Dietary probiotic and synbiotic supplementation starting from maternal gestation improves muscular lipid metabolism in offspring piglets by reshaping colonic microbiota and metabolites

ABSTRACT Probiotics and synbiotics have been intensively used in animal husbandry due to their advantageous roles in animals’ health. However, there is a paucity of research on probiotic and synbiotic supplementation from maternal gestation to the postnatal growing phases of offspring piglets. Thus, we assessed the effects of dietary supplementation of these two additives to sows and offspring piglets on skeletal muscle and body metabolism, colonic microbiota composition, and metabolite profiles of offspring piglets. Pregnant Bama mini-pigs and their offspring piglets (after weaning) were fed either a basal diet or a basal diet supplemented with antibiotics, probiotics, or synbiotics. At 65, 95, and 125 days old, eight pigs per group were euthanized and sampled for analyses. Probiotics increased the intramuscular fat content in the psoas major muscle (PMM) at 95 days old, polyunsaturated fatty acid (PUFA) and n-3 PUFA levels in the longissimus dorsi muscle (LDM) at 65 days old, C16:1 level in the LDM at 125 days old, and upregulated ATGL, CPT-1, and HSL expressions in the PMM at 65 days old. Synbiotics increased the plasma HDL-C level at 65 days old and TC level at 65 and 125 days old and upregulated the CPT-1 expression in the PMM at 125 days old. In addition, probiotics and synbiotics increased the plasma levels of HDL-C at 65 days old, CHE at 95 days old, and LDL-C at 125 days old, while decreasing the C18:1n9t level in the PMM at 65 days old and the plasma levels of GLU, LDH, and TG at 95 days old. Microbiome analysis showed that probiotic and synbiotic supplementation increased colonic Actinobacteria, Firmicutes, Verrucomicrobia, Faecalibacterium, Pseudobutyrivibrio, and Turicibacter abundances. However, antibiotic supplementation decreased colonic Actinobacteria, Bacteroidetes, Prevotella, and Unclassified_Lachnospiraceae abundances. Furthermore, probiotic and synbiotic supplementation was associated with alterations in 8, 7, and 10 differential metabolites at three different age stages. Both microbiome and metabolome analyses showed that the differential metabolic pathways were associated with carbohydrate, amino acid, and lipid metabolism. However, antibiotic supplementation increased the C18:1n9t level in the PMM at 65 days old and xenobiotic biodegradation and metabolism at 125 days old. In conclusion, sow-offspring’s diets supplemented with these two additives showed conducive effects on meat flavor, nutritional composition of skeletal muscles, and body metabolism, which may be associated with the reshaping of colonic microbiota and metabolites. However, antibiotic supplementation has negative effects on colonic microbiota composition and fatty acid composition in the PMM. IMPORTANCE The integral sow-offspring probiotic and synbiotic supplementation improves the meat flavor and the fatty acid composition of the LDM to some extent. Sow-offspring probiotic and synbiotic supplementation increases the colonic beneficial bacteria (including Firmicutes, Verrucomicrobia, Actinobacteria, Faecalibacterium, Turicibacter, and Pseudobutyrivibrio) and alters the colonic metabolite profiles, such as guanidoacetic acid, beta-sitosterol, inosine, cellobiose, indole, and polyamine. Antibiotic supplementation in sow-offspring's diets decreases several beneficial bacteria (including Bacteroidetes, Actinobacteria, Unclassified_Lachnospiraceae, and Prevotella) and has a favorable effect on improving the fatty acid composition of the LDM to some extent, while presenting the opposite effect on the PMM.

