Effects of Dietary Probiotics and Acidifiers on the Production Performance, Colostrum Components, Serum Antioxidant Activity and Hormone Levels, and Gene Expression in Mammary Tissue of Lactating Sows
, Chaohua Xu 1,2,†, Jingjing Wang 2, Chengjun Hu 1,3, Fengjie Ji 1,3, Jiajun Xie 2, Yun Yang 1,3, Xilong Yu 2, Xinping Diao 2,* and Renlong Lv 1,3,*Round 1
Reviewer 1 Report
Wu et al. had studied Effects of Dietary Probiotics and Acidifiers on the Production Performance, Colostrum Components, Serum Antioxidant and Hormone Levels, Gene Expression in Mammary Tissue of Lactating Sows. They concluded that the basal diet was mixed with 200 mL/d probiotics and 0.5% acidifiers could improve production performance, colostrum components, serum antioxidant and hormone levels of lactating sows. This study provide a more theoretical basis for the dietary probiotics and acidifiers application in the diet of lactating sows and to make the sustainable and healthy development of pig farming. However, there are some problem in the structure and description of the manuscript. My major concerns are listed as following.
1. Indicate the breed of sows used in the study.
2. Indicate composition and proportion of acidifier.
3. What is the form of Probiotics? Powder or Liquid?
4. Discussion. The description of Discussion should compare the finding between other publications and the present study, but not describe the the meaning of the detection indicator.
5. Discussion. The results for interactions on detection indicators between Acidifier and Probiotics should be discussed.
6. Conclusion. The conclusion is not accurate. In the present study, the optimal dosage of Acidifier and Probiotics can not be given. In addition, the conclusion is vague, which acidifier and probiotics should be indicated.
Author Response
Dear Reviewer,
Thank you for comments concerning our manuscript entitled Effects of Dietary Probiotics and Acidifiers on the Production Performance, Colostrum Components, Serum Antioxidant Activity and Hormone Levels, Gene Expression in Mammary Tissue of Lactating Sows, ID: animals-2343043.
Comments Wu et al. had studied Effects of Dietary Probiotics and Acidifiers on the Production Performance, Colostrum Components, Serum Antioxidant and Hormone Levels, Gene Expression in Mammary Tissue of Lactating Sows. They concluded that the basal diet was mixed with 200 mL/d probiotics and 0.5% acidifiers could improve production performance, colostrum components, serum antioxidant and hormone levels of lactating sows. This study provide a more theoretical basis for the dietary probiotics and acidifiers application in the diet of lactating sows and to make the sustainable and healthy development of pig farming. However, there are some problem in the structure and description of the manuscript. My major concerns are listed as following.
Response: Thank you. Your comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researcher. We have studied comments carefully and have made a correction which we hope meet with approval. Revised portions are marked in “Track Changes” model in the paper.
Concern 1：Indicate the breed of sows used in the study.
Response: Thank you. And we revised it in the M&M parts.
Gestating and lactating sows: Hainan Local breed sows (Tunchang), second litter, with the same genetic background and birthing date. All sows were artificially inseminated with semen from Duroc boars.
Concern 2：dicate composition and proportion of acidifier.
Response: Thank you. And we revised it in the M&M parts.
Acidifiers were supplied by Chongqing U-Power Biotechnology Co., Ltd. mainly containing 18% phosphate, 12% ammonium formate, 15% citric acid, 15% fumaric acid, 10% lactic acid, and 30% buffer system.
Probiotics, in the liquid form, were provided by Shenzhen Baiaofei Biotechnology Co., LTD. It was a mixture of Lactic acid bacteria, Yeast, and the effective strains and live bacteria numbers were as follows: the content of Lactobacillus fermentum was 1×108 CFU/mL, and the Basidiomycetous Yeast content was 8×107 CFU/mL.
Concern 3：What is the form of Probiotics? Powder or Liquid?
Response: Thank you. And we revised it in the M&M parts.
Probiotics, in the liquid form, were…
Concern 4：Discussion. The description of Discussion should compare the finding between other publications and the present study, but not describe the the meaning of the detection indicator.
Response: Thank you. We have modified some of them in the discussion parts.
Concern 5：Discussion. The results for interactions on detection indicators between Acidifier and Probiotics should be discussed.
