Oxalic Acid Supplementation in Different Hemicellulose Diets Affects In Vitro Rumen Fermentation by Regulating Nutritional Digestibility, Microbial Diversity and Metabolic Pathways

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript investigates the effects of oxalic acid supplementation in different hemicellulose diets on in vitro rumen fermentation, microbial diversity, and metabolic pathways. Overall, the study is well-designed and the experimental procedures are described in detail. However, there are several areas that need improvement before publication.

1.      The literature review in the introduction section can be further expanded to include more recent and relevant studies in the field, which can help to better position the research and highlight the innovation of the study.

2.      In the 2 × 7 full-factor experimental design, it is not clear whether the selection of the two hemicellulose levels (10.3% and 17%) and the seven oxalic acid doses (0, 2.5, 5, 10, 20, 40, 80 mg/kg DM) was based on prior research or theoretical considerations. There should be a more detailed explanation for the rationality of these choices. The sample size determination method is not described. It is necessary to clarify how the number of replicates per group (four replicates) was decided to ensure the statistical power of the experiment.

3.      When collecting rumen fluid, the details of the rigid PVC tube used, such as its diameter and length, are not provided. These details could potentially affect the representativeness of the collected sample. The method of maintaining an anaerobic environment by injecting CO₂ outside the rumen lacks information on the flow rate and duration of CO₂ injection. Without this information, it is difficult to assess the stability of the anaerobic environment during the experiment.

4.      For the gas chromatograph used to measure fatty acids and total volatile fatty acid content, the specific conditions of the instrument, such as the temperature program, carrier gas flow rate, and injection volume, are not detailed. These parameters can significantly influence the accuracy and reproducibility of the measurements.

5.      The choice of primers (ACTCCTACGGGAGGCAGCA and GGACTACHVGGGTWTCTAAT) for sequencing the bacterial 16S rDNA gene should be justified. Are these primers commonly used for rumen bacteria analysis? Were any tests conducted to evaluate their specificity and coverage?

6.      In tables and figures, several values are labeled with different letters (e.g., a, b, c), indicating statistical differences. However, the manuscript does not explain the statistical tests used or the criteria for these letters. Please provide a detailed statistical analysis section.

7.      Some captions for figures and tables are not descriptive enough. They should provide a clear and concise explanation of what the figure or table depicts without referring to the text. Furthermore, some figures are too blurry to read.

8.      Figure 1 is complex and contains multiple data points without clear differentiation. Please break this figure into subfigures or provide additional clarification to make the data more accessible.

9.      The manuscript claims that oxalic acid supplementation has varying effects on different hemicellulose diets. However, it does not clearly define what constitutes "low" and "high" hemicellulose levels. Please provide specific percentage values or a reference range for these terms.

10.   When discussing the effects of oxalic acid on rumen pH and the comparison with previous findings, specific references to previous studies should be provided in more detail. Merely stating that the results are consistent with previous findings is not sufficient. In the discussion of the relationship between ammonia nitrogen levels and protein degradation, the specific metabolic pathways and enzymes involved in the conversion of feed proteins to ammonia nitrogen in the presence of oxalic acid should be elaborated. This would enhance the understanding of the underlying mechanisms.

Comments on the Quality of English Language

no

Author Response

The manuscript investigates the effects of oxalic acid supplementation in different hemicellulose diets on in vitro rumen fermentation, microbial diversity, and metabolic pathways. Overall, the study is well-designed and the experimental procedures are described in detail. However, there are several areas that need improvement before publication.

Comments 1: The literature review in the introduction section can be further expanded to include more recent and relevant studies in the field, which can help to better position the research and highlight the innovation of the study.