Probiotics and synbiotics, as common feed additives in livestock production, are widely used to enhance the animals' health and performance.The authors present an interesting question in this paper: Can dietary probiotics and synbiotics supplementation improve muscular lipid metabolism in offspring piglets by reshaping colonic microbiota and metabolites?There is a large amount of presented but not seated in any statements or data that would make the work meaningfully applicable to swine production.My detailed assessment as follows: 1. Please provide the information of the probiotics and synbiotics products used in the present study, e.g., purity, source, composition, etc. 2. Please change [12000] to [12,000], use commas when numbers exceed four digits.Please check and correct the full text.3. What amounts of feed were provided daily?Restricted or ad libitum? 4. Feed intake and body weight must be provided?5. Given the nature of the dietary intervention (probiotics and synbiotics supplementation) and the expectation of major changes in microbiota composition and metabolism, measuring small intestinal microbiota seems critical in this study.Why only colonic microbiota was measured?6.Which ANOVA was used for statistical analysis?One/two way? 7. How about the growth performance of the offspring piglets after dietary supplementation?What about mortality rate during the experimental period from 35 to 125 days?8. Line 100 "Sows were housed in individual pens (2.2 × 0.6 m) during gestation" in the pigging of the trail what is the number of sow month gestation?9. How long did the sows receive feed with experimental supplements?Whole pregnancy or less?It needs to be added.10.Lines 113-114: although the supplier, ingredient, and feeding method of these additives were consistent with the previous study (12), it is worth giving the name of the probiotic and prebiotic here.11.What were the criteria for eight piglets for sampling (L118-119)?12.What about the environmental conditions surrounding the sows and piglets?For example, season, temperature, relative Detailed comments: 1.What amounts of feed were provided daily?Restricted or ad libitum?Response: The sows were fed with 0.8, 1.0, 1.2, 1.5, and 2.0 kg of pregnancy diets from days 1-15, 16-30, 31-75, 76-90, and 91-105 of pregnancy, respectively; fed with 1 kg of pregnancy feed diets a week before parturition and ad libitum access after three days of parturition; and fed with 2.4 kg of lactation diets until weaning.The offspring pigs had ad libitum to feed at all times.We have added these details in the revised manuscript (L117-121 and L129-130).
2. What about the sexual distribution of the experimental pigs?Male/female?Provide more details.Response: One male and one female piglet close to the average BW per litter were selected and transferred to the nursery house for the subsequent feeding trial.We have added this detail in the revised manuscript (L124-125).
3. Was there any vaccination program for sows and offspring piglets during the trial?Response: Feeding and management (including vaccination program) for sows and offspring piglets were carried out according to the standard operations of commercial pig farms.We have added these in the revised manuscript (L143-415).
4. What were the criteria for eight piglets for sampling (L118-119)?Response: Eight offspring pigs per group (one pig from each pen with an average BW of the pen) at each time point (65, 95, and 125 day-old) were selected for sampling.We have added these details in the revised manuscript (L148-149).3. What amounts of feed were provided daily?Restricted or ad libitum?Response: The sows were fed with 0.8, 1.0, 1.2, 1.5, and 2.0 kg of pregnancy diets from days 1-15, 16-30, 31-75, 76-90, and 91-105 of pregnancy, respectively; fed with 1 kg of pregnancy diets a week before parturition and ad libitum after three days of parturition; and fed with 2.4 kg of lactation diets until weaning.The offspring pigs had ad libitum access to feed at all times.We have added these details in the manuscript (L117-121 and L129-130).
4. Feed intake and body weight must be provided?Response: The growth performance (including BW, ADG, ADFI, and F/G) of offspring pigs have been reported in our previous study ( 5. Given the nature of the dietary intervention (probiotics and synbiotics supplementation) and the expectation of major changes in microbiota composition and metabolism, measuring small intestinal microbiota seems critical in this study.Why only colonic microbiota was measured?Response: The colon is the main site for microbial fermentation, so this study mainly measured colonic microbiota.
6. Which ANOVA was used for statistical analysis?One/two way?Response: one-way ANOVA was used for statistical analysis.We have added this in the revised manuscript (L254).
7 10.Lines 113-114: although the supplier, ingredient, and feeding method of these additives were consistent with the previous study (12), it is worth giving the name of the probiotic and prebiotic here.
Response: We added the information of probiotic and prebiotic in the manuscript.In addition, we also answered this in the first question.
Response: Eight offspring pigs per group (one pig from each pen with an average BW of the pen) at each time point (65, 95, and 125 d-old) were selected for sampling.We have added these details in the revised manuscript (L148-149).
12. What about the environmental conditions surrounding the sows and piglets?For example, season, temperature, relative humidity, the nature of housing, and the form of feed provided?Response: The sows were feeding in the autumn and winter, and the piglets were feeding in the spring and summer.Feeding and housing management were performed according to the standard operations of commercial pig farms.