Response: Thank you. We could not find references on the combined use of acidifiers and probiotics in livestock and poultry. We can only infer the effect of the combination of the two from the effect of their use alone, and analyze the reasons for the effect.
Concern 6：Conclusion. The conclusion is not accurate. In the present study, the optimal dosage of Acidifier and Probiotics can not be given. In addition, the conclusion is vague, which acidifier and probiotics should be indicated.
Response: Thank you. We have modified them in the conclusion parts.
In this study, the optimum dosage is 200 mL/d probiotics and 0.5% acidifiers mixture added to the diets of lactating sows, which can improve the production performance, colostrum components, serum antioxidant activity and hormone levels, and gene expression in the mammary tissue of lactating sows.
Special thanks to you for your good comments.
Hongzhi Wu,
Author Response File: 
Author Response.docx
Reviewer 2 Report
Dear authors,
The main problem is that you do not mention, what species of lactic bacteria and yeast were used in this study and what were the concentrations of the single components for the acidifier. So I am asking the authors to add more specific information about probiotic and acidifier composition.
In addition, the manuscript has grammatical errors and needs correction
Why did you choose Glyceraldehyde -3-phosphate dehydrogenase as a housekeeping gene? and why only one housekeeping gene?
Since you design the primers, how did you validate them?
line 173_replace the word ''sow'' with the word ''porcine''
In the statistical analysis, did you perform kolmogorov smirnov test, to define if your data follows normal distribution?
In the discussion section you mention acidifiers or probiotics_ As example: Ma et al.(36) found that acidifiers could significantly reduce the serum_ In my opinion you should mention the components of the acidifier or probiotics, not generally acidifiers or probiotics, because different components have different effects.
line 321_ 328 delete the word could, there and everywhere_ Ma et al.(36) found that acidifiers could significantly reduce the serum--> Ma et al. fould that acidifiers significantly reduced the serum.
You conclude that ''In this study, the optimum dosage is 200 mL/d probiotics and 0.5% acidifiers mixture added to the diets of lactating sows.'' In my opinion you should mention the positive interaction effect of the probiotic and acidifier in these doses.
Author Response
Dear Reviewer,
Thank you for comments concerning our manuscript entitled Effects of Dietary Probiotics and Acidifiers on the Production Performance, Colostrum Components, Serum Antioxidant Activity and Hormone Levels, Gene Expression in Mammary Tissue of Lactating Sows, ID: animals-2343043. Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researcher. We have studied comments carefully and have made a correction which we hope meet with approval. Revised portions are marked in “Track Changes” model in the paper.
Concern 1：The main problem is that you do not mention, what species of lactic bacteria and yeast were used in this study and what were the concentrations of the single components for the acidifier. So I am asking the authors to add more specific information about probiotic and acidifier composition.
Response: Thank you. And we revised them as your suggestions.
Acidifiers were supplied by Chongqing U-Power Biotechnology Co., Ltd. mainly containing 18% phosphate, 12% ammonium formate, 15% citric acid, 15% fumaric acid, 10% lactic acid, and 30% buffer system.
Probiotics, in the liquid form, were provided by Shenzhen Baiaofei Biotechnology Co., LTD. It was a mixture of Lactic acid bacteria, Yeast, and the effective strains and live bacteria numbers were as follows: the content of Lactobacillus fermentum was 1×108 CFU/mL, and the Basidiomycetous Yeast content was 8×107 CFU/mL.
Concern 2：In addition, the manuscript has grammatical errors and needs correction
Response: Thank you. We modified the grammatical errors. Hope it meets your requirements.
Concern 3：Why did you choose Glyceraldehyde -3-phosphate dehydrogenase as a housekeeping gene? and why only one housekeeping gene?
Response: Thank you. GAPDH, an enzyme in glycolysis, is widely distributed in cells of various tissues. The GAPDH gene has a highly conserved sequence and is expressed at a high level in almost all tissues. The protein expression level in the same cell or tissue is generally constant, and is not affected by the induction substances such as partial recognition sites and Buddha wave fat. In order to ensure the rigor of the experiment, GAPDH and β-actin two housekeeping genes are required in our laboratory, but GAPDH is used as the housekeeping gene habitually in writing (Figure 1 in this manuscript), because they have the same expression trend. Now we have added the relevant content in the M&M parts, and showed the gene expression results with β-actin as the internal reference in this letter (Figure 2).