Response 1: Thank you for pointing this out. We agree with this comment. Therefore, we have added recent and relevant studies in the field in the introduction section in line 73-94，“Oxalic acid is one of the main effective substances in yeast culture. The cell wall components in yeast culture can regulate the microbial community in rumen, promote the growth of beneficial microorganisms and inhibit the reproduction of harmful microorganisms, so as to optimize the rumen fermentation process. Sheep can adapt to having 1% oxalic acid in their diet, but a 2% increase in oxalic acid is harmful and affects growth performance, fiber and CP digestibility, rumen function, and the microbial ecosystem within the sheep's rumen[7]. Studies have shown that the effects of oxalic acid in monogastric animals (such as pigs and poultry) are more complex, especially the absorption and deposition of minerals. Oxalic acid can combine with calcium, magnesium and other minerals to form insoluble oxalate[13], which affects the bioavailability of these minerals, may lead to mineral deficiency, affecting animal bone health and growth and development. For example, in poultry, oxalic acid intake can affect bone and egg laying performance by affecting oxalic acid absorption and metabolism in the small intestine, reducing calcium absorption[14].In terms of gut microbes, oxalic acid intake can have an impact on the microbial community. Oxalic acid may negatively affect microbial metabolic processes such as rumen fermentation and cecal fermentation in ruminants and monogastric animals by altering intestinal pH and microbial environment[15]. At the same time, oxalic acid metabolism also affects the liver and kidney function of animals, especially in the case of long-term intake, oxalic acid accumulation may lead to kidney stones and other mineral metabolism related diseases[6].”

Comments 2:  In the 2 × 7 full-factor experimental design, it is not clear whether the selection of the two hemicellulose levels (10.3% and 17%) and the seven oxalic acid doses (0, 2.5, 5, 10, 20, 40, 80 mg/kg DM) was based on prior research or theoretical considerations. There should be a more detailed explanation for the rationality of these choices. The sample size determination method is not described. It is necessary to clarify how the number of replicates per group (four replicates) was decided to ensure the statistical power of the experiment.

Response 2: According to the NRC 2007 nutrient requirements of small ruminants (Ruminants NRC. Nutrient requirements of small ruminants: Sheep, goats, cervids, and new world camelids. USA: The National Academies Press, Washington DC (2007).), the NDF content was 26%, and based on the practical experience of the ranch in China, two levels of hemicellulose were selected (10.3% and 17%) in this experiment. The diet with 10.3% hemicellulose content was defined as a low hemicellulose level (HM10.3%), and the diet with 17% hemicellulose content was defined as a high hemicellulose level (HM17%).

Our previous study found that (Chen X et al.Mechanistic insights into rumen function promotion through yeast culture (Saccharomyces cerevisiae) metabolites using in vitro and in vivo models[J].Frontiers in Microbiology, 2024.) adding 625 mg / kg yeast culture to the diet can improve the rumen fermentation efficiency of sheep. It was found that oxalic acid was one of the functional substances of yeast culture, and the content of oxalic acid in yeast culture was 0.36 %, and the content of oxalic acid in diet was 2.254 mg / kg. Therefore, seven oxalic acid doses (0, 2.5, 5, 10, 20, 40, 80 mg/kg DM) was set based on this previous study. In this experiment, there were 4 replicates in each batch, 3 batches in each group, and a total of 12 replicates, which could ensure the feasibility and accuracy of the experiment.

Comments 3:  When collecting rumen fluid, the details of the rigid PVC tube used, such as its diameter and length, are not provided. These details could potentially affect the representativeness of the collected sample. The method of maintaining an anaerobic environment by injecting CO₂outside the rumen lacks information on the flow rate and duration of CO₂ injection. Without this information, it is difficult to assess the stability of the anaerobic environment during the experiment.

Response 3: According to your suggestion, the related information was added in the text, line125-127, we use the rigid PVC tube with a diameter of 2 cm and a length of 80 cm. CO2 was continuously injected into each container for 30 seconds at a flow rate of 5ml/s.

Comments 4: For the gas chromatograph used to measure fatty acids and total volatile fatty acid content, the specific conditions of the instrument, such as the temperature program, carrier gas flow rate, and injection volume, are not detailed. These parameters can significantly influence the accuracy and reproducibility of the measurements.

Response 4: According to your suggestion, the related information was added in the text, line149-155, “The analysis of volatile fatty acids (VFA) and six major volatile fatty acids (acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid) were carried out using an Agilent 7890 A gas chromatograph (Agilent Technologies, Santa Clara, California, United States) and a 50m (internal diameter 0.32mm) CP-Wax Chrompack silica-fused capillary column (Varian, Palo Alto, California, United States). The initial and final oven temperatures were set at 65℃ and 190℃, rising 20℃ per minute, split ratio: 50:1; Sample size :1μL[18].”

