Evaluation of the Effect of In Ovo Applied Bifidobacteria and Lactic Acid Bacteria on Enteric Colonization by Hatchery-Associated Opportunistic Pathogens and Early Performance in Broiler Chickens

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript explores the impact of in ovo administration of specific probiotics (Bifidobacterium saeculare and lactic acid bacteria) on reducing gut colonization by opportunistic pathogens and improving early growth performance in broiler chickens. The study, encompassing four trials, provides robust evidence on the efficacy of these probiotics in enhancing beneficial bacterial colonization and suppressing gram-negative bacteria. Notably, combinations such as LAB46 + B2-2 demonstrated significant benefits. The manuscript's strengths lie in its comprehensive experimental design and statistical analyses, addressing a critical area in poultry production.

 

SIMPLE SUMMARY

The manuscript lacks a Simple Summary, which is an essential component for scientific papers, particularly in multidisciplinary journals. This section is crucial for summarizing the study's significance in a straightforward and accessible way, targeting a broader audience, including non-specialists, policymakers, and stakeholders in the poultry industry.

 

ABSTRACT

The abstract provides a clear summary of the study, outlining the problem, objectives, methods, and main findings. The mention of the specific probiotic treatments and their effects adds value.

Weakness:

It lacks sufficient quantitative detail, such as the exact percentage reductions in pathogen colonization or improvements in body weight gain. Adding specific numerical results would improve the scientific rigor and utility of the abstract.

 

INTRODUCTION

The introduction establishes the significance of controlling microbial colonization in hatcheries and highlights the potential of probiotics for improving poultry gut health. It is well-supported by references and provides a logical flow to the research objectives.

Weaknesses:

• While the literature review is thorough, the rationale for selecting specific probiotics (e.g., LAB46 and B. saeculare) is insufficiently discussed.
• The hypothesis is implied but not explicitly stated.

 

MATERIALS AND METHODS

The methodology is comprehensive, detailing experimental design, probiotic preparation, and bacterial recovery processes.

Weaknesses:

• Some procedural details, such as the validation of the contamination model, are missing.
• The justification for using specific doses and delivery methods for probiotics is not well-explained.
• The description of treatment groups is overly detailed, making it harder to follow without summarization. (To enhance clarity, consider including an explanatory figure or flowchart that summarizes the treatment groups and experimental design. A visual representation would aid in simplifying complex information, allowing readers to quickly understand the differences between treatments and the overall study design.)

 

The statistical methods are generally appropriate for the study design and well-applied. However, the lack of adjustments for multiple comparisons, insufficient details for the Chi-square analysis, and missing effect size metrics limit the robustness of the conclusions. Addressing these issues would improve the transparency and reliability of the manuscript's statistical analysis.

1. Multiple Comparisons Adjustment:
• Incorporate a correction method (e.g., Bonferroni, Tukey) to adjust for multiple comparisons during post hoc testing.
2. Transparency in Chi-Square Analysis:
• Include observed and expected frequencies or contingency tables in the supplementary materials to enhance clarity.
3. Improve Meta-Analysis:
• Provide details on heterogeneity metrics and weighting procedures to strengthen the meta-analysis.
4. Address Non-Significant Findings:
• Discuss potential reasons for non-significant results to ensure balanced interpretation of the data.

 

RESULTS

The results section presents extensive data, including bacterial recovery, performance metrics, and statistical significance. The use of tables and figures is appropriate.

 

DISCUSSION

The discussion links the results to existing literature and explores potential mechanisms behind the observed effects. The emphasis on selecting appropriate strains is valuable.

You need to expand on the practical implications, address inconsistencies across trials, and propose future research directions to investigate long-term effects.

 

CONCLUSION

The conclusion effectively summarizes the study's key outcomes and their significance.

Include a concise statement about the broader applicability of the findings and potential for commercial implementation. Avoid redundancy with the discussion.

Author Response

Thank you so much for taking the time to review the manuscript.  Please find our responses in the attachment under "Reviewer 1" section and in the revised manuscript draft. We appreciate you.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

In the conducted studies, the authors assessed whether the administration of non-pathogenic microorganisms (bifidobacteria and Lactobacillaceae) in ovo protects chickens from the multiplication of opportunistic microorganisms. The influence of beneficial bacteria on the weight gain of chickens was also assessed. The experiment was conducted in several trials using a few hatcheries. I have generally positive impressions after reading this manuscript. It contains a lot of information, but some issues require explanation and a clearer presentation.

