanations in the manuscript. Line 69-73: Depending on the magnitude of the reduction in feed intake, and given the importance of folate, high doses of folic acid (FA), as 3.0 -9.0 mg/kg were often added to piglet diets in production. However, few studies have reported the exact effect of dietary folic acid supplementation on promoting growth and improving immunity in weaned piglets.

Suggestion 3.     Why were the immunological state and growth performance of weanling piglets taken into consideration? in the introduction, please clarify them.

We have added explanations in the manuscript. Line 66-69: After weaning, the supply of milk-derived folic acid stops and feed intake decreases, resulting in insufficient folic acid in piglets, which may aggravate the immune stress of weaned piglets (Liu et al., 2013; Wang et al., 2021). Line 71-73: However, few studies have reported the exact effect of dietary folic acid supplementation on promoting growth and improving immunity in weaned piglets.

Suggestion 4.     Line 75: Please provide the initial body weight±SD of the piglets.

We revised as: Line 89-91： The piglets (with an average initial body weight of 6.93 ± 0.07 kg) were equally assigned into 36 cages with a similar initial body weight of 3 piglets per cage.

Suggestion 5.     Why do the FA levels not seem to be consistent? Why is 3.0 mg/kg suddenly higher than 0.33 mg/kg? please clarify

We have added explanations in the manuscript. Line 91-97: FA 0.3 mg/kg was the level recommended by NRC (2012). In production, based on the different understanding of the possible important functions of folic acid supplementation, the dosage of folic acid supplementation in the diet of weaned piglets is often much higher than the NRC recommended amount, usually between 3.0 and 9.0 mg/kg. Therefore, in order to fully determine the possible effects of folic acid supplementation, we set the above higher folate addition levels and increased the high level of folic acid addition to 15.0 mg/kg.

Suggestion 6.     How were FA levels incorporated into the diets? Explain these, please

We would Explain as: The observed values of dietary FA that were lower than the calculated values could potentially be attributed to the expenses associated with FA during feed processing, as well as the determination method utilized (Molloy and Scott, 1997; Guay et al., 2002). However, given the similar gradient and high correlation between the observed and calculated values of dietary FA, it can be concluded that the experimental treatments employed in this study were meaningful and valid. (Line 482-487)

Suggestion 7.     What underlying processes result in the immunological and growth alterations in piglets fed FA levels?  The underlying causes of these changes must be thoroughly discussed in the discussion section.   

We have done our best to revised it. In the discussion section we discussed the underlying causes of these changes as well as we can.

Thank you for your review and suggestions again.

Kind regards,

Dr. Gao

11.14.2023

Reviewer 2 Report

The manuscript "Effects of dietary folic acid supplementation on growth performance and immune parameters in weanling piglets" can be considered to be published on Agriculture if an intensive revision is made. Please find my comments as below:

- Reference format must be followed the journal guidance

- L60: the author investigate the effects of dietary fatty acid (FA) instead of folic acid

- L61: "at varying levels" is unnecessary

- Section 2.1: Number or ID of  of the approval must be provided

- L76: Please detail why author select these dose of FA, particularly the level of FA jumped from 0.3 to 3 mg/kg, then 15 mg.

- L79: address of the company needs to be  included

- L81: Please explain why the levels of vitamin were at 200% of NRC requirement

- L82: basal diet or experimental diets?

- Unit of calculated nutrients must be added (Table 1)

- L96: Feed intake, not feed disappearance

- Table 2, the parameters needs to be grouped in the same period. Current presentation is hard to follow.

- The diets must be presented as the amount of FA supplementation, instead of total dietary FA. There is inconsistent in FA level. The basal diet contain 0.35 mg/kg, however, when added 3.0, 6.0, 9.0 or 15.0 mg/kg FA, the total FA were 2.19, 5.18, 7.51 and 10.81 mg/kg. They were even much less than added amounts.

- The supplementation of FA did not influence growth performance parameters, except G/F with the diet supplemented with 9 mg/kg FA.

- In term of immune functions, the level of FA only improved IFN-γ, and there was no different among supplementation levels, but in the conclusion, the authors concluded 0.66 mg (corresponded to 0.3 mg supplementation) was needed for period from 21 to 45 days of age.

- In the conclusion, the authors mentioned FA level of 6.82 and 5.42 mg/kg but these levels did not appear in any sections of the paper, both in FA supplementation or total dietary levels.

- L353: authors concluded "a dietary FA level of 6.82 mg/kg 353 from 21 to 28 days of age" but the results from Table 2 did not support this. There was no significant effects of FA level on all parameters during 21 to 28 days of age.

Author Response

Dear Sr.

   We appreciate your review and suggestions of our manuscripts. We take into account all of the suggestions you provided. I corrected and revised some points. I would be happy to reply to your suggestions one by one, as shown below.

     Reference format must be followed the journal guidance

 We have revised reference format following the journal guidance.

- L60: the author investigate the effects of dietary fatty acid (FA) instead of folic acid

We corrected it.

- L61: "at varying levels" is unnecessary

We corrected and deleted it

- Section 2.1: Number or ID of the approval must be provided

We provided as: All experiments involving swine were carried out in strict accordance with the Guide for the Care and Use of Laboratory Animals Monitoring Committee of Sichuan Province, China, and the protocols approved by the Sichuan Agricultural University Animal Ethical and Welfare Committee (Approval NO. 20180021).

- L76: Please detail why author select these doses of FA, particularly the level of FA jumped from 0.3 to 3 mg/kg, then 15 mg.

We have added explanations in the manuscript. Line 91-97: FA 0.3 mg/kg was the level recommended by NRC (2012). In production, based on the different understanding of the possible important functions of folic acid supplementation, the dosage of folic acid supplementation in the diet of weaned piglets is often much higher than the NRC recommended amount, usually between 3.0 and 9.0 mg/kg. Therefore, in order to fully determine the possible effects of folic acid supplementation, we set the above higher folate addition levels and increased the high level of folic acid addition to 15.0 mg/kg.

- L79: address of the company needs to be included

We have revised as Line 104-105: FA supplied by Bayer Sichuan Animal Health Co., Ltd (at No. 189, Section 1, Changcheng Road, Shuangliu District, Chengdu).

- L81: Please explain why the levels of vitamin were at 200% of NRC requirement

We have added explanations in the manuscript. Line 108-109: to avoid other vitamin dosage restrictions becoming contributing factors.

- L82: basal diet or experimental diets?

We corrected it as basal diet.

- Unit of calculated nutrients must be added (Table 1)

We added the Unit of calculated nutrients in Table 1.

- L96: Feed intake, not feed disappearance

We corrected it as: Feed intake

- Table 2, the parameters need to be grouped in the same period. Current presentation is hard to follow.

We corrected it as:

, instead of total dietary FA. There is inconsistent in FA level. The basal diet contain 0.35 mg/kg, however, when added 3.0, 6.0, 9.0 or 15.0 mg/kg FA, the total FA were 2.19, 5.18, 7.51 and 10.81 mg/kg. They were even much less than added amounts.

We revised it, and the diets were presented as the amount of FA supplementation.

We would Explain as: The observed values of dietary FA that were lower than the calculated values could potentially be attributed to the expenses associated with FA during feed processing, as well as the determination method utilized (Molloy and Scott, 1997; Guay et al., 2002). However, given the similar gradient and high correlation between the observed and calculated values of dietary FA, it can be concluded that the experimental treatments employed in this study were meaningful and valid.

- The supplementation of FA did not influence growth performance parameters, except G/F with the diet supplemented with 9 mg/kg FA.

We would Explain as: There were significant differences of G/F from 29 d to 45 d of age (p < 0.01) and from 21 d to 45 d of age (p < 0.01) among the groups. Compared with the control group, the weanling piglets fed diets supplemented FA 3.00 mg/kg or 9.00 mg/kg had better G/F from 29 d to 45 d of age and G/F from 21 d to 45 d of age respectively (p < 0.05).

- In term of immune functions, the level of FA only improved IFN-γ, and there was no different among supplementation levels, but in the conclusion, the authors concluded 0.66 mg (corresponded to 0.3 mg supplementation) was needed for period from 21 to 45 days of age.