Comments 5: The choice of primers (ACTCCTACGGGAGGCAGCA and GGACTACHVGGGTWTCTAAT) for sequencing the bacterial 16S rDNA gene should be justified. Are these primers commonly used for rumen bacteria analysis? Were any tests conducted to evaluate their specificity and coverage?

Response 5: The choice of primers (ACTCCTACGGGAGGCAGCA and GGACTACHVGGGTWTCTAAT) for sequencing the bacterial 16S rDNA gene were justified. Which were commonly used for rumen bacteria analysis.The 16S rDNA gene is highly conserved in bacteria and has a certain variable region, which makes it an important target for bacterial classification and identification. This pair of primers can be used in the analysis of rumen bacteria. There are complex and diverse bacterial communities in the rumen. These primers can amplify the partial sequence of 16S rDNA of rumen bacteria, so as to identify and analyze the diversity of rumen bacteria. The specificity of the test was evaluated, and the sparse curve results showed that the test data were reliable.

 

Comments 6: In tables and figures, several values are labeled with different letters (e.g., a, b, c), indicating statistical differences. However, the manuscript does not explain the statistical tests used or the criteria for these letters. Please provide a detailed statistical analysis section.

Response 6: We have provided a detailed statistical analysis section in tables and figures. “a,b,c,d,e,f, Different superscripts in the same row imply that their mean values are significantly different (p <0.05), and the value decreases from a to f.”

Comments 7: Some captions for figures and tables are not descriptive enough. They should provide a clear and concise explanation of what the figure or table depicts without referring to the text. Furthermore, some figures are too blurry to read.

Response 7: We have provided a clear and concise explanation for the figures and tables.

Comments 8: Figure 1 is complex and contains multiple data points without clear differentiation. Please break this figure into subfigures or provide additional clarification to make the data more accessible.

Response 8: We have provided additional clarification to make the data more accessible in Figure 1.

Comments 9: The manuscript claims that oxalic acid supplementation has varying effects on different hemicellulose diets. However, it does not clearly define what constitutes "low" and "high" hemicellulose levels. Please provide specific percentage values or a reference range for these terms.

Response 9: According to the NRC 2007 nutrient requirements of small ruminants (Ruminants NRC. Nutrient requirements of small ruminants: Sheep, goats, cervids, and new world camelids. USA: The National Academies Press, Washington DC (2007).), the NDF content was 26%, and based on the practical experience of the ranch in China, two levels of hemicellulose were selected (10.3% and 17%) in this experiment. The diet with 10.3% hemicellulose content was defined as a low hemicellulose level (HM10.3%), and the diet with 17% hemicellulose content was defined as a high hemicellulose level (HM17%).

Comments 10: When discussing the effects of oxalic acid on rumen pH and the comparison with previous findings, specific references to previous studies should be provided in more detail. Merely stating that the results are consistent with previous findings is not sufficient. In the discussion of the relationship between ammonia nitrogen levels and protein degradation, the specific metabolic pathways and enzymes involved in the conversion of feed proteins to ammonia nitrogen in the presence of oxalic acid should be elaborated. This would enhance the understanding of the underlying mechanisms.

