Characterizing the dynamics of the rumen microbiota, its metabolites, and blood metabolites across reproductive stages in Small-tailed Han sheep

ABSTRACT Different reproductive stages of mammals involve complex biological processes, and the intestinal microbiota, as an endocrine organ or an “invisible organ,” is involved in the regulation of hormone levels, immune function, and metabolism. However, the effects of the rumen microbiota, its metabolites, and blood metabolites on the reproductive performance of ruminants remain unclear. This study revealed that the Prevotella abundance increased significantly during pregnancy (P < 0.01); the Fibrobacter abundance increased significantly during lactation (P < 0.05); and rumen microbial carbohydrate metabolism, glucose biosynthesis, and metabolic functions were significantly enriched during pregnancy (P < 0.05). Microbial metabolic profile analysis showed that the differentially abundant microbial metabolites during pregnancy and lactation were mainly enriched in the biosynthesis of ubiquinone and other terpenoid quinones, and there was a certain correlation with the microbiota. Among them, sapindoside A was increased during pregnancy, nicotinamide riboside and β-cryptoxanthin were reduced during pregnancy, and L-tryptophan was significantly increased during lactation. In addition, the volatile fatty acid levels in lactation were significantly higher than those in non-pregnancy and pregnancy (P < 0.05), and the NH3-N content during pregnancy was significantly higher than that during lactation and non-pregnancy (P < 0.05). Moreover, there were differences in the serum metabolite levels at different reproductive stages, and similar metabolites existed when comparing the rumen metabolites, which were mainly enriched in arachidonic acid metabolism, vitamin B6 metabolism, and ABC transporter protein, resulting in significantly higher serum IgA and IgM levels during lactation than during non-pregnancy and pregnancy (P < 0.05). IMPORTANCE Our study illustrates the succession of the rumen microbiota and its metabolites in Small-tailed Han sheep at different reproductive stages. Among them, Firmicutes and Prevotella, which are related to energy metabolism, increased in abundance during pregnancy, while Fibrobacter, a fiber-degrading bacterium, increased in abundance during lactation. At the same time, the microbial metabolic profile and serum metabolic profile characteristics of different reproductive stages were revealed, and some functional pathways and metabolites related to energy and immunity were found. This study provides a reference for the health management of ruminants during non-pregnancy, pregnancy, and lactation.

Some key points: • Many of the figures need to revised and made larger or moved to the supplementary material • Abbreviations needed to be explained and used consistently throughout the manuscript • The discussion could be abbreviated as much of it is a repetition of the results I had begun making some edits to the Word doc (see file), but this is probably something that the authors should complete.
Reviewer #3 (Comments for the Author): This manuscript evaluated the ewe gut microbiota and metabolome during non-pregnancy, pregnancy, and lactation.The authors evaluated the correlation between microbiota and metabolites during these three physiological stages, and identified some important microorganisms and metabolites, which may have some health indication for sheep pregnancy and lactation.In general, this article provided some decent information for the microbiome and metabolome of ewe gut during these three physiological stages.However, the authors seem to exaggerate the effects of certain bacteria and metabolites in the ewe gut during pregnancy/lactation and confuse the causation and correlation effects.For example, the conclusion drawn by the authors are not convincing (Line 498-499).The roles of Fibrobacter during pregnancy is not proved in this study, since no in vitro or in vivo experiments were performed to evaluate the effects of these bacteria.The authors performed only amplicon metagenomics and MS-based metabolomics, and the evidence is lacking for this conclusion.Another example in the discussion section (Line 325-327), Firmicutes carry genes related to energy metabolism, and their abundance increased during pregnancy.But this information alone does not provide basis that firmicutes help host digest and absorb nutrients, which is a general and bold statement without evidence.Similar statements can be found throughout the discussion section 350,(376)(377)(378).The argument on the association of blood antibody levels (IgA, IgG, and IgM) and the gut microbiome is weak.If the three reproductive stages have different levels of blood antibodies and different gut microbiota, this information alone does not provide association between blood antibody and gut microbiota.Additionally, the authors seem to lack the general microbiology knowledge, and they used the sequencing analyses software denoted names, instead of the correct genus names throughout the article.Please make sure to remove any suffix from sequencing analysis.For example, Line 146-147, "Prevotella_1", "Rikenellaceae_RC9_gut_group", so on and so forth.Please confirm the species of "SP3-e08" (Line 252).The authors should provide higher quality figures for Fig 5,6,7,9.It is important to make figures self-explanatory, and the figure captions should be more informative.Please make sure to explain relevant abbreviations in the figure captions as well.For example, explain abbreviation "Gp", "Lp", "Np" in figure captions.The authors are suggested to review the manuscript carefully and make sure to use capitalization and italics properly.
Here are some additional specific suggestions for the authors: Line 130: typo "OTUs" Line 199, 282: "Venn diagram" Line 339: It is not correct to call Helicobacter and Erysipelotrichaceae pathogenic.Some species may be pathogenic to ewes, but not all genus/family are pathogenic.Line 376-378: This indication is not valid based on the information provided by the authors.The "need" for vitamin supplements and its importance for pregnancy/lactation requires additional references or experiments.Line 418-420: "ensure the health" is too strong a claim.Provide either provide literature for these claims or change the language.Line 631: change "gate" to "phylum" Figure 1

