Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors analyzed the nutrients of chestnut biochar and evaluated the effect of chestnut biochar on growth performance, diarrhea incidence, fecal digestibility and serum antioxidant capacity of weaned piglets. This study demonstrated that the inclusion of 1% biochar as a functional ingredient in the diet of post-weaning piglets could enhance the health status of piglets, which may provide the evidence to improve the swine industry and achieve sustainable development. The manuscript is well written and the topic is sound. However, there are some issues need to be solved.

 

1. Please explain how is possible that piglets with 28 days age could be 9.42 kg, and why the replicate amount of each dietary group was different?
2. I think the chemical analysis and electron microscopy characterization of chestnut biochar should be moved to M&M section, and Table 2 should be listed before Table 1. In addition, the analyzed nutritional contents of experimental diets should be inserted in Table 1.
3. Tables 5-7 should be re-drown, there are so mess. Please keep the same decimal numbers in each Table,
4. I do not think it is necessary to use MIXED procedure in this study, and a GLM procedure should be fine.
5. The economic convenience of using chestnut biochar should be discussed in Introduction or Discussion section.

Author Response

Dear Review,

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

General Comments

The manuscript contains several issues that require attention before it can be considered for publication. In particular, the statistical analysis methods need careful revision, and the experimental design, including animal numbers per pen, requires clarification. Specific concerns are detailed below.

Line-by-Line Comments

• Line 14: Please clarify whether this is considered a substantial challenge for swine producers. Are there any regulatory guidelines or consequences currently established?
• Line 16: It is unclear what is meant by "resource" in this context. Please specify.
• Line 17: The term “employment” is typically used in reference to human subjects. Consider using alternative terminology more appropriate for animal studies.
• Lines 111 & Related Design Questions:
• Why do the number of pigs and pens differ between treatments?
• How many pigs were housed per pen?
• Was pen considered the experimental unit for all measurements, including average daily gain (ADG) as noted in Line 147?
• Diet Composition:
  The crude protein (CP) levels appear low for weanling pigs. Please provide standardized ileal digestible (SID) lysine and energy values for the experimental diets.
• Line 151: Clarify how fecal scoring was performed. Additionally, what procedures were followed if pigs did not defecate during the observation window?
• Line 154: Is the fecal scoring system capable of distinguishing between nutritional and pathological causes of diarrhea?
• Line 165: Were the bacterial culture conditions appropriate for both facultative and non-facultative anaerobes? Were the media specifically formulated to support the growth of target organisms?
• Line 198: Only one experimental unit appears to have been used for growth performance. Please justify this design or consider re-analysis.
• Line 202: Was the time factor also included in the model for growth performance? If so, please describe how time was handled (fixed or random).
• Line 212: What was used as the random factor in the mixed model? If time was treated as a fixed effect, the initial results shouldn’t be used as covariance.
• Line 213: Which specific traits were analyzed using median-based pairwise comparisons?
• Line 216: The dry matter content of the biochar appears to be low. Please confirm and explain if this affected formulation or results.
• Line 254: Suggest changing the section heading to “Growth Performance” for consistency and clarity.
• Line 255: Please explain the rationale for examining the time effect on growth performance. Were any pigs removed during the study period?
• Figure 2: Was diarrhea frequency used in the study an appropriate method? The results on days 0 and 28 show inconsistencies between diarrhea frequency and fecal dry matter. Please discuss this discrepancy.
• Line 277: Were day 0 values used as covariates in the statistical analysis? If so, re-analysis may be needed, as this affects interpretation. Also, explain the observed diminishing treatment effect by day 28.
• Line 285: How was nutrient digestibility on day 0 assessed? What diets were pigs fed during the adaptation period? If dry matter digestibility was unaffected, how can the increase in crude protein digestibility be explained?
• Line 289: Why are only day 0 mineral results presented? If the goal was to assess mineral utilization, why was mineral digestibility not calculated and reported?
• Line 364: Please discuss the bioavailability of minerals and other nutrients in biochar. Does biochar have binding affinity for positively charged dietary ions?
• Line 417: Recheck the nutrient requirements referenced from the NRC.
• Line 430: This argument is currently weak. The supporting evidence cited appears to pertain more to the biochar source than to a generalized mechanism. Consider strengthening this section with additional references or a more cautious interpretation.
• Line 442: If nitrogen digestibility increased in the treatment group, why was this not reflected in weight gain? Please address this apparent disconnect.