Response 10: We have provided in more details as followings: line581-601, “Studies have shown that certain strains in the rumen, such as Bacillus and Clostridium, can produce a variety of proteases capable of specifically recognizing and cleaving peptide bonds in proteins. The Tan study[27] highlighted that proteases synthesized by rumen microbes are crucial for the initial digestion of proteins. Specifically, the subtilisin produced by Bacillus subtilis can efficiently hydrolyze feed proteins into smaller polypeptides. The polypeptide is subsequently hydrolyzed into amino acids by peptidases, which can be classified into endopeptidases and exopeptidases. Endopeptidases specifically target internal peptide bonds within the polypeptide chain, cleaving it into shorter peptide fragments. Exopeptidase hydrolyzes amino acids sequentially from the terminus of the peptide chain [30]. The addition of oxalic acid alters the amino acid metabolism pathway in certain rumen microorganisms. Bacteria capable of degrading oxalic acid, when utilizing it as a carbon or energy source, may activate a series of enzymes involved in amino acid metabolism. According to relevant studies, these bacteria derive energy through oxalic acid metabolism and drive the deamination of amino acids. Under the catalysis of amino acid deaminases, amino acids undergo deamination to produce ammonia nitrogen and corresponding keto acids [31]. For instance, alanine reacts with alanine deaminase to generate pyruvate and ammonia nitrogen. Pyruvate subsequently participates in microbial energy metabolism, providing energy for growth and reproduction, while ammonia nitrogen is released into the rumen environment, leading to changes in ruminal ammonia nitrogen levels [32].”

4. Response to Comments on the Quality of English Language

Point 1: The English could be improved to more clearly express the research.

Response 1: please check the English language editing certificate as follows:

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

22: addition of 10 mg/kgDM oxalic --> addition of 10 mg/kg DM oxalic 

 

The introduction is well-structured and provides sufficient background. However, consider elaborating on the practical implications of oxalic acid supplementation in different livestock systems to enhance the reader's understanding of its real-world applications.

 

While the factorial design is sound, a more detailed justification for the chosen oxalic acid dosage levels would strengthen the study. Including a rationale for the 10 mg/kg DM focus in comparison to other doses could clarify its significance.

 

The methods are described comprehensively. Adding a brief discussion on how the in vitro results translate to in vivo conditions would increase the study's relevance.

 

 

Results are presented clearly, but the discussion could benefit from deeper comparisons with existing literature. Highlighting discrepancies or confirming agreements with prior studies would contextualize the findings better.

 

The conclusions are aligned with the results, but adding more actionable recommendations for ruminant dietary formulation would enhance the practical utility of the study.

 

 

The figures and tables effectively support the results. Ensure that all abbreviations are defined clearly within the legends to improve readability.

 

 

Consider including a section on future research directions, such as long-term effects of oxalic acid supplementation or its economic implications in ruminant production systems.

Author Response

Comments 1: 22: addition of 10 mg/kgDM oxalic --> addition of 10 mg/kg DM oxalic

Response 1：Line 22, “addition of 10 mg/kgDM oxalic ”was revised to “addition of 10 mg/kg DM oxalic ”.

Comments 2: The introduction is well-structured and provides sufficient background. However, consider elaborating on the practical implications of oxalic acid supplementation in different livestock systems to enhance the reader's understanding of its real-world applications.

Response 2: Thank you for pointing this out. We agree with this comment. Therefore, we have added the practical implications of oxalic acid supplementation in different livestock systems in the introduction section. In line 73-94，“Oxalic acid is one of the main effective substances in yeast culture. The cell wall components in yeast culture can regulate the microbial community in rumen, promote the growth of beneficial microorganisms and inhibit the reproduction of harmful microorganisms, so as to optimize the rumen fermentation process. Sheep can adapt to having 1% oxalic acid in their diet, but a 2% increase in oxalic acid is harmful and affects growth performance, fiber and CP digestibility, rumen function, and the microbial ecosystem within the sheep's rumen[7]. Studies have shown that the effects of oxalic acid in monogastric animals (such as pigs and poultry) are more complex, especially the absorption and deposition of minerals. Oxalic acid can combine with calcium, magnesium and other minerals to form insoluble oxalate[13], which affects the bioavailability of these minerals, may lead to mineral deficiency, affecting animal bone health and growth and development. For example, in poultry, oxalic acid intake can affect bone and egg laying performance by affecting oxalic acid absorption and metabolism in the small intestine, reducing calcium absorption[14].In terms of gut microbes, oxalic acid intake can have an impact on the microbial community. Oxalic acid may negatively affect microbial metabolic processes such as rumen fermentation and cecal fermentation in ruminants and monogastric animals by altering intestinal pH and microbial environment[15]. At the same time, oxalic acid metabolism also affects the liver and kidney function of animals, especially in the case of long-term intake, oxalic acid accumulation may lead to kidney stones and other mineral metabolism related diseases[6].”

