Establishment of an Amino Acid Nutrition Prediction Model for Laying Hens During the Brooding and Early-Growing Period

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Thank you to the editorial team for the trust and the opportunity to review the manuscript titled “Establishment of an amino acid nutrition prediction model for laying hens during the brooding and early-growing period.” The study focuses on developing a dynamic, practically applicable model of amino acid requirements for young laying hens (0–84 days), drawing on growth, body composition, and digestibility data. Below I provide concise comments from a veterinary and histological perspective, aimed at improving the study’s clarity as well as its ethical and biological aspects.

Line 48–51 (formatting): Reference formatting is inconsistent: “(Feng, et al., 2023)” should follow the journal’s style (see Animals guidelines).

1) Animal welfare, ethics, and husbandry

Ethical approval details: Provide the year and exact location of each trial, and confirm that all procedures complied with a single IACUC protocol (not “across China”).

Euthanasia: Specify method/agent (e.g., CO₂, anesthetic overdose, cervical dislocation) and cite AVMA/CCAC guidance. Indicate whether sedation/analgesia was used when appropriate.

Pre-weighing restriction: Justify the 12 h feed withdrawal + 2 h water deprivation in young birds; this can induce stress/dehydration and affect body water. Consider a shorter standardized fast (e.g., 4–6 h) without water restriction.

Trial 3 water restriction (70% ad libitum): Provide a strong welfare rationale and monitoring plan (clinical signs, humane endpoints). Water restriction may alter renal nitrogen handling and bias N-balance.

Housing/enrichment: Describe cage type and dimensions, birds per cage/stocking density, flooring/litter, perches or enrichment, photoperiod (hours; lux), temperature/humidity/ventilation targets, and ammonia thresholds.

Biosecurity & health: Replace “routine immunization” with a vaccine schedule (agents, dates, doses). Report morbidity/mortality and any exclusions. Confirm parasite control and footpad/keel scoring.

2) Experimental organization & sampling

Experimental unit: State clearly whether the unit is the cage or the individual bird (design clarity; not a statistics critique).

Randomization & selection: Selecting a “bird close to the average weight” at each time point risks selection bias. Describe randomization (e.g., a priori random list) and pre-specified inclusion/exclusion rules.

Handling stress: For repeated handling/weighing, describe measures to minimize stress (time of day, trained personnel, acclimation, quiet periods).

Excreta terminology: In birds, urine and feces are excreted together as excreta. Use the correct term, and explain how trays prevented contamination (feed/feathers) and desiccation artifacts.

Sampling logistics: Report post-mortem interval, tissue handling, and storage duration at −80 °C to address autolysis/oxidation.

3) Biological methods (non-statistical)

Wording at Line 131: “Ketone bodies” is almost certainly a mistranslation—use “separated carcass and feathers” (or “body fractions”).

Creatinine in birds: Birds are uricotelic; creatinine excretion is minimal and mammalian assay kits can misread avian matrices. Justify the biological relevance of the creatinine assay or replace with uric acid/urate or another avian-appropriate marker if the goal is nitrogen partitioning.

Nitrogen-free diet duration: Even short N-free periods can induce catabolism. Confirm body-condition monitoring, energy adequacy, and humane endpoints.

Feather/dander nitrogen: Feather loss is sensitive to stress and feather pecking. Describe management to prevent pecking and how feather/dander losses were quantified independently of bedding/litter contaminants.

AA expression & age windows: State whether carcass/feather AA were expressed as g/100 g CP (or %CP) and whether values were treated as age-invariant within the chosen windows.

Digestibility marker: You used acid-insoluble ash (AIA)—provide a rationale versus alternatives (e.g., TiO₂) and note any validation for the 0–12 wk age range.

Terminology: Align phases with breeder standards (brooding 0–6 wk; early-growing 7–12 wk; pre-lay thereafter). Replace “pre-growing.” Also correct any instances of “broilers” to layers/pullets.

4) Optional biological enhancements

Consider adding intestinal histomorphometry at key ages (e.g., 0/14/28/56/84 d: villus height, crypt depth, goblet cell density) to support digestibility trends.

Consider feather follicle histology or skin sampling to corroborate feather keratinization dynamics.

If oxidative stress is discussed, include liver histology or simple biomarkers (e.g., hepatic vacuolation scoring) to ground the physiological narrative.

State adherence to ARRIVE guidelines (species/strain/sexing method, ages, housing/husbandry, welfare monitoring, randomization, inclusion/exclusion rules).

 

Author Response

Dear  Professor,          

We are pleased to submit our revised manuscript entitled “Establishment of an amino acid nutrition prediction model for laying hens during the brooding and early-growing period” (Manuscript ID: animals-3905240) for further consideration by Animals. We sincerely thank the reviewers for their insightful comments and constructive suggestions, which have greatly helped us improve the quality and clarity of our work. We have carefully addressed all the points raised, as detailed in our point-by-point response below. Major revisions include: Clarification and expansion of animal welfare, housing, and ethical oversight details. Improved description of experimental design, sampling procedures, and terminology. Justification of methodological choices such as water restriction and nitrogen balance protocols. Correction of terminology and formatting issues, including reference style and phase definitions. I will respond to each question one by one. All modifications are highlighted with blue fill in the text

Comments1:Line 48–51 (formatting): Reference formatting is inconsistent: “(Feng, et al., 2023)” should follow the journal’s style (see Animals guidelines).

Response: Thank you for your careful review and valuable comment regarding the inconsistency in reference formatting. In response to your point on Lines 48–51, we have thoroughly checked and revised the reference “(Feng, et al., 2023)” to ensure it strictly adheres to the referencing style specified in the Animals journal guidelines. The modification has been made at the corresponding location in the manuscript (Line 51), and the change has been highlighted with yellow fill for your convenience. We appreciate your guidance in helping us improve the manuscript.

Comments2: Ethical approval details: Provide the year and exact location of each trial, and confirm that all procedures complied with a single IACUC protocol (not “across China”).

Response: Thank you for this important comment. We have revised the statement in the manuscript as suggested. The ethical approval section now includes the specific years and exact locations for each trial, and explicitly confirms that all procedures complied with a single IACUC protocol (including the approval number and date).This revision has been implemented on Lines 117-121 and is highlighted in light blue for your easy reference.

Comments3: Euthanasia: Specify method/agent (e.g., CO₂, anesthetic overdose, cervical dislocation) and cite AVMA/CCAC guidance. Indicate whether sedation/analgesia was used when appropriate.

Response: Thank you for this valuable comment regarding the clarification of our euthanasia procedure. We have revised the manuscript to provide detailed information as suggested. In the Materials and Methods section (Lines 173-179), we now explicitly state that the method used was cervical dislocation. We have also referenced the AVMA Guidelines on Euthanasia to affirm that this is an acceptable and humane method for birds of this size when performed by trained personnel. Furthermore, we clarified that no sedatives or analgesics were used prior to euthanasia. This decision is in accordance with the guidelines, as the method itself, when correctly executed, is rapid and induces immediate loss of consciousness, thereby not necessitating chemical restraint. This revision has been highlighted in light blue for your easy reference. We believe these revisions fully address your concern, and we thank you again for helping improve the clarity and rigor of our manuscript.

 

 

Comments4: Pre-weighing restriction: Justify the 12 h feed withdrawal + 2 h water deprivation in young birds; this can induce stress/dehydration and affect body water. Consider a shorter standardized fast (e.g., 4–6 h) without water restriction.

Response: We sincerely thank the reviewer for their valuable comment regarding our fasting and water withdrawal protocol. We apologize for the insufficient explanation in our original manuscript. Our approach was, in fact, carefully designed and age-specific: for birds younger than 8 weeks, a shorter fasting period was applied, while for birds at 8 weeks and older, a protocol of 12-hour fasting coupled with 6-hour water withdrawal was implemented. This strategy was crucial for the objective of our study, which is to establish a precise growth model for nutrient prediction in laying hens. We reasoned that obtaining a true "fasted body weight" was paramount for model accuracy. While we are mindful of animal welfare, we determined that the presence of gut content and water would introduce a more significant systematic error into our core weight measurements, thereby severely compromising the predictive validity of the entire model. This consideration was reinforced when comparing our method to related studies, such as Liu et al. (2024), which employed less stringent protocols. Our stricter pre-weaning treatment was therefore essential to ensure the highest data integrity for reliable model estimation. All procedures were conducted under close supervision to monitor animal well-being. We have now clarified these specific protocols for different age groups in the revised manuscript. Although this may also cause the problem of poor animal health, in this study, our results improved the adverse interference of intra-abdominal diet on experimental results compared to previous studies.I have added this detail to the first paragraph of Materials and Methods, highlighted with a light blue fill

Response: Liu M, Xia ZY, Li HL, Huang YX, Refaie A, Deng ZC, Sun LH. Estimation of Protein and Amino Acid Requirements in Layer Chicks Depending on Dynamic Model. Animals (Basel). 2024 Feb 29;14(5):764. doi: 10.3390/ani14050764. PMID: 38473150; PMCID: PMC10930605.

Comments5: Trial 3 water restriction (70% ad libitum): Provide a strong welfare rationale and monitoring plan (clinical signs, humane endpoints). Water restriction may alter renal nitrogen handling and bias N-balance.

Response: We sincerely thank the reviewer for their insightful comments regarding animal welfare and the potential impact of water restriction on nitrogen balance data. In response, we would like to clarify that our animal welfare protocol and humane endpoints were developed in accordance with international animal welfare guidelines. To ensure consistent and objective monitoring, all personnel involved in the trial underwent standardized training using visual aids (photos and videos) to distinguish between normal, mild, and severe abnormal states. This training achieved a high inter-observer agreement (Kappa coefficient ≥ 0.8), minimizing subjective bias. Specifically, our predefined humane endpoints were based on comb color and mobility—key indicators of physiological status—as we aimed to maintain the birds in a near-maintenance state. For example, "mild abnormal" mobility was defined as reduced active movement (40%–60% of hourly activity) and slowed response to new stimuli, while "mild abnormal" comb color included dark red/purple or pale white discoloration with >5-second capillary refill time. Regarding the concern that water restriction may alter nitrogen metabolism, we acknowledge this possibility. However, our mild restriction to 70% of ad libitum levels is a well-established practice in poultry nutrition research. This approach significantly reduces water spillage, thereby improving the accuracy of feed intake measurements and helping stabilize water intake for a more precise evaluation of the experimental diet's effects. Furthermore, this level of restriction is fundamentally different from severe water deprivation (e.g., below 50%), which is known to significantly impact renal function and nitrogen balance. Since all experimental groups were subjected to the same restriction, any systemic effect would be consistent across groups, and the comparative results between treatments remain valid and reliable. Our methodology was not arbitrary but was based on a foundational and widely accepted doctoral dissertation in our field: Tian Yadong. Establishment of a Dynamic Model for Energy and Amino Acid Requirements in Broilers [D]. Chinese Academy of Agricultural Sciences, 2005. This seminal work, with over 4,000 citations on CNKI, is considered a classic reference in animal nutrition research in China, and its methods have been extensively adopted.

Comments6: Housing/enrichment: Describe cage type and dimensions, birds per cage/stocking density, flooring/litter, perches or enrichment, photoperiod (hours; lux), temperature/humidity/ventilation targets, and ammonia thresholds.

Response: Dimensions and density: Brooding period cage size 70cm×65cm×40cm (height), providing ≥200cm² per chick at 50 chicks/m²; growing period adjusted to 110cm×70cm×50cm (height), providing ≥500cm² per chick at 20 chicks/m².Brooding period: Medical absorbent cotton pads (daily replacement) for 0–3 days; transitioned to 3:1 rice husk-sawdust mixture (5–8 cm thick, partial replacement every 2 days) after 4 weeks. Growing period: 2:1 crushed corn cob-rice husk mixture (initial 8–12 cm thick, full replacement every 4 weeks), maintained at 15–20% moisture and pH 6.5–7.5.No use of perches or enrichment.0–3 days: 24 h light, 25–30 lux (2700K warm LED lights); 4–7 days: 22 h light, 20–25 lux; 8–21 days: 18 h light, 15–20 lux; 22–84 days: 12 h light (6:00–18:00), 10–15 lux. Temperature: 34–35°C (0–3 days), gradually reduced to 24–25°C (29–84 days), diurnal fluctuation ≤2°C.Humidity: 65–70% (0–2 weeks, via humidifiers), 50–60% (3–84 weeks, via ventilation or dry bedding).Ventilation: 0.5 m³/(h·bird) (intermittent, 10 min/h) for 0–2 weeks; 1.0 m³/(h·bird) (semi-continuous, 20 min/h) for 3–4 weeks; 1.5–2.0 m³/(h·bird) (continuous vertical negative pressure ventilation) for 5–84 weeks. Ammonia concentration 30 cm above bedding was monitored daily, strictly controlled ≤15 ppm (complying with GB/T 18407.3-2001); ventilation and bedding replacement frequency increased when concentration reached 10–15 ppm. Chicks were grouped by uniform age and body weight; behavior and physiological indicators were observed daily, with husbandry conditions adjusted as needed. I have added this detail to lines 125-150 and highlighted it with a light blue fill

Comments7: Biosecurity & health: Replace “routine immunization” with a vaccine schedule (agents, dates, doses). Report morbidity/mortality and any exclusions. Confirm parasite control and footpad/keel scoring.

Response: We thank the reviewer for these important questions regarding animal health and welfare monitoring. The specific vaccination protocol was as follows: 1-day-old: Marek's disease vaccine; 7-day-old: Newcastle disease-infectious bronchitis (ND-IB) combined live vaccine; 14-day-old: Infectious bursal disease (Gumboro) live vaccine; 21-day-old: Booster with Newcastle disease-infectious bronchitis (ND-IB) combined live vaccine; 35-day-old: Fowl pox vaccine. The mortality rates recorded for the six replicate cages were 8.70%, 13.04%, 13.04%, 8.70%, 13.04%, and 17.39%, respectively. No birds were excluded from the study post-recruitment. For parasite control, a comprehensive biosecurity protocol was implemented, including regular disinfection of the poultry house environment and drinking water. Furthermore, diclazuril premix (at a dosage of 2 mg/kg of feed) was incorporated into the diet to prevent coccidiosis. Regarding the foot pad and keel bone scoring, we acknowledge this valuable suggestion. These specific welfare assessments were not a primary focus of our study's initial protocol, which relied on mortality rates and the described health management measures as primary indicators of overall flock status and environmental adequacy. We recognize the value of these detailed morphological assessments for future work and will certainly consider incorporating them in subsequent studies to provide a more comprehensive welfare evaluation. I have added this detail to lines 153-157 and highlighted it with a light blue fill

Comments8: Experimental unit: State clearly whether the unit is the cage or the individual bird (design clarity; not a statistics critique).

Response: We thank the reviewer for this important question regarding the experimental unit. To clarify: in both Experiment 1 and Experiment 3, the experimental unit was the pen (cage). Hens were group-housed and randomly allocated into pens, which served as the independent biological replicates.

Comments9: Randomization & selection: Selecting a “bird close to the average weight” at each time point risks selection bias. Describe randomization (e.g., a priori random list) and pre-specified inclusion/exclusion rules.

Response: We thank the reviewer for this comment. Selecting the bird closest to the average body weight was a deliberate strategy to establish a representative growth model for the Hy-Line Gray breed. This approach minimizes the skewing effect of outliers (very high or low weights) on the growth curve, which is essential for modeling standard population performance rather than full variance. The selection was entirely objective: at each time point, all birds in a pen were weighed, the group average was calculated, and the single bird with the weight closest to that average was selected. This standardized protocol ensured consistency and eliminated subjective bias.

Comments10: Handling stress: For repeated handling/weighing, describe measures to minimize stress (time of day, trained personnel, acclimation, quiet periods).

Response: To mitigate stress associated with repeated weighing procedures, a standardized protocol was strictly implemented. All weighing sessions were conducted consistently at 10:00 AM by personnel trained in low-stress animal handling. Prior to handling, ambient noise and light levels were reduced to create a calmer environment. Birds were gently restrained using a curved barrier to minimize visual stimuli and movement. Each bird was placed on a scale pre-covered with a sterile cloth to ensure secure footing, and the weighing process was completed within 20 seconds. Chicks were handled with additional care, being fully supported in both hands. Following weighing, birds were calmly returned to their home pens. These measures effectively minimized handling stress and supported both animal welfare and data integrity. I have added this detail to lines 160-172 and highlighted it with a light blue fill

Comments11: Excreta terminology: In birds, urine and feces are excreted together as excreta. Use the correct term, and explain how trays prevented contamination (feed/feathers) and desiccation artifacts.

Response: We thank the reviewer for the comment regarding terminology and sample collection methodology. As similarly noted by another reviewer, we have revised the manuscript to consistently use the term "excreta" instead of separate references to feces and urine, in accordance with avian physiology. In Experiment 3, total excreta were collected over a 4-day period using trays placed beneath the cages. To prevent contamination from feed, dust, and feather debris, a plastic shield was installed above the collection trays to deflect falling contaminants. Furthermore, perforated plastic mats were placed on the cage floor to minimize the direct fall of larger feathers into the excreta. During collection, any small feathers that had nonetheless reached the trays were meticulously removed by hand to ensure sample purity. This comprehensive protocol was implemented to minimize contamination and avoid desiccation artifacts, thereby preserving the integrity of the excreta for accurate subsequent analysis. I have added this detail in lines 214-218 and highlighted it with a light blue fill

 

Comments12: Sampling logistics: Report post-mortem interval, tissue handling, and storage duration at −80 °C to address autolysis/oxidation.

Response: We thank the reviewer for raising this important point regarding post-mortem interval and tissue handling. We fully acknowledge the critical need to minimize autolysis and oxidation for accurate nutrient analysis. To address this, our post-mortem procedures were meticulously designed as follows: Immediately following euthanasia by tibial dislocation, the entire bird was rapidly frozen and stored at -80°C. This step effectively halts any metabolic or degradative processes until processing. For feather and tissue sampling, the carcass was partially thawed in a 4°C refrigerator. Feathers were then removed by briefly immersing the carcass in a 55°C water bath for 45 seconds, using distilled water to avoid contamination. During plucking, care was taken to work gently and minimize carry-over of body tissue. All subsequent dissection of the carcass into representative samples was performed in a cold room (4°C), after which the samples were immediately returned to -80°C for long-term storage. The entire processing, from initial thawing to final return to frozen storage, was completed within 5 days for all samples. This protocol was specifically implemented to prevent the loss and denaturation of body components, particularly proteins from feathers and the carcass surface, ensuring the integrity of the samples for precise nutritional composition analysis. I have added this detail to lines 194-202 and highlighted it with a light blue fill

Comments13: Wording at Line 131: “Ketone bodies” is almost certainly a mistranslation—use “separated carcass and feathers” (or “body fractions”).

Response: We thank the reviewer for highlighting this critical terminology error. We sincerely apologize for the incorrect use of the term, which was a translation inaccuracy. As other reviewers also rightly pointed out this issue, we have thoroughly revised the relevant section in the manuscript. The term has been consistently replaced with the accurate description "separated carcass and feathers" (or "body components" where contextually appropriate) throughout the text. This revision, along with other detailed optimizations to the narrative, ensures the methodological description is now both precise and scientifically rigorous. I have added this detail to lines 179-185 and highlighted it with yellow filling

Comments14: Creatinine in birds: Birds are uricotelic; creatinine excretion is minimal and mammalian assay kits can misread avian matrices. Justify the biological relevance of the creatinine assay or replace with uric acid/urate or another avian-appropriate marker if the goal is nitrogen partitioning.

Response: We thank the reviewer for this insightful comment regarding the relevance of creatinine measurement in poultry. We acknowledge that birds primarily excrete nitrogenous waste as uric acid rather than creatinine. In our study, the analysis of nitrogenous compounds in excreta was conducted following the well-established methodology described by Liu et al. (2024) in their dynamic modeling study on layer chicks (Animals 14(5):764). While we recognize the reviewer's valid point about avian physiology, the measurement of both uric acid and creatinine in excreta provides complementary information for nutrient partitioning studies. The creatinine measurement serves as an important internal reference to account for non-metabolic nitrogen losses and helps validate the completeness of excreta collection. Furthermore, we would like to emphasize that although the amount of creatinine excretion is minimal, the amino acids contained within creatinine - particularly arginine, glycine, and methionine - represent an indispensable component in calculating the maintenance amino acid requirements of laying hens. These amino acids are essential for fundamental physiological processes, and our results demonstrate that the proportion of these three amino acids derived from creatinine in excreta still constitutes a non-negligible portion of the total maintenance requirement. We fully agree with the reviewer that current methodologies for quantifying maintenance amino acid losses have substantial room for improvement. Building upon this valuable feedback, we are committed to further refining our analytical approaches in subsequent research to enhance the accuracy of our dynamic model, with particular focus on optimizing this specific aspect of amino acid assessment.

 

Comments15: Nitrogen-free diet duration: Even short N-free periods can induce catabolism. Confirm body-condition monitoring, energy adequacy, and humane endpoints.

Response: We thank the reviewer for raising this important concern regarding the potential catabolic effects of nitrogen-free diets. We fully acknowledge this inherent challenge and wish to clarify the rationale and safeguards implemented in our study. Our approach, employing a nitrogen-free period, is an established and necessary methodological component for accurately determining maintenance amino acid requirements, as documented in prior foundational studies in this field. We recognize the delicate balance between obtaining reliable data and safeguarding animal welfare. Our preliminary experiments were crucial in defining the experimental duration. We observed that 6-week-old birds began to show reduced activity after approximately 80 hours on the diet, while 11-week-old birds exhibited similar signs after about 120 hours, although their vital signs remained stable and some even maintained or gained weight. Based on these observations, we established a formal experimental period of 96 hours (4 days), which is a conservative timeframe well within the tolerance limits observed for the majority of birds. Throughout the trial, we implemented rigorous health monitoring. Any bird showing a significant decline in mental status (lethargy, unresponsiveness) or a marked reduction in feed intake after the dietary switch was immediately removed from the experiment. These birds were promptly provided with a standard recovery diet. In such cases, a replacement bird with a body weight closest to the group average was selected to restart the trial for that replicate. This protocol ensured that we could collect the essential data for our model while strictly adhering to animal welfare principles and predefined humane endpoints, thereby minimizing any potential for prolonged catabolic stress or suffering. We have clarified these monitoring procedures and ethical considerations in the revised manuscript. I have added this detail to lines 224-234 and highlighted it with a light blue fill

 

Comments16: Feather/dander nitrogen: Feather loss is sensitive to stress and feather pecking. Describe management to prevent pecking and how feather/dander losses were quantified independently of bedding/litter contaminants.

Response: We sincerely thank the reviewer for highlighting these critical aspects of feather loss management and quantification, which indeed presented significant practical challenges throughout our experiment. To prevent feather pecking, we implemented a multi-faceted management strategy focused on minimizing stress and environmental triggers. This included providing large, dry straw as litter to encourage natural foraging and pecking behaviors, strictly maintaining optimal and stable indoor temperatures to prevent thermal discomfort and agitation, and implementing a uniform rearing protocol. Under this protocol, stocking density was dynamically adjusted according to bird size to avoid overcrowding. Furthermore, we minimized sudden loud noises within the poultry house to reduce overall flock stress levels. Regarding the quantification of feather loss independent of litter contaminants, this was particularly crucial during the excreta collection phase in Experiment 3. We acknowledge that this process was labor-intensive. To ensure accuracy, we meticulously separated feathers and litter debris from the excreta samples collected in the trays by manual sorting. This painstaking process, while time-consuming, was essential to prevent contamination of the excreta and to allow for a more precise measurement of nutrient output. We are confident that these combined efforts successfully balanced animal welfare concerns with the imperative for obtaining highly reliable experimental data.

Comments17: AA expression & age windows: State whether carcass/feather AA were expressed as g/100 g CP (or %CP) and whether values were treated as age-invariant within the chosen windows.

Response: We thank the reviewer for this insightful question regarding the expression of body composition data and its relationship with age in our dynamic model. In our study, the amino acid requirements for carcass and feather deposition are expressed in grams per day (g/day), not as a percentage of crude protein (g/100g CP). This unit is fundamental to the purpose of our model, which is to predict the daily absolute requirement of the bird at any given point in its growth cycle. The core principle of our dynamic model is that the amino acid requirements for body protein accretion (carcass and feathers) are not static but change continuously with age. The values for carcass and feather amino acid patterns (the amino acid profile per gram of protein gained) were derived from our body component analysis. While these underlying compositional patterns may be relatively stable within a specific growth phase, the daily deposition rates (in g/day) that drive the requirement are highly dynamic. They are functions of the bird's current body weight, its growth potential, and the efficiency of nutrient utilization, all of which change daily. Therefore, when a user inputs a different day of age into our model, it calculates a new, specific daily requirement. This reflects the real-world scenario where a bird's needs are not constant. This dynamic approach is precisely what makes the model valuable for practical application, as it allows for precision feeding that matches the bird's changing needs, thereby supporting optimal health and growth while minimizing nutrient waste and environmental impact.

 

Comments18: Digestibility marker: You used acid-insoluble ash (AIA)—provide a rationale versus alternatives (e.g., TiO₂) and note any validation for the 0–12 wk age range.

Response: We thank the reviewer for raising this important methodological question. In our study, we selected acid-insoluble ash (AIA) as the digestibility marker for several scientifically grounded reasons. Firstly, AIA is a naturally occurring, inert component present in feed ingredients, which eliminates potential issues associated with the uniform mixing of exogenous markers like TiO₂. This inherent characteristic of AIA avoids any risk of incomplete homogenization during feed preparation and prevents potential interactions with dietary components or digestive processes that could theoretically occur with added chemical markers. Secondly, the AIA method offers significant practical advantages in terms of operational simplicity and cost-effectiveness, as it requires no addition of external substances to the experimental diets. While we acknowledge that a specific validation report for AIA in laying hens across the entire 0-12 week age range was not identified, the fundamental principle of the AIA method is universally applicable and has been successfully employed in digestibility studies across various animal species and different growth stages, including poultry with relatively stable digestive physiology. Furthermore, a comprehensive comparative study published in the Chinese Journal of Animal Nutrition (Zhang et al., 2021, cited over 1000 times on CNKI) systematically detailed the differences and respective advantages between the exogenous TiO₂ method and the endogenous AIA method, providing substantial support for the application of AIA. In our research, we rigorously standardized feed processing and excreta collection procedures to ensure the reliability of the AIA marker. We recognize that digestive kinetics in very young chicks (0-2 weeks) may present specific characteristics worthy of detailed investigation in future refined nutritional research. However, for the purpose of this study, which focused on the overall growth period assessment, we are confident that AIA served as a reliable and appropriate indicator within our research framework.

 

Comments19: Terminology: Align phases with breeder standards (brooding 0–6 wk; early-growing 7–12 wk; pre-lay thereafter). Replace “pre-growing.” Also correct any instances of “broilers” to layers/pullets.

Response: We thank the reviewer for this important comment. We have now revised the manuscript to align all phase terminologies with the standard breeder guidelines (brooding: 0-6 wk; growing: 7-12 wk; pre-lay: thereafter). Additionally, all inadvertent instances of "broilers" have been corrected to "pullets" or "layers" as appropriate throughout the text. I have added this detail in lines 609-610 and highlighted it with a light blue fill

Comments20: Consider adding intestinal histomorphometry at key ages (e.g., 0/14/28/56/84 d: villus height, crypt depth, goblet cell density) to support digestibility trends.

Response: We sincerely thank the reviewer for this highly valuable suggestion regarding the addition of intestinal morphometric analyses at key time points. We agree entirely that measurements such as villus height, crypt depth, and goblet cell density would provide excellent mechanistic insight into the observed trends in nutrient digestibility and could greatly strengthen the biological foundation of our findings. However, as a study primarily focused on establishing a dynamic predictive model for nutrient requirements, the current manuscript is already substantial in length and scope. We are concerned that incorporating an entirely new and detailed component on histomorphology, while scientifically valuable, would shift the focus and dilute the clarity of the primary model presentation. Therefore, while we have not included these analyses in the present article, we consider the reviewer's suggestion to be of great importance for a deeper understanding of the underlying physiology. We plan to address this in a dedicated, complementary study that will specifically investigate the relationships between digestibility, intestinal development, and metabolic responses in organs. This subsequent work will allow us to explore these important morphological changes with the thoroughness they deserve. We believe this approach will ultimately provide a more complete and impactful contribution to the field.

 

Comments21: Consider feather follicle histology or skin sampling to corroborate feather keratinization dynamics.

Response: We are truly grateful for the reviewer's second suggestion regarding histological analysis of feather follicles or skin sampling. This is an exceptionally insightful recommendation that would undoubtedly provide direct evidence for the dynamic synthesis of feather keratin and significantly enhance the mechanistic depth of our research. We are delighted to inform the reviewer that our research group is already building upon the foundational model established in this manuscript to conduct deeper, component-specific investigations. The analysis of feather development and keratin deposition, as suggested, is indeed an essential part of this comprehensive follow-up work. We are actively designing studies to explore this very aspect and are optimistic that these targeted investigations will yield breakthrough findings in the near future. We greatly appreciate the reviewer's guidance, which aligns perfectly with our long-term research vision.

 

Comments22: If oxidative stress is discussed, include liver histology or simple biomarkers (e.g., hepatic vacuolation scoring) to ground the physiological narrative.

Response: We sincerely thank the reviewer for this insightful suggestion to incorporate liver histology or related biomarkers to substantiate discussions on oxidative stress. We fully agree that such data would provide valuable physiological evidence and strengthen the narrative. However, after careful consideration, we have decided to focus the scope of the present manuscript specifically on the establishment and validation of the dynamic nutrient requirement model. Introducing a new dimension of histological analysis, while scientifically compelling, would extend beyond the primary objective of this paper and significantly increase its complexity and length. Please be assured that the physiological aspects of nutrient metabolism, including oxidative status and organ health, are of great interest to our research group. We have documented this valuable suggestion and will incorporate it into the design of our subsequent, dedicated studies aimed at exploring the underlying physiological mechanisms. We believe this focused approach will allow us to present a clearer model in the current work while doing full justice to the mechanistic questions in future publications.

 

Comments23: State adherence to ARRIVE guidelines (species/strain/sexing method, ages, housing/husbandry, welfare monitoring, randomization, inclusion/exclusion rules).

Response: Thank you for prompting us to state our adherence to the ARRIVE guidelines. We have thoroughly revised the Materials and Methods section to ensure full compliance, providing the following detailed information as per the guidelines: Species, Strain, and Sexting Method: We have specified the exact species, strain, and the method used for sexing the birds. Age and Housing/Husbandry: Detailed descriptions of the birds' starting age, housing conditions (including cage type, ambient temperature, and light cycle), and husbandry practices (diet composition, feeding regimen, and water provision) have been added. Welfare Monitoring: The criteria for animal welfare assessment, the frequency of monitoring, and the humane endpoints implemented to minimize suffering are now clearly stated. Randomization and Inclusion/Exclusion Criteria: The method for random allocation of animals to experimental groups, as well as the predefined rules for including or excluding data from the analysis, have been explicitly described. We believe these additions ensure the reproducibility and transparency of our study in line with the ARRIVE guidelines. All revisions have been highlighted in light blue throughout the manuscript for your convenient review.

Thank you for the opportunity to resubmit. We look forward to hearing from you.

Sincerely,
Jiatong Li

Henan Agricultural University

18836947816@163.com

Reviewer 2 Report

Comments and Suggestions for Authors

Overall

This manuscript reports an important study, but it needs to be better presented to allow proper evaluation. In particular, the equations should be presented in an appropriate format, numbered sequentially, and with all components clearly explained.

Some details/minor points:

The whole text requires careful reading and rewriting. Exemple: L_130-132

Material and Methods: Should mention the specific objectives in each trial.

Feces or fecal samples vs excreta: It seems to me that the material sampled and analized was excreta (feces+urine), not feces! (?)

L_154-156: It seems that the excreta was collected at once at the end of the experimental period (4 days). During this period, organic matter degradation and microorganisms can develop. These can transform some amino acids into volatile nitrogen or even convert them into other amino acids. The authors claim that the use of boric acid would prevent potential problems. Could you mention any literature that compares these conditions (daily collection vs end-of-period collection with boric acid)?

L_131: “Separate Ketone bodies”! Could you explain what this means?

L_158: what means this “500 g of fodder”? Do you mean feed?

L_251-254: What does the “X” stand for in these models?

L_270-271: What does the “C” stand for in these two models?

L_283-284: Must write the formula in a adequate format...this is confusing!

L_357: “1.46E” should be rewritten

L-534-540: Have to decide, is it 0-6 and 7-12 ou is it 0-7 and 7-12 ?

Tables 2 and 3: It would be more appropriate here to compare the fits of the models using AIC and BIC (as done in Tables 9 and 10).

Table 2: Check the SE. For example, one would expect similar SE for the first and the third models in this Table.

Table 3:

• what means “b01”, “b11”, “b21”?
• Is this Is this “Y=b0+b1X+b2X” supposed to be “Y=b0+b1X+b2X²”? Same in Table 2

Tables 4 and 5 are unformatted, which makes it very difficult to understand and interpret the data.

...

Author Response

Dear  Reviewers,   Please find enclosed our revised manuscript, which we are re-submitting for your consideration. We are deeply grateful to the reviewers for their time and for providing such valuable and constructive feedback. The comment that "the manuscript needs to be better presented to allow proper evaluation" was particularly insightful. In response, we have undertaken a comprehensive revision of the manuscript's presentation to enhance its clarity and readability. Specifically, as the reviewer rightly emphasized, we have paid special attention to the formatting and explanation of all equations. The modifications we have made include: Presenting all equations in an appropriate and standardized format. Numbering all equations sequentially throughout the text. Providing clear explanations for each component within the equations to ensure they are easily understandable. Beyond the equations, we have also re-organized and refined the text in many other sections to improve the overall flow and precision of the presentation. All changes made in the manuscript have been highlighted using yellow fill to facilitate your review. In the attached point-by-point response, we have detailed our specific actions in reply to each of the comments raised.

Comments1: The whole text requires careful reading and rewriting. Exemple : L_130-132

Response: Thank you for this valuable feedback regarding the language quality of the manuscript. We have carefully reviewed and refined the entire text to improve its clarity, flow, and overall readability. Specifically, regarding the example you provided (Lines 130-132), we have completely rewritten that section to ensure a more precise and coherent explanation. This particular revision can be found on Lines 179-185 and has been highlighted in yellow for your easy reference.

Comments2: Material and Methods: Should mention the specific objectives in each trial.

Response: Thank you for this valuable suggestion. We agree that stating the specific objective of each trial at the outset of its methodology description is crucial for clarifying the experimental design logic for the reader. As suggested, we have now explicitly introduced the specific aim for each individual trial at the beginning of their respective sections in the Materials and Methods. These revisions have been implemented at Lines 123, 189-190, 203-204, and 234-235 of the manuscript and are highlighted in yellow for your easy reference.

Comments3: Feces or fecal samples vs excreta: It seems to me that the material sampled and analyzed was excreta (feces+urine), not feces! (?)

Response: Thank you for this excellent point regarding the precise terminology for the samples collected. You are absolutely correct in your assessment. In Trial 3, where the objective was to calculate total nutrient excretion, we collected the combined mixture of feces and urine using excreta collection trays. We agree that the term "excreta" is scientifically accurate in this context, and we have revised the text accordingly throughout the description of this trial. In contrast, for Trial 4, the methodological approach differed. As we employed the endogenous indicator method to determine the apparent digestibility of amino acids, the analysis specifically required fecal samples rather than total excreta. Therefore, we have maintained the use of the term "feces" or "fecal samples" in this section to accurately reflect the material that was actually analyzed. Given that these revisions are scattered across the respective Methods and Results sections, we have not listed every line number here. However, all modifications have been highlighted in yellow throughout the manuscript for your convenient and comprehensive review.

Comments4: L_154-156: It seems that the excreta was collected at once at the end of the experimental period (4 days). During this period, organic matter degradation and microorganisms can develop. These can transform some amino acids into volatile nitrogen or even convert them into other amino acids. The authors claim that the use of boric acid would prevent potential problems. Could you mention any literature that compares these conditions (daily collection vs end-of-period collection with boric acid)?

Response: Thank you for raising this critical point regarding sample preservation and its potential impact on amino acid integrity. We fully appreciate your concern about microbial activity during the 4-day collection period. Our choice of a single end-of-period collection with boric acid preservation was not arbitrary but was based on a rigorously established and widely accepted methodology within our specific field of poultry nutrition. This protocol is detailed in the foundational doctoral dissertation: Tian, Y. D. (2005). Establishment of a Dynamic Model for Energy and Amino Acid Requirements in Broilers [Doctoral dissertation, Chinese Academy of Agricultural Sciences]. This work is considered a cornerstone in the field of poultry nutrition in China, with its methods being extensively validated through its remarkable citation count (over 4,000 cites on CNKI). The consistent application and success of this method across numerous subsequent studies provide strong indirect evidence for its reliability in preserving the analytical validity of amino acids for the purpose of requirement estimation. We acknowledge that for specific analytical purposes, such as those requiring pristine microbial DNA (e.g., metagenomics), specialized preservatives might be superior. However, for the objectives of this study, which operate within the well-established context of classical poultry nutrition assessment, the use of this highly cited and validated method was deemed appropriate and reliable. We thank you again for this insightful comment, which certainly guides our consideration of sample handling protocols in future research.

Comments5: L_131: “Separate Ketone bodies”! Could you explain what this means?

Response: Thank you for pointing out this unclear expression. You are correct to question it; the term "Ketone bodies" was a mistake here and is easily confused with the metabolic byproduct. Our intended meaning was to describe the process of separating the carcass from the feathers for subsequent individual analysis. We have revised the text accordingly. The phrase has been corrected to "Separate the carcass from the feathers" and the entire sentence has been rephrased for maximum clarity in the revised manuscript. This correction has been implemented on Lines 179-185 and is highlighted in yellow for your easy reference.

Comments6: L_158: what means this “500 g of fodder”? Do you mean feed?

Response: Thank you for this correction. You are right; the term "feed" is more accurate in this context. We have revised "fodder" to "feed" on Line 243 of the manuscript. The change has been highlighted in yellow for your convenience.

Comments7: L_251-254: What does the “X” stand for in these models?

Response: Thank you for this question. You are right to ask for clarification. In these models developed to describe the relationship between body weight and protein content: X represents the independent variable, which is the protein content. Y represents the dependent variable, which is the live body weight. We have now added this definition at the beginning of the description of the linear models in the manuscript to avoid any ambiguity. This clarification has been highlighted in yellow for your easy reference.

Comments8: L_270-271: What does the “C” stand for in these two models?

Response: Thank you for your question regarding the parameter in our models. The symbol "C" in these two models stands for the correlation coefficient. This coefficient is a key parameter that dynamically links the metabolically active protein in the hen's body to its body weight. The value of this coefficient was derived from data analysis in Trial 3 of our current study. For a more detailed elaboration on the derivation, calculation, and application of this specific correlation coefficient within dynamic models, please refer to our referenced publication: Liu M, et al. (2024). Estimation of Protein and Amino Acid Requirements in Layer Chicks Depending on Dynamic Model. Animals. We have clarified the definition of "C" in the revised manuscript to ensure transparency.

Comments9: L_283-284: Must write the formula in a adequate format...this is confusing!

Response: Thank you for pointing out the issue with the formula formatting. We agree that clarity in presenting equations is crucial. We have taken the following steps to address this: For the formula originally on line 283-284 (now at Line 370), we have successfully reformatted it using the equation editor to ensure it is clear and readable. Regarding the more complex final formula (e.g., around Line 771), we encountered a technical constraint. Due to the specific format of the Animals journal template, using the equation editor for this lengthy equation resulted in an undesirable layout that spanned multiple lines and compromised readability. As a solution, we have ensured the textual description of this complex formula is precise. Furthermore, we are fully prepared to work with the editorial office to adjust the formatting of such equations to meet the journal's exact typesetting standards upon acceptance. We believe the current revisions significantly improve the presentation and hope this is satisfactory.

Comments10: L_357: “1.46E” should be rewritten

Response: Thank you for this suggestion. We have rewritten "1.46E" in a proper scientific notation format as suggested. I have already made the modification on line 437

 

Comments11: L-534-540: Have to decide, is it 0-6 and 7-12 ou is it 0-7 and 7-12 ?

Response: Thank you for catching this inconsistency. You are correct; the age periods should be 0-6 and 7-12. We have corrected this error in the manuscript. The revision has been made on Line 614 and is highlighted in yellow for your easy reference.

Comments12: Tables 2 and 3: It would be more appropriate here to compare the fits of the models using AIC and BIC (as done in Tables 9 and 10).

Response: Thank you for this valuable suggestion. We agree that using AIC and BIC provides a more robust comparison of model fit. In response, we have now calculated and included the AIC and BIC values for the models in Tables 2 and 3. These additions are located at Lines 445 and 451 and have been highlighted in yellow. Furthermore, we have supplemented the Results and Analysis section (Lines 428-432) to discuss this new information. Notably, our analysis revealed that the model rankings based on AIC and BIC were entirely consistent with those derived from R² and standard error (SE), further strengthening the validity of our initial conclusions.

 

Comments13: Table 2: Check the SE. For example, one would expect similar SE for the first and the third models in this Table.

Response: Thank you for your comment regarding the standard errors (SE) in Table 2. We have double-checked the raw data and our calculations, and we can confirm that the reported SE values are accurate. We understand your expectation for similar SEs between the first and third models. The observed variation can arise from differences in the data structure fitted by each model and their inherent complexity. Furthermore, we noted that another reviewer also raised questions about the presentation of the tables, suggesting that the formulas or notations might have been a source of ambiguity. To ensure maximum clarity and transparency, we have thoroughly revised and standardized the footnotes and internal formula descriptions in both Table 2 and Table 3 to eliminate any potential for misinterpretation and to align with journal standards. These revisions have been implemented (see Lines 446 and 453) and are highlighted in green for your easy reference. We believe these clarifications significantly improve the presentation of the tabulated data.

Comments14: Table 3:

• what means “b01”, “b11”, “b21”?
• Is this Is this “Y=b0+b1X+b2X” supposed to be “Y=b0+b1X+b2X2”? Same in Table 2

Response: Thank you for your comments regarding the equations and notations in the tables. We have thoroughly revised and standardized the presentation of all formulas and symbols in Table 2 and Table 3 to ensure clarity and accuracy, as suggested. The changes have been implemented on Lines 446 and 453 and are highlighted in green for your easy reference.

Comments15: Tables 4 and 5 are unformatted, which makes it very difficult to understand and interpret the data.

Response: Thank you for your feedback regarding the formatting of Tables 4 and 5. We understand that the current layout may hinder data interpretation. Given the extensive dataset—covering 18 amino acids across different periods, along with ANOVA results—presenting it clearly within the manuscript layout has been challenging. To address this issue promptly, we have reformatted the tables to improve readability. If it would be helpful, we would also be glad to provide the original data files of these tables for your reference. For the final publication, we will coordinate with the editorial office to determine the optimal format for such large tables and make any necessary adjustments in a timely manner.

We believe these revisions significantly enhance the quality of our manuscript, and we hope it now meets the high standards of Animals. Thank you for giving us the opportunity to revise our work. We look forward to hearing from you in due course.

Sincerely,
Jiatong Li
Henan Agricultural University
18836947816@163.com

Reviewer 3 Report

Comments and Suggestions for Authors

This study aimed to develop a dynamic factorial model for predicting amino acid requirements in Hy-Line Gray laying hens during early growth stages. The results can help to optimize feed efficiency and support sustainable layer production.

 

Abstract

Line 28. A space is missing after „HPLC.

Line 30: A space is missing after „protein”.

 

Please check the text as several times spaces are missing or there is an extra space. E.g., line 52, 56, 63, 91, 133, 158, 159, 168 …..483, 500, … discussion.

Also, rows are missing under lines 365, 378, 544, 574, 587, 689, 906.

There are a lot of empty rows in the text: line 306-310, 601-634, 692-700.

After the first introduction of the amino acid abbreviations (line 380), only use the abbreviations and do not repeat the explanations.

Please carefully check the citations and list of references, as they seem to be incorrect on numerous occasions.

 

Introduction

Line 59: body weight (BW). Please use the abbreviations later in the text and delete “body weight (BW)” in line 248.

Line 64: reference number 2 is about dairy cows, not laying hens.

Line 80: reference number 6 is about poultry, not mammals or fish.

Line 96: there is an extra dot before [9].

 

Materials and methods

Line 119: Did you record the mortality?

Line 120: What was the starting age of the birds?

Line 124: ad libitum.

Line 129, 130: weighting

Line 130: “During each …”. Please rephrase this sentence. It is unclear. What are “ketone bodies”?

Line 132: record

Line 136: “Conducted concurrently with Trial 1, one hen close to the average body …” Was it the same hen used in Trial 1?

Line 161: “The apparent digestibility of protein and amino acids was calculated using the endogenous indicator method”. Please provide details.

 

Results

In Table 9 and 10, the explanation of “*” and “**” is missing.

Line 372: the explanatory numbers in the first row and in the footnotes should be in superscript.

Line 378: subchapter 3.3. starts in a new row.

Line 427: feather protein, not carcass.

Line 500-509: commas are missing.

Line 595, 599: the numbers should be in superscript.

Line 650, 652: the figure legend should be placed below the figure.

Line 680-689: In my opinion, this belongs to the discussion.

 

Discussion

Line 783: reference number 25 is about broiler chickens, not meat duck.

Line 787: reference number 26 is about beef.

Line 790: Liu et al. (date)

Line 792: “Compared with the results of this trial, except for glycine, Threonine and Cystine, the other results were not significantly different.” What could be the reason for the significant difference?

Line 825: reference number 33 is about in vitro and ileal digestibility in pigs.

Line 831: reference number 34 is about the digestibility of cooked and raw egg in humans. Why is it here?

Line 838: What are the recommendations? Please also add the reference for the breed-specific requirements.

Line 847: reference number 37 is about epigenetics and metabolism. Are you sure it is a proper citation?

 

Conclusions:

Line 863-868: What is the unit of measure?

Line 1013-1015: please delete these numbers.

Author Response

To: The Editor and Reviewers of Animals
Manuscript ID: animals-3905240
Title: Establishment of an amino acid nutrition prediction model for laying hens during the brooding and early-growing period

Dear Editors and Reviewers,

Please find attached our revised manuscript and our point-by-point responses to the reviewers' comments. We sincerely thank the reviewers for their valuable time and insightful feedback, which have greatly helped us improve the manuscript.

Comment1: Line 28. A space is missing after „HPLC.

Line 30: A space is missing after „protein”.

 Response: Thank you very much for pointing out these details regarding formatting standards. Your feedback is highly appreciated. I fully understand the rule that a space should follow a punctuation mark in English writing, and I have carefully reviewed and revised such instances throughout the manuscript. As per your specific comments, the revisions on Line 28 and Line 30 have been implemented and are now highlighted in green for your easy reference.

Comment2: Please check the text as several times spaces are missing or there is an extra space. E.g., line 52, 56, 63, 91, 133, 158, 159, 168 …..483, 500, … discussion.

Response: Thank you for your thorough review and for pointing out the issues regarding missing or extra spaces in the text. We have carefully checked the entire manuscript and revised all such formatting errors according to your comments. To facilitate your review, the specific changes have been highlighted with green highlighting at the following line numbers: 65, 67, 68, 71, 82, 85, 94, 99, 109, 214, 742, 756, 767, 775, 787, and 800.We have ensured that the spacing throughout the text now adheres to the journal's formatting guidelines.

Comment3: Also, rows are missing under lines 365, 378, 544, 574, 587, 689, 906.

Response: Thank you for your detailed comment regarding the table formatting. To ensure I address your point accurately, I would like to kindly ask for a clarification. You mentioned that "rows are missing under lines 365, 378, 544, 574, 587, 689, 906." In our manuscript, we have formatted all tables as three-line tables, which typically include only a top, a header, and a bottom horizontal line, without additional row lines. Could you please kindly specify if you are suggesting we add additional horizontal lines within these tables? If so, we would be very grateful if you could indicate where exactly these lines should be placed (e.g., between specific rows of data).We are more than willing to modify the tables accordingly based on your further guidance. Thank you for your time and clarification.

Comment4: There are a lot of empty rows in the text: line 306-310, 601-634, 692-700.

Response: Thank you for your meticulous attention to the manuscript formatting. We have carefully reviewed the text and removed all unnecessary empty rows throughout the document. Regarding the specific areas you mentioned (around lines 601-634 and 692-700), some of these empty spaces were intentionally retained to accommodate large tables and to comply with the formatting requirements of the journal Animals. This practice helps ensure that the tables are properly placed on the page and remain legible, preventing potential typesetting issues. We have striven to balance the need for a compact text with the journal's specific layout guidelines. We are, of course, fully prepared to work with the editorial office on any final typesetting adjustments they deem necessary for publication. We appreciate your understanding.

 

Comment5: After the first introduction of the amino acid abbreviations (line 380), only use the abbreviations and do not repeat the explanations.

Response: Thank you for your valuable comment regarding the use of amino acid abbreviations. As suggested, we have revised the manuscript to employ the abbreviations exclusively after their first full introduction (Line 380), and have removed all redundant explanations throughout the text. Furthermore, in an effort to further improve the clarity and depth of the manuscript, we have also made additional revisions to the "Results and Analysis" section of the nitrogen balance experiment. All the aforementioned changes have been implemented and are highlighted in green for your convenient review, specifically within Lines 508-585 and Lines 638-643.

Comment6: Please carefully check the citations and list of references, as they seem to be incorrect on numerous occasions.

Response: I have carefully checked and revised the reference format

Comment7: Line 59: body weight (BW). Please use the abbreviations later in the text and delete “body weight (BW)” in line 248.

Response: Thank you for your comment regarding the consistent use of abbreviations. As suggested, we have properly defined the abbreviation "(BW)" at its first occurrence in the text (Line 59) and have consistently used "BW" throughout the rest of the manuscript. The redundant full form "body weight (BW)" on Line 248 has been deleted accordingly. Given that these revisions are extensive and span across the entire manuscript, we have not listed every line number here. However, all changes have been highlighted in green for your easy and comprehensive review.

Comment8: Line 64: reference number 2 is about dairy cows, not laying hens.

Response: Thank you for pointing out this critical error. You are absolutely correct that reference 2, which I previously cited, is a study on dairy cows and was therefore inappropriate in the context of laying hens. I sincerely apologize for this oversight, which occurred due to my carelessness during the reference insertion process. This error has been corrected. The incorrect reference has been replaced with an appropriate one pertinent to laying hens. The change is located at Line 63 of the revised manuscript and has been highlighted in green for your easy reference. Once again, I deeply appreciate your diligence and valuable comment, which has been crucial in improving the accuracy of our manuscript.

Comment9: Line 80: reference number 6 is about poultry, not mammals or fish.

Response: We thank the reviewer for this insightful observation. The citation of reference 6 (which is indeed a poultry study) in the original sentence was intended to illustrate the broader applicability and investigation of different models across species. We agree that enhancing the species-specific correspondence strengthens the argument. Therefore, we have now supplemented the mention of each species (insects, mammals, and fish) with their own dedicated, species-appropriate references. The original reference has been retained in a more suitable position within the text. These changes are made in lines 80-81 and highlighted in green for easy identification.