Concern 4：Since you design the primers, how did you validate them?
Response: Thank you. We modified the validate in the M&M parts.
RT-qPCR products were cloned into pMD18-T vector (TaKaRa) and sequenced by the Sanger method. The sequencing results were compared with the gene sequences in NCBI, and the genes amplified by the primers in Table 2 were verified as the target genes according to the alignment sequence results.
Figure 1. The relative expression levels of genes in mammary tissue of lactating sows. (A) The data of relative expression levels of PRLP in GC, GP, GA, and GM were 1.00±0.01c, 1.12±0.02b, 1.71±0.02a, 1.72±0.03a, individually; (B) The data of relative expression levels of LALBA from in GC, GP, GA and GM were 1.00±0.01, 0.98±0.02, 0.99±0.02, 1.01±0.03, individually; (C) The data of relative expression levels of AKT1 in GC, GP, GA and GM were 1.00±0.01, 1.02±0.01, 0.97±0.03, 0.98±0.03, individually; (D) The data of relative expression levels of β4GALT1 in GC, GP, GA and GM were 1.00±0.01, 0.97±0.04, 1.03±0.03, 1.02±0.02, individually; (E) The data of relative expression levels of FASN in GC, GP, GA, and GM were 1.00±0.01b, 1.31±0.01a, 1.33±0.02a, 1.32±0.02a, individually; (F) The data of relative expression levels of GLUT1 in GC, GP, GA and GM were 1.00±0.01, 1.01±0.02, 0.97±0.04, 0.99±0.02, individually.
Figure 2. The relative expression levels of genes in mammary tissue of lactating sows. (A) The data of relative expression levels of PRLP in GC, GP, GA, and GM were 1.00±0.01c, 1.30±0.05b, 1.82±0.06a, 1.83±0.04a, individually; (B) The data of relative expression levels of LALBA from in GC, GP, GA and GM were 1.00±0.01, 1.03±0.03, 0.96±0.03, 1.02±0.03, individually; (C) The data of relative expression levels of AKT1 in GC, GP, GA and GM were 1.00±0.01, 1.01±0.01, 0.99±0.02, 0.99±0.02, individually; (D) The data of relative expression levels of β4GALT1 in GC, GP, GA and GM were 1.00±0.01, 0.99±0.01, 1.03±0.04, 1.02±0.03, individually; (E) The data of relative expression levels of FASN in GC, GP, GA, and GM were 1.00±0.01b, 1.40±0.05a, 1.42±0.06a, 1.43±0.04a, individually; (F) The data of relative expression levels of GLUT1 in GC, GP, GA and GM were 1.00±0.01, 1.01±0.03, 0.99±0.03, 1.02±0.03, individually.
Concern 5：line 173_replace the word ''sow'' with the word ''porcine''
Response: Thank you. We have modified it.
Concern 6：In the statistical analysis, did you perform kolmogorov smirnov test, to define if your data follows normal distribution?
Response: Thank you. Yeah, all data in this study was performed with the Kolmogorov-Smirnov test to check if they followed a normal distribution.
Concern 7：In the discussion section you mention acidifiers or probiotics_ As example: Ma et al.(36) found that acidifiers could significantly reduce the serum_ In my opinion you should mention the components of the acidifier or probiotics, not generally acidifiers or probiotics, because different components have different effects.
Response: Thank you. We modified them in the discussion parts.
Concern 8：line 321_ 328 delete the word could, there and everywhere_ Ma et al.(36) found that acidifiers could significantly reduce the serum--> Ma et al. fould that acidifiers significantly reduced the serum.
Response: Thank you. We checked and modified them in the discussion parts.
Concern 9：You conclude that ''In this study, the optimum dosage is 200 mL/d probiotics and 0.5% acidifiers mixture added to the diets of lactating sows.'' In my opinion you should mention the positive interaction effect of the probiotic and acidifier in these doses.
Response: Thank you. We checked and modified them in the conclusion parts.
In this study, the optimum dosage is 200 mL/d probiotics and 0.5% acidifiers mixture added to the diets of lactating sows, which can improve the production performance, colostrum components, serum antioxidant activity and hormone levels, and gene expression in the mammary tissue of lactating sows.