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This manuscript evaluated the ewe gut microbiota and metabolome during nonpregnancy, pregnancy, and lactation.The authors evaluated the correlation between microbiota and metabolites during these three physiological stages, and identified some important microorganisms and metabolites, which may have some health indication for sheep pregnancy and lactation.
In general, this article provided some decent information for the microbiome and metabolome of ewe gut during these three physiological stages.
However, the authors seem to exaggerate the effects of certain bacteria and metabolites in the ewe gut during pregnancy/lactation and confuse the causation and correlation effects.For example, the conclusion drawn by the authors are not convincing (Line 498-499).The roles of Fibrobacter during pregnancy is not proved in this study, since no in vitro or in vivo experiments were performed to evaluate the effects of these bacteria.The authors performed only amplicon metagenomics and MS-based metabolomics, and the evidence is lacking for this conclusion.Another example in the discussion section (Line 325-327), Firmicutes carry genes related to energy metabolism, and their abundance increased during pregnancy.But this information alone does not provide basis that firmicutes help host digest and absorb nutrients, which is a general and bold statement without evidence.Similar statements can be found throughout the discussion section 350,(376)(377)(378).The argument on the association of blood antibody levels (IgA, IgG, and IgM) and the gut microbiome is weak.If the three reproductive stages have different levels of blood antibodies and different gut microbiota, this information alone does not provide association between blood antibody and gut microbiota.
Additionally, the authors seem to lack the general microbiology knowledge, and they used the sequencing analyses software denoted names, instead of the correct genus names throughout the article.Please make sure to remove any suffix from sequencing analysis.
The authors should provide higher quality figures for Fig 5,6,7,9.It is important to make figures self-explanatory, and the figure captions should be more informative.Please make sure to explain relevant abbreviations in the figure captions as well.For example, explain abbreviation "Gp", "Lp", "Np" in figure captions.
The authors are suggested to review the manuscript carefully and make sure to use capitalization and italics properly.
Here are some additional specific suggestions for the authors: Line 130: typo "OTUs" Line 199, 282: "Venn diagram" Line 339: It is not correct to call Helicobacter and Erysipelotrichaceae pathogenic.Some species may be pathogenic to ewes, but not all genus/family are pathogenic.
Line 376-378: This indication is not valid based on the information provided by the authors.The "need" for vitamin supplements and its importance for pregnancy/lactation requires additional references or experiments.Line 418-420: "ensure the health" is too strong a claim.Provide either provide literature for these claims or change the language.
Line 631: change "gate" to "phylum" caption: missing information.What is the meaning of different color and shape of objects in Fig 1C? Fig 3 is not informative, and the authors did not explain the meaning of this figure properly in the caption.What is the LDA scores of other OTUs, and why only show the OTUs from group "Gp"?What about the other two groups?Staff Comments:

Figure 1
Figure 1 caption: missing information.What is the meaning of different color and shape of objects in Fig 1C?

Fig 3
Fig 3 is not informative, and the authors did not explain the meaning of this figure properly in the caption.What is the LDA scores of other OTUs, and why only show the OTUs from group "Gp"?What about the other two groups?