From the content of the manuscript it results that in the studies the negative control (NC) was embryos to which nothing was administered in ovo. I believe that the control should also be eggs to which physiological fluid should be administered instead of bifidobacteria and Lactobacillaceae and potential pathogens. The process of puncturing the egg itself affects further hatching parameters.

L69 - DOE18 and L85 - DOH - please explain

L69-89 - I suggest that the experimental plan be presented more clearly in a table (divided into trials and hatcheries and the number of eggs in the groups and number of and the number of embryos used for microbiological analyses) or graphically; The number of eggs used in each trial and in each group must be clearly specified.

L77 - Information about the hatchery is unclear; should it be understood that in one/a given hatchery eggs were used for the NC and PM groups, and eggs for the other experimental groups came from another hatchery?

Did the eggs in each hatchery come from different laying farms? Did the eggs used for a given trial (all experimental groups) come from the same laying farm? These issues are important, because eggs from different environments can have different microbiological contaminations. From my own experience, I know that eggs are not sterile (this has been confirmed by many authors), they can contain different bacteria. Hence, a different initial profile of microbiological contamination of eggs could have influenced the final result of the study.

L75-78 - Why was only the hatchery used for sample 1 marked, i.e. GQF 1550. Please also provide the symbols/tags of the other hatcheries. For what purpose did the authors provide the number of hatching eggs in each hatchery? Were all eggs, e.g. 240 from the first hatchery, given in ovo preparations?

L91 - Where were the LAB18 and LAB46 strains isolated from? Do these strains meet the criteria for probiotics? Has their antimicrobial susceptibility and presence of resistance genes been determined?

L97, 100, 103 etc.. - please correct the temperature unit symbol to °C

L105 - Did the authors have permission to administer pilocarpine intraperitoneally to turkeys? Where did the idea come from to administer Bifidobacterium strains isolated from another bird species to chickens? (possible lack of biological matching). Were the Bifidobacterium strains tested for the presence of resistance genes?

L121, 132 and many others - 200ul - add space, please

L143-148 - Was physiological fluid injected into the eggs from the control group instead of a bacterial suspension? How many eggs in total were used in the study?

L154 - Where exactly were the E. coli and Enterococcus strains isolated from? Were these strains characterized? Were their virulence profiles determined? How did the two E. coli strains differ from each other, apart from the source of isolation?

L216 - Were in vitro analyses performed before the in vivo experiment to determine the interactions between individual LAB strains and bifidobacterium? It is possible that the LAB18 strain produces a bacteriocin that has an inhibitory effect on B3-4. Similarly, it was also necessary to initially check in vitro whether LAB and bifidobacterium strains inhibit the growth of opportunistic strains.

Table 3 - the abbreviations BW, BWG, FCR should be explained either in the title or below the table.

Table S1 - I believe that this table contains interesting information and it should be placed in the main text

There are many reports on the effect of administering probiotic strains in ovo. I think that the discussion can be expanded - e.g. what species of Lactobacillus were administered in ovo by other authors?

L321 - observed 2x

Author Response

Thank you for taking time out of your busy schedule to review our manuscript. Please find our responses in the attachment (Reviewer 2 section) and in the revised manuscript.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

SIMPLE SUMMARY

I appreciate the authors' efforts in revising their manuscript and addressing the provided feedback. However, I would like to note that my suggestion regarding the inclusion of a Simple Summary was not incorporated. This section is a key component of scientific papers, particularly in multidisciplinary journals, as it enhances accessibility by summarizing the study’s significance in a clear and concise manner for a broader audience, including non-specialists, policymakers, and stakeholders in the poultry industry. Despite this omission, I acknowledge the improvements made and commend the authors for their commitment to refining their work.

ABSTRACT

I appreciate the authors' response regarding the word limit constraints in the abstract. However, in many cases, it is possible to slightly exceed the word limit, especially when including essential quantitative data that significantly enhance the study’s clarity and impact. Certain numerical values, such as p-values and key percentage changes, are crucial for drawing the reader’s attention and providing a quick yet rigorous overview of the study’s significance. Including specific figures related to pathogen reduction or body weight gain would improve the scientific rigor of the abstract without necessarily omitting critical information. Carefully selecting and prioritizing the most relevant numbers can strengthen the abstract’s utility while maintaining conciseness.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

I accept the Authors' explanations and corrections.

I suggest that information about the number of eggs used in each trial be added to Table 1 of the supplement.

L93 - Information about the origin of strains LAB18 and LAB46 (Flora-Max B11, company?) must be included in the M&M chapter in the section "Lactic acid bacteria".

The issue of the probable inhibitory effect of strain LAB18 (bacteriocin?) on strain B3-4 should be considered in the Discussion chapter.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf