Assessment of the Effects of Stocking Density on Laying Hens Raised in Colony Cages: Part II—Egg Production, Egg Quality, and Welfare Parameters

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The effect of stocking density in conventional cages, later enriched/furnished/colony cages, has been studied for decades. The focus then shifted to compare cages and alternative systems, and recently to compare different alternative systems. The legislation (especially in EU) is pushing towards complete ban of cages. Therefore the research question of this paper is questionable. On the plus side, combination of production, health and behavioural parameters was used to assess studied groups.

The authors completely ignored the fact, that together with stocking density, they changed also group size. Group size effect is not considered or mentioned in the text at all.

Part 2.6 - Condition of keel bone, toes, footpad, comb, beak, and feathers can not be considered "Welfare tests". Keel bone scoring on two (or three?) point scale has low resolution. In cages deformities and bends, rather than fractures, are more common. Were those recorded?

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

The aim of your study was to determine whether stocking density affects egg production and quality from laying hens. Stocking density and the welfare of laying hens are of great importance to producers, retailers and consumers, so this is important research. The choice between bird welfare and productivity requires some solutions/compromises. Your research points to such possibilities.
The manuscript is well prepared, the introduction introduces the reader to the topic. The methodology is elaborate, includes a great deal of measurement, the results are meticulously described. The discussion and summary explain the results well. Overall, I have no major comments. I have included minor suggestions in the text of the manuscript.

Comments for author File: Comments.pdf

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Line 19: "Furthermore, feather scores decreased as 19 density increased." What does a decrease in feather scores mean? Is it good or bad?
Line 47: A short explanation of the difference between battery cages and those cages would be helpful
Line 47-52: Does the UEP use the word 'optimize'? If not - I think it is a little bit misleading. A cage of this recommended size is not equal to an optimized animal welfare
Line 115: Please explain 'USDA'
Line 124: Please clarify if egg quality was assessed by measuring one egg per week per replicate or six eggs per week per replicate.
Line 146: Please clarify if the same birds were blood sample at all occasions 
Line 181: Please re-phrase "The latency to feed test was performed by taking the novel object presented in Figure 1 and placing two in the feeder". It is not clear how many objects that was placed in the feeder.
Line 204: Please clarify if this was before or after culling
Line 270: Please explain "grade A eggs"
Line 276: Please explain "grade B eggs"
Line 279-285: Please specify S, M, L and XL with actual measurements
Table 4-10: Please explain why statistical significance <0.05 is indicated with different letters instead of for example one letter or a star. The reader does not understand the difference between the letters.
Line 399-400: Please provide references on that stress in commercial hybrids alters egg production.
Line 540: affected

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

The study is very comprehensive and clearly reflects a substantial amount of work. It presents data that could be of value for the industry. However, the manuscript requires a major revision in data analysis and to improve clarity, consistency, and overall presentation.

In the title and throughout the article, you refer to 'stocking density.' However, the treatments are expressed in cm² per hen, which aligns more accurately with “space allowance” (since stocking density generally refers to kg/m2). Could you please harmonize the terminology accordingly along the document?

L60: Stress can be considered part of either welfare or health, depending on what it refers to—emotional stress or oxidative stress. Moreover, current recommendations suggest that animal welfare should primarily be assessed through animal-based measures (e.g., keel bone damage, footpad dermatitis, novel object test, etc), complemented by metabolic indicators or hormonal markers. In my view, oxidative stress is more closely related to health, as it can lead to inflammation. I suggest rewriting the paragraph to reflect these considerations more accurately and harmonize then along the document.

L68: It would be more appropriate to use “keel bone damage” instead of “breakages,” as “damage” encompasses both fractures and deviations, providing a more comprehensive and accurate description.

L72: Heterophil and lymphocyte ratio instead of levels?

L74: It would be more accurate to use “feather pecking” instead of “cannibalism.” Cannibalism is a more severe outcome that typically follows uncontrolled feather pecking, which can escalate to bleeding and even death. Using “feather pecking” better reflects the earlier stage of the behavior and allows for a more precise assessment of welfare concerns.

Section Material and methods: could you please consider re-arranging the subsections? Specifically, the fearfulness test is an integral part of the welfare assessment, as it provides insight into the emotional welfare of the hens in addition to their physical welfare. Incorporating this distinction would enhance the clarity and depth of your methodology.

L86-88: Please, add code and date.

L92-95: Decimals are meaningful in this context. Additionally, please provide information on the genetics of the hens and their initial body weight. It would also be helpful to clarify how the hens were reared during the pullet phase—specifically, whether they were raised in colony cages or on the floor—as this background can influence their adaptation and welfare outcomes.

Table 1: I would appreciate it if the values could be rounded to three significant figures for consistency.

L115-117: Please, briefly explain, for non-American readers, what the grades and size classifications according to the USDA entail. Providing this clarification would ensure that the methodology is accessible and understandable to a wider range of readers.

L117-119: Move to section about statistical analysis

L121: Please specify the reasons of injury or sickness you found. This is fully relevant for the welfare/health assessment.

L146: Could you please specify the type of needles used, including their characteristics and brand? Providing this information would support reproducibility and help other researchers replicate your study accurately.

L148: Substitute H/l for H:L to be consistent along the document.

L155-156: Please, specify the time and the g and the centrifuge used.

L158: Please, specify the kit used.

L192-193: What kind of damage? Fracture, deviation. How many fractures and where? Why to score it as 0 or 2 and not 0 or 1 (absence/presence)?

L193: Could you please clarify whether your assessment evaluated toe pecking behavior or the occurrence of missing toes?

L195: Could you please clarify whether you evaluated bumblefoot, as it is an important welfare consideration for hens raised in colony cages?

L197-198: could you please clarify whether the evaluation distinguishes between healed and unhealed conditions? This distinction is particularly important when assessments are conducted at multiple timepoints throughout the animals’ lifespan to ensure accurate welfare evaluations

L197-199: Something is wrong in this sentence.

L189-202: would it be possible to include illustrative images of the scoring system used? This could enhance clarity and provide additional context for readers.

L204: Could you please specify the method used for euthanizing the birds?

L234-243: The statistical approach used does not appear to be appropriate for a study of this nature. For production and egg quality variables, a mixed model for repeated measures would be more accurate and better reflect the experimental design. The model should include space allowance and hen age, as well as their interaction, as fixed factors, and incorporate the id of the cage as random effect to account for repeated measurements. Welfare parameters, given their scoring system, should not be analyzed using linear models but rather with multinomial logistic models. The analysis of mortality should not be neglected here. Additionally, for histology, gut health, and gene expression data, it would be beneficial to treat the cage ID within each treatment as a random effect. This approach would help account for potential clustering of data and improve the robustness of the analysis.

Table 2 to 6: Please, revise if the space allowance is truly expressed in inches2 or cm2.

Table 3: I would appreciate it if the values could be rounded to three significant figures for consistency. Is It possible to have % above 100% in hen-day and hen-housed production?

L526: It might be feasible but requires training and testing to check intra and inter-observer repeatability.

L526-538: Could you please add the appropriate references to support these claims?

L518: unknown?

Discussion: Hen welfare in colony cages should not be assessed based on performance or physical injuries. While physical harm is a concern, cages typically induce significant emotional stress, primarily manifested as fear responses due to the restricted environment and potential aggressions from conspecifics. This poor emotional state often leads to abnormal behaviors, such as feather pecking or cannibalism, which stem from frustration. The inability to perform natural behaviors such as nesting, perching, foraging, and dust bathing further exacerbates the mental and emotional distress experienced by the hens. It is crucial to recognize that animal welfare encompasses both physical and emotional well-being. I encourage a deeper exploration of the psychological aspects of cage confinement. Expanding on this issue will provide a more holistic understanding of hen welfare in cage systems.

Conclusions: harmonise between space allowance or stocking density.

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 4 Report

Comments and Suggestions for Authors

• The abstract can be improved for clarity and focus. It would be helpful to begin by stating that stocking density is one of the major welfare concerns in laying hens across all production systems, which is why lawmakers, retailers, and consumers are increasingly concerned about this issue. Following this, the aim of the study is mentioned as identifying the effects of stocking density on white egg layer production and egg quality. However, since welfare and health are also evaluated in the study, why not include this as part of the study's objective? Aligning the welfare aspect with the first sentence would create a more cohesive and comprehensive presentation of the study’s goals. Please, remove also the decimals from the stocking densities tested. Additionally, the abstract does not briefly mention the materials and methods used or what specific aspects were analyzed in the study. Talk about reducing stocking density and not increasing space in the abstract and along the paper.

 