Special thanks to you for your good comments.
Hongzhi Wu,
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
The optimal dosage of Acidifier and Probiotics should be given by conducting a trial designed with 5 inclusion levels of Acidifier and Probiotics at least. In the present study, the optimal dosage of Acidifier and Probiotics can not be given for sows.
Author Response
Dear Reviewer,
Thank you for comments concerning our manuscript entitled Effects of Dietary Probiotics and Acidifiers on the Production Performance, Colostrum Components, Serum Antioxidant Activity and Hormone Levels, Gene Expression in Mammary Tissue of Lactating Sows, ID: animals-2343043. Your comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researcher. We have studied comments carefully and have made a correction which we hope meet with approval. Revised portions are marked in “Track Changes” model in the paper.
Comments: The optimal dosage of Acidifier and Probiotics should be given by conducting a trial designed with 5 inclusion levels of Acidifier and Probiotics at least. In the present study, the optimal dosage of Acidifier and Probiotics can not be given for sows.
Response: Thank you. The experiment adopted a two-factor random design. Two levels of probiotics (0, 200 mL/d), Acidifiers (0, 0.5%), and 200 mL/d probiotics + 0.5% acidifiers were selected to investigate the effects of probiotics and acidifiers on the production performance, colostrum components, serum antioxidant activity and hormones levels, gene expression in mammary tissue of lactating sows. The dosage of probiotics and acidifiers used in this study was the best recommended dosage for the lactating sows, and the GM group is a combination of the two best recommended additions. The conclusions of this experiment are drawn under the conditions of this experiment design. In the future, we will set up a single factor test with 5 or more gradients according to your suggestion to detect the optimal addition amount of each substance, and then set different gradient compatibility based on the combination of the optimal addition amount to determine the optimal combination ratio of the probiotics and acidifiers. And thank you again for you comments.
Special thanks to you for your good comments.
Hongzhi Wu,
Reviewer 2 Report
Dear authors,
Thank you very much for the response. I think the text was corrected to a large extent. Conserning, housekeeping genes. Since you have determined two housekeeping genes, considering, take the geometric mean of the 2 genes and normalize with that.
Best regards
Author Response
Dear Reviewer,
Thank you for comments concerning our manuscript entitled Effects of Dietary Probiotics and Acidifiers on the Production Performance, Colostrum Components, Serum Antioxidant Activity and Hormone Levels, Gene Expression in Mammary Tissue of Lactating Sows, ID: animals-2343043. Your comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researcher. We have studied comments carefully and have made a correction which we hope meet with approval. Revised portions are marked in “Track Changes” model in the paper.
Comments: Thank you very much for the response. I think the text was corrected to a large extent. Conserning, housekeeping genes. Since you have determined two housekeeping genes, considering, take the geometric mean of the 2 genes and normalize with that.
Response: Thank you. We have taken the geometric mean of the 2 housekeeping genes and normalize with that as the following Figure 1. And we have modified it in the corresponding part of the manuscript.
Figure 1. The relative expression levels of genes in mammary tissue of lactating sows. (A) The data of relative expression levels of PRLP in GC, GP, GA, and GM were 1.00±0.01c, 1.21±0.03b, 1.77±0.04a, 1.78±0.03a, individually; (B) The data of relative expression levels of LALBA from in GC, GP, GA, and GM were 1.00±0.01, 1.01±0.02, 0.98±0.02, 1.02±0.03, individually; (C) The data of relative expression levels of AKT1 in GC, GP, GA, and GM were 1.00±0.01, 1.02±0.01, 0.98±0.02, 0.99±0.02, individually; (D) The data of relative expression levels of β4GALT1 in GC, GP, GA and GM were 1.00±0.01, 1.01±0.02, 1.00±0.02, 1.01±0.02, individually; (E) The data of relative expression levels of FASN in GC, GP, GA, and GM were 1.00±0.01b, 1.34±0.03a, 1.38±0.03a, 1.38±0.03a, individually; (F) The data of relative expression levels of GLUT1 in GC, GP, GA and GM were 1.00±0.01, 1.01±0.02, 0.98±0.02, 1.01±0.02, individually.
Special thanks to you for your good comments.
Hongzhi Wu,
Author Response File: Author Response.docx