We revised it.

- In the conclusion, the authors mentioned FA level of 6.82 and 5.42 mg/kg but these levels did not appear in any sections of the paper, both in FA supplementation or total dietary levels.

We revised: The present study revealed significant quadratic effects of dietary FA levels on the growth performance of weanling piglets. The results indicated that to optimize average daily gain (ADG) or feed efficiency, dietary FA levels of 6.58 or 6.82 mg/kg were needed from 21 d to 28 d of age, and 5.78 or 5.42 mg/kg were needed from 29 d to 45 d of age, respectively. From the quadratic regression curve with the highest confidence (R²=0.72, p < 0.01), the dietary FA level needed to optimize G/F was 5.83 mg/kg from 21 d to 45 d of age. Therefore, we recommend that the suitable dietary FA level for feed efficiency was 5.83 mg/kg in weaned piglets weighing between 7 to 11 kg (from 21 d to 45 d of age) in this study. Notably, these levels were higher than the current recommendation of 0.3 mg FA/kg diet for 5-10 kg piglets by NRC (2012) [8]. Results of our successive trials (data were not given) indicated that the level of FA supplemented into diet would be declined following with the growth of piglet.

- L353: authors concluded "a dietary FA level of 6.82 mg/kg from 21 to 28 days of age" but the results from Table 2 did not support this. There were no significant effects of FA level on all parameters during 21 to 28 days of age.

We revised the Conclusions: In summary, our study demonstrates that a sufficient dietary FA level of 0.66 mg/kg or more is essential for both growth performance and immune function of weaned piglets from 21 to 45 days of age. Moreover, to optimize feed efficiency, the dietary FA level of 5.83 mg/kg was recommended for weaned piglets weighing between 7 to 11 kg. These findings have important implications for the development of optimal nutritional strategies for weaned piglets, as well as for the promotion of their health and well-being.

Thank you for your review and suggestions again.

Kind regards,

Dr. Gao

11.14.2023

Reviewer 3 Report

This was a nicely presented manuscript studying the effect of FA on piglet performance and immunity.

My specific concerns/suggestions are listed below:

L28 change "serum" to "peripheral blood"

L31 perhaps "optimal" would be a better work than "suitable"?

L31-33 needs to be re-written, it is unclear.

L773-74 please include the level of FA in the sow diet.

2.3-2.4 why weren't all of the piglets bled? If would have provided more information and possibly have brought the P values to significance.

2.5 much of the information in this section and associated results aren't contained in the abstract.

Table 2 vs Table 3. I am confused by Table 3 because significant differences were not found between the FA treatments groups shown in Table 2 thus I am not sure why the data from Table 3 is useful? If it remains in the manuscript a better explanation for what was used in the calculation should be included.

L 186 suggest changing "bigger" to "greater"

Some references describing actual immune activity rather than just cell numbers or cytokine concentrations would be interesting.

There are some small corrections to be made but overall the manuscript was very well-written.

Author Response

Dear Sr.

   We appreciate your review and suggestions of our manuscripts. We take into account all of the suggestions you provided. I corrected and revised some points. I would be happy to reply to your suggestions one by one, as shown below.

L28 change "serum" to "peripheral blood"

We corrected it following your suggestion.  

L31 perhaps "optimal" would be a better work than "suitable"?

We corrected it following your suggestion.

L31-33 needs to be re-written, it is unclear.

We revised it to be:  In conclusion, an adequate dietary FA level of 0.66 mg/kg or more is necessary to improve the immunity and growth performance of weaned piglets from 21 to 45 days of age. The suitable dietary FA level for feed efficiency was 5.83 mg/kg in weaned piglets weighing between 7 to 11 kg.  

L73-74 please include the level of FA in the sow diet.

We corrected it following your suggestion.

2.3-2.4 why weren't all of the piglets bled? If would have provided more information and possibly have brought the P values to significance.

The suggestion was good. But we were not able to do it in the trial due to constraints.

2.5 much of the information in this section and associated results aren't contained in the abstract.