Author Response

Dear Review,

Please see the attachment.

 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The idea of using charcoal in animal production is not new, but as you clearly present in your introduction, this concept has recently gained new justification. These types of feed additives belong to the group used for non-specific prevention of diarrhea. They can be especially recommended during periods of expected high stress, particularly the peri-weaning period. During this time, we often observe disruptions in hydrochloric acid secretion and other digestive fluids, leading to increased pH levels in the stomach and intestines. This, in turn, raises the risk of the occurrence and development of post-weaning diarrhea. When recommending such feed additives, we can apply them as a general principle—such as for weaner feeding—as you did in your experiment. However, since you mentioned the use of antibiotics during the nursery phase, it is worth adding that this additive could also be used and recommended for local, non-specific treatment. It is important to highlight its impact on feed quality, especially during storage.

I appreciated your presentation and explanations. You recommend a special chestnut biochar and describe its characteristics well. Based on your results (showing no disruption in digestion), we can consider increasing the concentration of this additive up to 1.5%.

Regarding the adaptation period: could you please clarify why you used a 7-day adaptation period? This feed should be particularly recommended immediately after weaning. Even if another feed is used post-weaning, this additive should still be included. Were these piglets moved to another farm?

Is the chestnut biochar produced on an industrial scale?

I agree that accurate evaluation of this feed ingredient requires long-term studies. This experiment should be repeated with a larger number of animals and across different farms. Additionally, although it is difficult when we aim to avoid deliberately infecting animals, it would be valuable to prepare a special feed mixture with a higher concentration of chestnut biochar to test during actual diarrhea outbreaks in specific pens, rooms, or technological groups to confirm its therapeutic properties.

To summarize, your work is interesting, well-prepared, and well-performed. However, the manuscript needs to be carefully reviewed again to correct missing punctuation marks. Please go through the entire text to add the necessary periods and commas.

Author Response

Dear Review,

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Lines 111 & Related Design Questions:

Why do the number of pigs and pens differ between treatments?

Authors: Thank you very much for your valuable observation. The trial was conducted in a commercial breeding facility equipped with 15 pens. To ensure uniform environmental conditions (both micro- and macroclimatic) within the hall and maintain consistency across the experimental groups, the authors decided to allocate a higher number of replicates to the treatment group. This allowed us to optimize pen usage while preserving the reliability of the experimental setup (Line: 113).

How many pigs were housed per pen?

 

Authors: Thank you for your useful observation. The number of pigs housed per pen ranged from 13 to 15, a choice determined by the availability of pens at the private farm where the trial was conducted. This detail has been clarified in the revised manuscript (lines 112–119). Specifically, piglets were housed as follows:

We would also like to point out that the number of animals per pen fully complied with current animal welfare legislation. According to Directive 2008/120/EC (and its national transpositions), the minimum floor space required per pig varies with body weight. Specifically, for piglets weighing between 7 kg and 35 kg, a minimum of 0.30 m² per animal is required, and for pigs over 35 kg, the minimum is 0.40 m² per animal.

In our case, considering the pen dimensions and the age and weight of the piglets, each pen provided sufficient space to legally accommodate up to 17 animals. Therefore, the housing conditions adopted not only complied with welfare standards but also ensured homogeneous environmental conditions for all experimental groups.

Q: Was the size of the pen consistent? IF it is the case, what was the number of pens per pen for each body weight block?

DietComposition:

The crude protein (CP) levels appear low for weanling pigs. Please provide standardized ileal digestible (SID) lysine and energy values for the experimental diets.

Authors: Thank you for your valuable observation. As correctly noted, the crude protein (CP) content in the experimental diets is relatively low compared to conventional formulations for weanling pigs. However, this was an intentional choice, aligned with recent nutritional strategies and regulatory trends aiming to reduce dietary crude protein levels in piglet diets.