• The manuscript should specify whether the colony cages used include any environmental enrichment elements such as nest boxes, perches, dust baths, or claw-shortening devices. If not, in what way do they differ functionally from conventional California cages, apart from accommodating more birds? This distinction is important for a proper assessment of their impact on animal welfare.
• L55: Differences of a few tenths of a cm² per bird are practically imperceptible. To improve clarity and readability, it is recommended to round all values to meaningful figures. Also, do these values correspond respectively to white and brown hens? If so, this should be explicitly stated in the text to avoid confusion.
• L55: The expression “optimize welfare” needs to be substantiated. Please specify based on which criteria welfare is considered optimized (e.g., natural behavior expression, mortality rates, productivity, corticosterone levels, etc.) Clarifying this would strengthen the statement and facilitate scientific evaluation.
• L58: In the European Union, colony cages as described are forbidden since 2012 independently of the stocking density used. Please, rephrase it.
• L59: Please convert the unit “in2” to “cm²” to ensure consistency with the rest of the manuscript and to comply with SI unit standards, particularly for an international readership.
• L60: The phrase “to achieve proper welfare” is misleading in this context. EU legislation establishes minimum requirements for the protection of animal welfare, but compliance with these standards does not necessarily guarantee that the welfare of laying hens is fully ensured. It would be more accurate to state that these are baseline conditions intended to prevent major welfare issues, rather than to actively promote high welfare.
• L66: Please consider introducing how animal welfare is currently assessed in poultry. Welfare encompasses both physical and mental health. You could clarify that your study will evaluate indicators such as footpad dermatitis, toe lesions, and keel bone damage to address physical welfare, and also include behavioral measures such as fear responses using the test you designed. After establishing this, you can then discuss how health is assessed as a component of overall welfare.
• L71: typographical error: the o in Oxidative should be lowercase.
• L76: The manuscript currently emphasizes stress and health, but it is important to explicitly include welfare as a broader concept that integrates both physical and psychological aspects. Please ensure that welfare is addressed as a distinct and comprehensive dimension in the assessment.
• L101-104: Please remove the decimal places from the values presented in these lines, as they do not add meaningful precision and may reduce readability. Rounding to whole numbers would be more appropriate and consistent with the level of accuracy required for this context.
• L221-222: Please specify in the methods section that you assessed both keel bone fractures and deviations. Additionally, in the discussion section, it should be clearly stated that only deviations were observed at the end of the study. This distinction is important for accurately interpreting the welfare implications.
• L129-130: “and had to be transformed for statistical analysis as the mortality data was not normally distributed” should go to statistical analysis section.
• L205: The title of Section 2.6 should be revised to something like “Welfare Assessment” in order to reflect that the section addresses both physical and emotional aspects of welfare. This would provide a more accurate and comprehensive framing of the content.
• L230-232: The description for score 3 includes a clear size criterion (≥5 cm in diameter) but score 2 lacks a defined reference for feather loss extent. Is there a diameter or specific area threshold that differentiates moderate wear (score 2) from slight (score 1)? Please clarify this in the methods to ensure reproducibility and consistency in scoring.
• Since you confirmed in your previous response that toe damage (wounds or breakages) and bumblefoot were assessed but no cases were found, please ensure this is clearly stated in the Materials and Methods Additionally, it should be mentioned in the Results and briefly discussed in the Discussion section that no such conditions were observed. This clarification will improve the transparency and completeness of your methodology and findings.
• L581: toe instead of tow?
• L593: Hens kept in cages often exhibit fewer physical injuries due to the limited and controlled nature of their environment. However, they commonly experience severe feather pecking, which is typically a redirected behavior resulting from their inability to express innate behaviors. This issue is further aggravated by the frustration associated with movement restriction, especially as stocking density increases. I believe this aspect should be more strongly emphasized in the discussion, as it represents a significant welfare concern in caged housing systems.
• In addition, comparing stocking density with lower densities to assess welfare and health is important, it is also worth noting that in laying hens, welfare could be evaluated based on their lifespan rather than just productivity levels. This is because certain genetic lines may continue producing eggs even under poor welfare conditions. Therefore, relying solely on productivity as an indicator of welfare might not fully capture the impact of stocking density on the hens’ overall well-being. This point should be discussed in the paper to provide a more comprehensive view of the factors influencing welfare. Although there is a forthcoming paper focused on behavioral and psychological aspects, it would still be valuable to briefly introduce this topic in the Discussion section of the current paper. This would clarify that welfare is multidimensional and that there are additional parameters to consider beyond those evaluated here, providing a more holistic view.
• L681: less space is higher stocking density.
• L689: feather pecking increased when the stocking density was high not low, right?. The phrase “perhaps due to an increase in cage abrasion” is speculative and lacks supporting evidence in the current manuscript. I recommend removing this part unless a source or data can substantiate the claim.
• L690: Allowing for more space per hen by reducing stocking density. To rephrase.
• Please include the stocking density expressed as cm²/hen in all relevant tables. Without this specification, the values could be confused with general space allowance (cm²), which may lead to misinterpretation.
• Conclusions: In the conclusions section, I recommend organizing the concepts according to the order in which they appear in the manuscript to improve clarity and coherence. Please also specify which parameters were affected by stocking density and which were not. Additionally, the conclusions could be strengthened by acknowledging that other relevant aspects of welfare remain to be evaluated in future studies, which would provide a more comprehensive understanding of the impact of stocking density on laying hens.

Author Response

Please see attachment

Author Response File: Author Response.pdf