We corrected following your suggestion. We added the information into the abstract. We revised it to be: Moreover, FA addition increased the serum IFN-γ level (p < 0.05), and tended to reduce the serum TNF-α/IL-4 (p < 0.1) and IgG (p < 0.1), but had no significant effect on serum IL-4, TNF-α, and nitric oxide.  

Table 2 vs Table 3. I am confused by Table 3 because significant differences were not found between the FA treatments groups shown in Table 2 thus I am not sure why the data from Table 3 is useful? If it remains in the manuscript a better explanation for what was used in the calculation should be included.

The present study revealed significant quadratic effects of dietary FA levels on the growth performance of weanling piglets. The results indicated that to optimize average daily gain (ADG) or feed efficiency, dietary FA levels of 6.58 or 6.82 mg/kg were needed from 21 d to 28 d of age, and 5.78 or 5.42 mg/kg were needed from 29 d to 45 d of age, respectively. From the quadratic regression curve with the highest confidence (R²=0.72, p < 0.01), the dietary FA level needed to optimize G/F was 5.83 mg/kg from 21 d to 45 d of age. Therefore, we recommend that the suitable dietary FA level for feed efficiency was 5.83 mg/kg in weaned piglets weighing between 7 to 11 kg (from 21 d to 45 d of age) in this study. Notably, these levels were higher than the current recommendation of 0.3 mg FA/kg diet for 5-10 kg piglets by NRC (2012) [8]. Results of our successive trials (data were not given) indicated that the level of FA supplemented into diet would be declined following with the growth of piglet.

L 186 suggest changing "bigger" to "greater"

We corrected it following your suggestion.

Thank you for your review and suggestions again.

Kind regards,  

Dr. Gao  

11.14.2023

Round 2

Reviewer 2 Report

The authors MUST change all FA levels mentioned in abstracts (example L27, 28), as well as entire manuscript to the supplementation levels which presented in the Method section (L112-114) in the tables.

The second column of Table 1 is NOT basal diet.

The term: Groups supplemented with different amounts in all table have to be changed to FA supplementation or something else similar

Table 3: Quadratic regressions of performance can only be generated for parameter which was found to be significant in table 2.

Conclusions were not reflected the resulted.

Author Response

Dear Sr.

   We appreciate your review and suggestions of our manuscripts. We take into account all of the suggestions you provided. We corrected and revised some points. We would be happy to reply to your suggestions one by one, as shown below.

Suggestion1：The authors MUST change all FA levels mentioned in abstracts (example L27, 28), as well as entire manuscript to the supplementation levels which presented in the Method section (L112-114) in the tables.

We corrected it following your suggestion.

Suggestion2：The second column of Table 1 is NOT basal diet.

We corrected it.

Suggestion3：The term: Groups supplemented with different amounts in all table have to be changed to FA supplementation or something else similar

We corrected it following your suggestion.

Suggestion4：Table 3: Quadratic regressions of performance can only be generated for parameter which was found to be significant in table 2.

Following your suggestion, we calculated again the Quadratic regressions of performance due to supplemented FA. The quadratic effects of supplemented FA levels on the growth performance (including G/F from 29 d to 45 d of age, and G/F from 21 d to 45 d of age) were significant (p < 0.01). The supplemented FA needed to optimize G/F were 7.00 mg/kg from 29 d to 45 d of age, or 7.55 mg/kg from 21 d to 45 d of age respectively. There are still discrepancies between these regression results and the measured results in Table 2. We have carefully considered and heeded the advice of you and reviewer 3 by removing Table 3 from the original manuscript.

Suggestion5：Conclusions were not reflected the resulted.

We corrected it as: In summary, our study demonstrates that FA at 0.30 mg/kg or more need to be added to the diet to improve the immunity and growth performance of weaned piglets from 21 to 45 days of age. The group supplemented with folic acid 9.00 mg/kg achieved the greatest ADG and the best feed efficiency in weaned piglets weighing between 7 to 11 kg in this study. These findings have important implications for the development of optimal nutritional strategies for weaned piglets, as well as for the promotion of their health and well-being.

Thank you very much.

Kind regards,

Dr. Gao

11.23.2023