Lowering crude protein in post-weaning diets has been shown to offer several advantages, including: i) a reduction in the incidence of gastrointestinal disturbances; ii) a decrease in nitrogen excretion, thereby reducing the environmental impact of pig production.

In support of this approach, several studies have demonstrated that reduced protein levels can promote gut health in piglets. High dietary protein levels may result in undigested protein reaching the hindgut, where it can be fermented by pathogenic bacteria, leading to the production of toxins that compromise the intestinal barrier. (doi:10.1016/j.livsci.2010.06.117; doi.org/10.1080/1828051X.2021.1952911).

In response to your request, we have added the standardized ileal digestible (SID) lysine and energy values of the experimental diets to the manuscript. Specifically, the total lysine content was 1.20%, corresponding to 1.05% SID lysine. The calculated metabolizable energy content was 3,220.88 kcal/kg.

We have updated the relevant section of the manuscript accordingly to ensure clarity and completeness. (Lines: 122-124)

Q: I do not intend to question the lowering of diet crude protein and SID lys, but I question the validity of the statement in line 120.

Line 212: What was used as the random factor in the mixed model? If time was treated as a fixed effect, the

initial results shouldn’t be used as covariance.

Authors: We thank the reviewer for the insightful comment. In our analysis, the random factor was the individual animal for parameters measured at the individual level, and the pen for variables recorded at the pen level (such as ADFI and FCR). Time was treated as a fixed effect, as was the treatment, along with their interaction (treatment × time). When baseline values were included as covariates (for serum oxidative markers or mineral content in feces) the analysis focused only on the treatment effect at subsequent timepoint. This approach was chosen to avoid redundancy in the model and ensure that the covariate adjustment did not overlap with the fixed time effect. We have clarified this methodological distinction in the materials and methods section (Lines: 213-215).

Q: Were the block factors exit? Was body weight balanced between treatments?

Figure 2: Was diarrhea frequency used in the study an appropriate method? The results on days 0 and 28 show inconsistencies between diarrhea frequency and fecal dry matter. Please discuss this discrepancy.

Authors: We appreciate the reviewer’s observation. Diarrhea frequency was used as a clinically relevant and widely adopted parameter in swine nutrition trials to assess the general health status of the gastrointestinal tract. While fecal dry matter (DM) provides a continuous and objective measurement, diarrhea scoring (including frequency) more directly reflects the animal’s clinical condition, which may not always correspond perfectly with DM values due to transient fluctuations. Additionally, it is important to note that in cases of more severe diarrhea, fecal sampling for DM analysis was often not possible, which may have contributed to the observed discrepancies between the two measures at certain time points, such as days 0 and 28.

Q: The Author’s statement confirms the arguable results from DM determination in the manuscript, which require addressing. Missing data from severe diarrhea pigs on DM altered the results of DM.

Line 285: How was nutrient digestibility on day 0 assessed?

Authors: The nutrient digestibility on day 0 was calculated with the same method (AIA method) used for d28

Q: Why assess the d 0 digestibility when the pigs hadn’t been exposed to experimental diets?

What diets were pigs fed during the adaptation period?

Authors: The adaptation period was of 7 days and the piglets was feeded with basal diet in according to farming practices.

Q: Please provide the nutrient content of the adaptation feed and specify if medication or heavy metals were being used or not.

If dry matter digestibility was unaffected, how can the increase in crude protein digestibility be explained?

Authors: We apologize for the lack of clarity in the original text. Nutrient digestibility on day 0 was assessed. The lack of effect on organic matter digestibility, despite an increase in CP digestibility, could be due to a selective enhancement of nitrogen utilization or increased efficiency in protein utilization activity induced by the biochar treatment. This may suggest a more efficient breakdown and absorption of protein without affecting the digestibility of other components that contribute to total OM.

 

Q: It still doesn’t make sense, given that dietary nitrogen is part of total dietary organic matter, unless other organic matter digestibility decreased.

 

 

 

Author Response

Dear Reviewer,

Please see the attachment

Author Response File: Author Response.pdf