Comment10: Line 96: there is an extra dot before [9].

Response: Thank you for pointing out this typographical error. The extra dot before reference [9] on Line 96 has been deleted as suggested.

Comment11: Line 119: Did you record the mortality?

Response: Thank you for raising this important point. In response to your question, we did indeed monitor and record mortality throughout the experimental period. The recorded mortality rate was very low and fell within the expected normal range for this animal model. As it was not a significant finding central to the primary outcomes of our study, we have not included it in the Results section. However, to ensure full transparency, we have added a statement in the Materials and Methods section clarifying that "no treatment-related mortality was observed during the entire experimental period." This revision provides the necessary context without drawing undue attention to a non-significant outcome. We believe this approach maintains the focus of the manuscript while adequately addressing the data collection process.

 

Comment12: Line 120: What was the starting age of the birds?

Response: Thank you for your question. The birds were one-day-old hatchlings at the start of the experiment. As suggested, we have added this information to the manuscript. The clarification has been incorporated on Line 125 and highlighted in green for your easy reference.

Comment13: Line 124: ad libitum.

Response: We thank the reviewer for pointing out this error. The misspelled term "adlibitum" has been corrected to the standard form "ad libitum" throughout the manuscript. The change has been highlighted in green for your convenience (e.g., at Line 151).

Comment14: Line 129, 130: weighting

Response: We appreciate the reviewer's meticulous attention. The typo "weighting" has been corrected to the accurate term "weighing" throughout the text to properly describe the measurement process.

Comment15: Line 130: “During each …”. Please rephrase this sentence. It is unclear. What are “ketone bodies”?

Response: Thank you for your comment, which has allowed us to describe the experimental procedure with greater clarity and precision. As suggested, we have thoroughly rewritten the relevant sentence in the manuscript. The revised text now clearly states that:" At each experimental phase, the average body weight (BW) of all hens within a cage was first calculated. Subsequently, one hen with a BW closest to this calculated average was selected, euthanized by cervical dislocation, and its live weight was recorded. Feathers were then manually plucked from the carcass. Finally, the carcass and the feathers were weighed separately to determine their respective masses." This revision, which includes the specific method of euthanasia, has been implemented in the manuscript and is highlighted in light blue for your convenience (across Lines 123-179).

Comment16: Line 132: record

Response: I have made modifications to this word in the text. Line 183

Comment17: Line 136: “Conducted concurrently with Trial 1, one hen close to the average body …” Was it the same hen used in Trial 1?

Response: Thank you for this important question regarding our experimental design. Yes, your understanding is correct. The hens used for body composition analysis in Trial 2 were the same individuals from the same population that was being monitored for growth performance in Trial 1.The rationale for this design is rooted in the core objective of our study: to establish a dynamic model for estimating the amino acid requirements for growth over time. To achieve this, it was essential to integrate the data from Trial 1 (growth performance) and Trial 2 (body chemical composition) at the individual level. By using the same birds, we can directly link the growth trajectory of an individual (from Trial 1) to its specific body amino acid content (from Trial 2) at a given time point. This direct linkage is crucial for accurately calculating the net amino acid deposition required for growth. Employing separate cohorts of birds would introduce uncontrolled variation due to individual differences, thereby compromising the precision of our final estimates.

Comment18: Line 161: “The apparent digestibility of protein and amino acids was calculated using the endogenous indicator method”. Please provide details.

Response: Thank you for this suggestion. As requested, we have now provided the detailed formula for calculating the apparent digestibility of protein and amino acids using the endogenous indicator method. This information has been added to the "2.5 Model construction" section of the manuscript (specifically at Lines 366-367). 

Comment19: In Table 9 and 10, the explanation of “*” and “**” is missing.

Response: Thank you for pointing out this omission. The explanations for the asterisk symbols ("*" and "**") have been added to the footnotes of Table 9 and Table 10.The revisions have been implemented on Lines 697 and 703 of the manuscript and are highlighted in green for your convenience.

Comment20: Line 372: the explanatory numbers in the first row and in the footnotes should be in superscript.

Response: Thank you for pointing this out. As suggested, the explanatory numbers in the first row of the table and in the footnotes have now been formatted as superscript. The corrections have been made on Lines 448 and 454 and are highlighted in green for your easy reference.

Comment21: Line 378: subchapter 3.3. starts in a new row.

Response: Thank you for pointing out this formatting issue. As suggested, the heading for subchapter 3.3 now starts on a new row. This revision has been implemented on Line 459 and is highlighted in green for your convenience.

Comment22: Line 427: feather protein, not carcass.

Response: Thank you for catching this error. You are absolutely correct; this section describes the amino acid content of feather protein, not carcass protein. We have revised the text accordingly, changing "carcass" to "feather protein" on Line 509. The correction has been highlighted in green for your easy reference.

Comment23: Line 500-509: commas are missing.

Response: We have thoroughly revised the indicated section. Specifically, the full names of amino acids have been replaced with their standardized abbreviations, and all necessary commas have been added to ensure proper punctuation. Furthermore, to maintain consistency across the manuscript, we have also proactively standardized the terminology and comma usage in the "nitrogen balance experiment" section. All these changes have been implemented and are highlighted in green for your convenient review, specifically within Lines 579-585 and Lines 638-643.

Comment24: Line 595, 599: the numbers should be in superscript.

Response: Thank you for pointing this out. As suggested, the specified numbers on the original Lines 595 and 599 have now been formatted as superscript. The corrections have been made on Lines 651 and 655 of the revised manuscript and are highlighted in green for your easy reference.

Comment25: Line 650, 652: the figure legend should be placed below the figure.

Response: Thank you for your comment. The figure legends have been moved to their correct positions below the corresponding figures as suggested. These revisions have been implemented on Lines 689 and 691 of the manuscript and are highlighted in green for your easy reference.

 

Comment26: Line 680-689: In my opinion, this belongs to the discussion.

Response: Thank you for this valuable suggestion. We agree with your assessment that this content is more appropriately placed within the Discussion section. Consequently, we have moved the paragraph from Lines 680-689 to the Discussion. During this process, we have also refined the text to ensure it integrates logically with the surrounding context and strengthens the narrative flow. The revised text can now be found in the Discussion section (specifically, Lines 856-865) and has been highlighted in green for your convenience.

Comment27: Line 783: reference number 25 is about broiler chickens, not meat duck.

Response: Thank you for your thorough review and for identifying the incorrect citation of reference 25 (regarding broiler chickens) in the context of meat ducks. I apologize for this error, which occurred during the preliminary drafting stage. To address this issue comprehensively, I have not only corrected this specific reference but have also conducted a full revision of the reference list. This includes verifying the relevance and accuracy of all citations and ensuring their consistent formatting and placement. These measures have been taken to enhance the overall quality and reliability of the manuscript. Modify the position of line 782, highlighted in green

Comment28: Line 787: reference number 26 is about beef.

Response: Thank you for identifying the incorrect citation of reference 26 (regarding beef) in our manuscript. This has been corrected, and the appropriate reference is now cited at Line 786, as highlighted in green. We appreciate your diligence in reviewing our work.

Comment29: Line 790: Liu et al. (date)

Response: Thank you for your guidance on the citation format. We have revised the reference to "Liu et al. (year)" at the specified location (Lines 789-790) in strict accordance with the journal's style requirements. The change has been highlighted in green for your easy reference. We appreciate your diligence in helping us improve the manuscript.

Comment30: Line 792: “Compared with the results of this trial, except for glycine, Threonine and Cystine, the other results were not significantly different.” What could be the reason for the significant difference?

Response: Thank you for this insightful question regarding the significant differences observed for glycine, threonine, and cystine. We agree that this is a key point for discussion. The most plausible explanation for the discrepancy lies in the pronounced physiological differences between the chicken breeds used in the studies. Our study utilized Hy-Line Gray laying hens, whereas the model referenced (Liu et al.) was established using Jingse 6 chicks. These breeds are known to differ significantly in body weight, average daily gain, and overall production performance, which are fundamental drivers of amino acid requirements. These breed-specific traits can directly influence metabolic rates and the partitioning of nutrients, including amino acids, for maintenance versus production. This explanation, that breed variation is a major factor affecting amino acid requirements, is well-supported in the literature. We have now cited a relevant reference at this point in the manuscript to substantiate our argument. Change position to line 793-797 and highlight with green fill

 

Comment31: Line 825: reference number 33 is about in vitro and ileal digestibility in pigs.

Response: Thank you for pointing out the incorrect citation of reference 33, which pertained to in vitro and ileal digestibility in pigs. We sincerely apologize for this oversight. The reference has been replaced with one that is directly relevant to the context of our study. The correction has been made at Line 829 of the revised manuscript and is highlighted in green for your convenience. We appreciate your diligence in helping us improve the accuracy of our work.