13.Why is there a difference in the amounts of probiotics, synbiotics, and antibiotics during the different trial periods?If the addition is to the feed, then the difference in the amount of feed consumed between the sow and the offspring is variable, and not the difference in the concentration of the feed additives used.
Response: There were differences in the requirements of sows and piglets (details provided in the "Animals and diets" part).The doses of the probiotics and synbiotics were as recommended by the manufacturers.
14. Are there any data on pregnant sows during the first period of the experiment, such as the rate of feed consumption, average weight of the offspring pigs at birth, as well as their body weight at weaning and mortality rate during lactation period as affected by feed additives used?Response: Response: The effects of these additives on pregnant and lactating sows, as well as suckling piglets, were reported in our previous studies.Thank you for the privilege of reviewing your work.Below you will find my comments and instructions from the mSystems editorial office.
Thank you for this revision, the reviewers were satisfied with your answers.Therefore, before accepting your manuscript for publication in mSystems, I need you to update the Data Availability section.
Please make the data available as explained in the ASM Data Policy, and as I asked before the revision.
Please return the manuscript within 60 days; if you cannot complete the modification within this time period, please contact me.If you do not wish to modify the manuscript and prefer to submit it to another journal, notify me immediately so that the manuscript may be formally withdrawn from consideration by mSystems.

Revision Guidelines
To submit your modified manuscript, log into the submission site at https://msystems.msubmit.net/cgi-bin/main.plex.Go to Author Tasks and click the appropriate manuscript title to begin.The information you entered when you first submitted the paper will be displayed; update this as necessary.Note the following requirements: • Upload point-by-point responses to the issues raised by the reviewers in a file named "Response to Reviewers," NOT in your cover letter.
• Upload a compare copy of the manuscript (without figures) as a "Marked-Up Manuscript" file.
• Upload a clean .DOC/.DOCX version of the revised manuscript and remove the previous version.
• Each figure must be uploaded as a separate, editable, high-resolution file (TIFF or EPS preferred), and any multipanel figures must be assembled into one file.
• Any supplemental material intended for posting by ASM should be uploaded with their legends separate from the main manuscript.You can combine all supplemental material into one file (preferred) or split it into a maximum of 10 files with all associated legends included.
For complete guidelines on revision requirements, see our Submission and Review Process webpage.Submission of a paper that does not conform to guidelines may delay acceptance of your manuscript.
Data availability: ASM policy requires that data be available to the public upon online posting of the article, so please verify all links to sequence records, if present, and make sure that each number retrieves the full record of the data.If a new accession number is not linked or a link is broken, provide mSystems production staff with the correct URL for the record.If the accession numbers for new data are not publicly accessible before the expected online posting of the article, publication may be delayed; please contact production staff (mSystems@asmusa.org)immediately with the expected release date.
Publication Fees: For information on publication fees and which article types are subject to charges, visit our website.If your manuscript is accepted for publication and any fees apply, you will be contacted separately about payment during the production process; please follow the instructions in that e-mail.Arrangements for payment must be made before your article is published.
ASM Membership: Corresponding authors may join or renew ASM membership to obtain discounts on publication fees.Need to upgrade your membership level?Please contact Customer Service at Service@asmusa.org.
The ASM Journals program strives for constant improvement in our submission and publication process.Please tell us how we can improve your experience by taking this quick Author Survey. 5. Given the nature of the dietary intervention (probiotics and synbiotics supplementation) and the expectation of major changes in microbiota composition and metabolism, measuring small intestinal microbiota seems critical in this study.Why only colonic microbiota was measured?Response: The colon is the main site for microbial fermentation, so this study mainly measured colonic microbiota.
6. Which ANOVA was used for statistical analysis?One/two way?Response: one-way ANOVA was used for statistical analysis.We have added this in the revised manuscript (L254).
7. How about the growth performance of the offspring piglets after dietary supplementation?What about mortality rate during the experimental period from 35 to 125 days?Response: The growth performance (including BW, ADG, ADFI, and F/G) of offspring pigs have been reported in our previous study (Zhu et al., 2023) 10.Lines 113-114: although the supplier, ingredient, and feeding method of these additives were consistent with the previous study (12), it is worth giving the name of the probiotic and prebiotic here.
Response: We added the information of probiotic and prebiotic in the manuscript.In addition, we also answered this in the first question.
11.What were the criteria for eight piglets for sampling (L118-119)?Response: Eight offspring pigs per group (one pig from each pen with an average BW of the pen) at each time point (65, 95, and 125 d-old) were selected for sampling.We have added these details in the revised manuscript (L148-149).
12. What about the environmental conditions surrounding the sows and piglets?For example, season, temperature, relative humidity, the nature of housing, and the form of feed provided?Response: The sows were feeding in the autumn and winter, and the piglets were feeding in the spring and summer.Feeding and housing management were performed according to the standard operations of commercial pig farms.
13.Why is there a difference in the amounts of probiotics, synbiotics, and antibiotics during the different trial periods?If the addition is to the feed, then the difference in the amount of feed consumed between the sow and the offspring is variable, and not the difference in the concentration of the feed additives used.
Response: There were differences in the requirements of sows and piglets (details provided in the "Animals and diets" part).The doses of the probiotics and synbiotics were as recommended by the manufacturers.
14. Are there any data on pregnant sows during the first period of the experiment, such as the rate of feed consumption, average weight of the offspring pigs at birth, as well as their body weight at weaning and mortality rate during lactation period as affected by feed additives used?Response: Response: The effects of these additives on pregnant and lactating sows, as well as suckling piglets, were reported in our previous studies.Ma C, Zhang W, Gao Q, Zhu Zhu Q, Azad MAK, Dong H, Li C, Li R, Cheng Y, Liu Y, Yin Y, Kong X. 2023.Sow-offspring diets supplemented with probiotics and synbiotics are associated with offspring's growth performance and meat quality.Int J Mol Sci 24:7668).The specific data is as follows: of the trail what is the number of sow month gestation?Response: Approximately 64 pregnant sows (within a month) were assigned for this trial.The average gestation period of sows was 114 ± 1.58 days.9. How long did the sows receive feed with experimental supplements?Whole pregnancy or less?It needs to be added.Response: The sows received feed with experimental supplements during gestation and lactation periods.We have added details in lines 114-115 this in the revised manuscript.
offspring's growth performance and meat quality.Int J Mol Sci 24:7668.8. Line 100 "Sows were housed in individual pens (2.2 × 0.6 m) during gestation" in the pigging Feed intake and body weight must be provided?Response: The growth performance (including BW, ADG, ADFI, and F/G) of offspring pigs have been reported in our previous study (Zhu Q, Azad MAK, Dong H, Li C, Li R, Cheng Y, Liu Y, Yin Y, Kong X. 2023.Sow-offspring diets supplemented with probiotics and synbiotics are associated with offspring's growth performance and meat quality.Int J Mol Sci 24:7668).The specific data is as follows: Sows were housed in individual pens (2.2 × 0.6 m) during gestation" in the pigging of the trail what is the number of sow month gestation?Response: Approximately 64 pregnant sows (within a month) were assigned for this trial.The average gestation period of sows was 114 ± 1.58 days.9. How long did the sows receive feed with experimental supplements?Whole pregnancy or less?It needs to be added.Response: The sows received feed with experimental supplements during gestation and lactation periods.We have added details in lines 114-115 this in the revised manuscript.
Q, Song M, Ding H, Yin Y, Kong X. 2020.Dietary synbiotic alters plasma biochemical parameters and fecal microbiota and metabolites in sows.J Funct Foods, 75:104221.Ma C, Gao Q, Zhang W, Zhu Q, Kong X. 2020.Effects of dietary lactobacillus and yeast fermentation broth on reproductive performance colostrum composition and plasma biochemical indexes of sows.Chinese Journal of Animal Nutrition 32(1):129-137.Ma C, Gao Q, Zhang W, Zhu Q, Tang W, Blachier F, Ding H, Kong X. 2020.Supplementing synbiotic in sows' diets modifies beneficially blood parameters and colonic microbiota composition and metabolic activity in suckling piglets.Front Vet Sci 7:575685.Ma C, Azad MAK, Tang W, Zhu Q, Wang W, Gao Q, Kong X. 2022.Maternal probiotics supplementation improves immune and antioxidant function in suckling piglets via modifying gut microbiota.J Appl Microbiol 133:515-528.15 Authors should re-format the references based on journal format See the instructions for authors Response: We have re-formatted the references based on journal format.