Comments 3: While the factorial design is sound, a more detailed justification for the chosen oxalic acid dosage levels would strengthen the study. Including a rationale for the 10 mg/kg DM focus in comparison to other doses could clarify its significance.

Response 3: Our previous study found that (Chen X et al.Mechanistic insights into rumen function promotion through yeast culture (Saccharomyces cerevisiae) metabolites using in vitro and in vivo models[J].Frontiers in Microbiology, 2024.) adding 625 mg / kg yeast culture to the diet can improve the rumen fermentation efficiency of sheep. It was found that oxalic acid was one of the functional substances of yeast culture, and the content of oxalic acid in yeast culture was 0.36 %, and the content of oxalic acid in diet was 2.254 mg / kg. Therefore, seven oxalic acid doses (0, 2.5, 5, 10, 20, 40, 80 mg/kg DM) was set based on this previous study.

At low doses ( 5 mg / kg DM ), oxalic acid had no significant effect on rumen microorganisms and fermentation parameters, and it was difficult to achieve the expected effects of changing rumen fermentation mode and improving hemicellulose degradation efficiency.When the amount of oxalic acid additive exceeds 50 mg/kg DM, it may have a toxic inhibitory effect on rumen microbes, disrupt the ecological balance within the rumen, lead to rumen fermentation disorders, and even have a negative impact on animal health.Thus, from a practical standpoint, in the context of ruminant animal breeding, considering the comprehensive improvement of feed cost, safety, and animal production performance, a dose of 10 mg/kg DM can effectively exert the positive regulatory effect of oxalic acid on rumen fermentation, while ensuring the safety and economy of the breeding process.

Comments 4:The methods are described comprehensively. Adding a brief discussion on how the in vitro results translate to in vivo conditions would increase the study's relevance.

Response 4: Thank you for pointing this out. A brief discussion on how the in vitro results translate to in vivo conditions that added in discussion. Line559-570，“In vitro rumen fermentation can simulate some rumen processes, yet differs from in vivo conditions. Factors like feeding behavior, gastrointestinal motility, metabolic regulation, and organ interactions in vivo are challenging to replicate in vitro. Nonetheless, in vitro results offer valuable insights for in vivo studies. They help in screening potential combinations of oxalic acid and hemicellulose diet and understanding their impact on fermentation indicators. Subsequent in vivo experiments can then target these findings. In vivo, growth performance, feed conversion, and nutrient digestibility can be observed to evaluate the practical application of the oxalic acid-hemicellulose interaction. Comparing in vitro and in vivo outcomes also aids in understanding rumen fermentation regulation mechanisms, such as how oxalic acid doses affect hemicellulose degradation by rumen microbes[26-28].”

Comments 5: Results are presented clearly, but the discussion could benefit from deeper comparisons with existing literature. Highlighting discrepancies or confirming agreements with prior studies would contextualize the findings better.

Response 5: We have added some comparisons in line 715-741, “This particular research project concentrated its efforts on examining the effects of oxalic acid supplementation on rumen fermentation processes, the diversity of microbial populations within the rumen, and the metabolic pathways that are influenced by this dietary addition. The study was specifically interested in how these factors are impacted when the diets contain varying levels of hemicellulose. The findings indicated that the supplementation of oxalic acid has the potential to enhance the production of volatile fatty acids (VFA), which are crucial for the energy metabolism of ruminants. However, the study also highlighted that the optimal dosage of oxalic acid required to achieve this boost in VFA production is not a one-size-fits-all solution; rather, it varies depending on the hemicellulose content present in the diet and the duration of fermentation. Through a systematic analysis of various combinations of these factors, the research provided a clearer understanding of the dose-response relationship.

Furthermore, the study revealed that oxalic acid supplementation has a significant impact on the relative abundance of different microorganisms within the rumen. The presence of varying levels of hemicellulose in the diet was found to modify the way oxalic acid influences these microbial communities. Utilizing metabolomic techniques, the research also shed light on the metabolic pathways that are commonly affected by oxalic acid supplementation. Specifically, it identified changes in carbohydrate digestion processes and choline metabolism, which are essential for the overall health and productivity of ruminant animals. In summary, this study not only confirmed previously known effects of oxalic acid but also uncovered its unique role and the intricate interactions it has with varying levels of hemicellulose in the diet. These insights are valuable for optimizing dietary strategies for ruminant animals, ensuring that they receive the most benefit from their feed while maintaining a healthy and efficient rumen fermentation process.”

Comments 6: The conclusions are aligned with the results, but adding more actionable recommendations for ruminant dietary formulation would enhance the practical utility of the study.

Response 6: The actionable recommendations for ruminant dietary formulation were added in the text in line742-765，“In the dietary formulation for ruminants, it is essential to consider various factors to determine the dosage adaptation scheme for hemicellulose and oxalic acid. For a diet low in hemicellulose (HM10.3%), it is recommended to add 10 mg/kg DM of oxalic acid (OA), which can significantly increase the concentration of rumen volatile fatty acids, supplying energy for animals. For instance, in beef cattle fattening, this can accelerate the fattening speed and shorten the cycle. For a high hemicellulose (HM17%) diet, during 6-hour fermentation, adding 10-20 mg/kg DM of OA is beneficial, while during 12-hour fermentation, either 10 mg/kg DM or 80 mg/kg DM is preferable to meet the energy requirements of different growth stages or production states. For example, during cow milk peak, this can improve milk production and quality. Additionally, the fermentation time also affects the formulation. In short-term fermentation scenarios, such as the early stage of rapid fattening of lambs, rumen fermentation can be rapidly optimized according to the aforementioned dosages. During long-term feeding, rumen fermentation parameters (such as volatile fatty acids and ammonia nitrogen concentrations) should be regularly monitored, and the oxalic acid dose should be flexibly adjusted according to the growth stage of the animal. For example, milk composition and rumen indexes should be dynamically adjusted through detection at different lactation stages of cows. Moreover, different breeds of ruminants (such as yaks, which have a unique ability to digest high-fiber diets) and the feeding environment should also be considered. The oxalic acid dose can be appropriately increased for energy supply in cold regions or winter, and may be reduced in hot and humid environments. When the quality of roughage is high, the oxalic acid dose should also be fine-tuned to achieve the best rumen fermentation and nutrient utilization effects[46].”

Comments 7:The figures and tables effectively support the results. Ensure that all abbreviations are defined clearly within the legends to improve readability.

Response 7: We have checked and revised the abbreviations are defined clearly within the legends.

Comments 8: Consider including a section on future research directions, such as long-term effects of oxalic acid supplementation or its economic implications in ruminant production systems.

Response 8: We have added future research directions in line 801-823, “In the field of ruminant nutrition research, there are three key directions that merit further exploration. First, considering the long-term effects of oxalic acid supplementation, while the short-term effects are currently understood, it remains unclear whether long-term supplementation will cause irreversible changes in rumen microbial communities. This includes the potential gradual extinction of certain microbial populations or a permanent shift in their dominant status. To address this, long-term tracking experiments, metagenomics, transcriptomics, and other technologies are necessary. These methods can dynamically monitor changes in microbial community structure and functional gene expression to determine their impact on the long-term stability of the rumen microbial ecosystem. Second, there is a need to explore the potential effects of long-term oxalic acid intake on the overall health of ruminants, an area that is currently under-researched. Subsequently, we can focus on its effects on the immune system, reproductive performance, and bone health, and comprehensively evaluate its impact on animal health through continuous monitoring of immune indicators, reproductive cycles, offspring quality, and detection of mineral content and metabolic indicators in blood and bone. Third, given the widespread use of feed additives in ruminant farming, it is significant to study the synergistic effects of oxalic acid with other common additives, such as probiotics, prebiotics, and enzyme preparations. In the future, multi-factor experiments can be designed to explore the effects of combined use on rumen fermentation, animal growth performance, and health status. Clarifying the synergistic mechanism will help develop more efficient and comprehensive feed additive formulations and provide optimized nutrition control schemes for the breeding industry.”

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I think this manuscript is ready for publication.

Comments on the Quality of English Language

no