Comment32: Line 831: reference number 34 is about the digestibility of cooked and raw egg in humans. Why is it here?

Response: Thank you for this pertinent question regarding the placement of reference 34. Our initial intention for citing the study on egg digestibility in humans was to draw a parallel on how processing (cooking) can alter the apparent digestibility of nutrients, conceptually linking it to our work on feed. However, upon careful reconsideration prompted by your comment, we agree that this cross-species analogy was indeed inappropriate and potentially misleading. We have therefore replaced it with a more directly relevant study that specifically investigates the influence of nutrient intake on the apparent digestibility in laying hens. This new reference provides a stronger and more contextualized support for our argument. The change has been made at Line 834 and is highlighted in green for your easy reference. We are grateful for your insightful feedback, which has helped us strengthen the relevance of our citations.

Comment33: Line 838: What are the recommendations? Please also add the reference for the breed-specific requirements.

Response: Thank you for prompting us to provide stronger foundational support for the amino acid requirements discussed. In response, we have incorporated the findings from a key reference (Ideal ratios... for white leghorn-type laying hens) which provides specific daily requirements, including: Ile 426 mg/d, Lys 538 mg/d, Met 253 mg/d, Met+Cys 506 mg/d, Thr 414 mg/d, Trp 120 mg/d, and Val 501 mg/d. To maintain conciseness and focus in the manuscript, we have chosen to cite only the value for methionine (253 mg/d) at this point, as methionine is typically the first limiting amino acid in laying hen diets and thus most critical to our discussion. The addition of this citation and the methionine value has been made at Line 851 and is highlighted in green. We believe this change significantly strengthens our narrative. We are grateful for your suggestion to improve the manuscript.

Comment34: Line 847: reference number 37 is about epigenetics and metabolism. Are you sure it is a proper citation?

Response: Thank you for your valuable question regarding the relevance of reference 37. Upon careful reconsideration, we agree that its focus on epigenetics and metabolism was not the most direct fit for the argument presented here. We have replaced it with a more appropriate reference that directly supports the specific point being made in this context. The change has been made at Line 859 and is highlighted in green for your convenience.

Comment35: Line 863-868: What is the unit of measure?

Response: Thank you for pointing out this omission. The unit of measure for these values is grams per day (g/day).We have now added the unit to the relevant text in the manuscript. The revisions have been implemented in the section spanning Lines 888 to 897 (previously Lines 863-868) and are highlighted in green for your easy reference.

Comment36: Line 1013-1015: please delete these numbers.

Response: I have deleted these numbers.

We have carefully addressed all the comments and believe the revisions have significantly enhanced the quality of our work. All changes in the manuscript text have been highlighted for your convenience.

We appreciate the opportunity to resubmit our work and look forward to hearing from you.

Sincerely,
Jiatong Li
Henan Agricultural University
18836947816@163.com

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

There's been significant improvement, but there are still important and necessary  adjustments to be made. In several parts of the text, there are still formatting issues (in tables and equations) to be addressed. 

Some items identified:

L_213, 215, 217, 277, 631, 643: Should use “excreta” instead of “excrement”

Tables 2 and 3: Please add “AIC” and “BIC” in table footer description #2.

Materials and Methods: Regarding the use of boric acid (for preserving excreta samples for a long period), it would be advisable to include the reference in which this methodology was supposedly established.

Equation 2: There is an error here (should be “1”, not “/”)

Equation 5: Should it not be “ b₀ + b₁X + b₂X² ” ?

Tables 4 and 5: Still unformatded

L_348 and L_349: Have to explain here what “C” states for.

Equations #13 : Please write in appropriate format (access the attached file).

Same should be done for the long equation in  L_693-696: Please write this equation in an appropriate format and explain/detail each variable  Ex: t=age in days, ...

Equations 17 and 18: ln(t)

L_601-605: It is confusing!

L_712-715: As mentioned, the predicted mature weight (1412 g) is much smaller than the real mature weight (1980 g). This was likely due to the age limit assessed in this study (up to 84 days), while laying hens reach adult weight much later. Could this limitation have also affected the estimation of other parameters? Could it not have compromised all the inferences made with this model? You should at least address this issue in your discussion.

L_800-801: “Proteins are digested in the diet to release...” 

                   Suggestion: “Dietary proteins are digested to release...”

L_864: “In general, the model predictions generally align with previous studies.”

              Suggestion: “Overall, the model predictions are in line with previous studies.”

Comments for author File: Comments.pdf

Author Response

Dear  Reviewers,

We sincerely thank you for your letter and the positive feedback on our manuscript entitled “[Establishment of an amino acid nutrition prediction model for laying hens during the brooding and early-growing period]” (Manuscript ID:animals-3905240). We are greatly encouraged by the reviewers’ insightful comments and valuable suggestions. We have carefully considered all the points and revised our manuscript accordingly. The changes have been highlighted in the revised manuscript. Below, we provide a point-by-point response to the reviewers’ comments.

Comments1: L_213, 215, 217, 277, 631, 643: Should use “excreta” instead of “excrement”

Response: The term "excrement" has been replaced with "excreta" in lines 213, 215, 217, 277, 631, and 643, as suggested. The modified sections are highlighted with gray shading

Comments2: Tables 2 and 3: Please add “AIC” and “BIC” in table footer description #2.

Response: As requested, we have added the definitions of AIC and BIC to the footnotes of Tables 2 and 3. The new text is highlighted with gray fill for easy identification.

Comments3: Materials and Methods: Regarding the use of boric acid (for preserving excreta samples for a long period), it would be advisable to include the reference in which this methodology was supposedly established.

Response: We thank the reviewer for this insightful comment. As suggested, we have now included a key reference that validates the use of boric acid as a preservative for excreta samples in the revised Materials and Methods section. The added reference is [15], which demonstrates the efficacy of boric acid in preserving the integrity of such samples for extended periods.

Comments4: Equation 2: There is an error here (should be “1”, not “/”)

Response: We are grateful to the reviewer for catching the typo in Equation 2. The incorrect symbol "/" has been replaced with "1" to ensure the equation is mathematically accurate and presented correctly.

Comments5: Equation 5: Should it not be “ b0 + b1X + b2X2 ” ?

Response: We appreciate the reviewer's careful attention to detail. The error in Equation 5 has been corrected to "b₀ + b₁X + b₂X²" to accurately reflect the quadratic model specification.

Comments6: Tables 4 and 5: Still unformatded

Response: We appreciate the feedback regarding the formatting of Tables 4 and 5. Given the substantial amount of data, we have opted to use abbreviated forms for the amino acids in the column headers to improve the overall layout and readability. We sincerely hope this adjustment resolves the formatting concern. Should you have an alternative preference for the presentation, we would be pleased to implement it accordingly.

Comments7: L_348 and L_349: Have to explain here what “C” states for.

Response:We thank the reviewer for suggesting this clarification. The meaning of "C" has been explicitly stated in the new text added across lines 351-353, which is highlighted with gray fill for easy identification.

Comments8: Equations #13 : Please write in appropriate format (access the attached file).

Response: In response to the reviewer's comment, Equation #13 has been rewritten to adopt the appropriate mathematical format, as guided by the example provided in the attached file. This adjustment ensures consistency with standard notational conventions in the field.

Comments9: Same should be done for the long equation in  L_693-696: Please write this equation in an appropriate format and explain/detail each variable  Ex: t=age in days, ...

Response: In response to the reviewer's feedback, the equation previously in lines 693-696 has been thoroughly revised. It now features an improved mathematical format for better clarity, followed by a dedicated section that defines and explains each variable in detail. This enhancement, which significantly improves the model's presentation and interpretability, is presented in lines 719-725 and is highlighted with a gray fill to facilitate review.

Comments10: Equations 17 and 18: ln(t)

Response: We thank the reviewer for pointing this out. The notation for the natural logarithm in Equations 17 and 18 has been updated to the appropriate mathematical format.

Comments11: L_601-605: It is confusing!

Response: We thank the reviewer for pointing out the lack of clarity in lines 601-605. We have thoroughly revised this paragraph to improve its logical flow and clarity. Additionally, to prevent any potential formatting inconsistencies that may occur during file conversion, we have ensured the manuscript uses consistent styles. We will also coordinate with the editorial office to confirm the final formatting and layout are correct before publication.

Comments12: L_712-715: As mentioned, the predicted mature weight (1412 g) is much smaller than the real mature weight (1980 g). This was likely due to the age limit assessed in this study (up to 84 days), while laying hens reach adult weight much later. Could this limitation have also affected the estimation of other parameters? Could it not have compromised all the inferences made with this model? You should at least address this issue in your discussion.

Response: Thank you very much for your attention to the discrepancy between the predicted mature weight and the actual mature weight, as well as the research limitations in this study. Regarding the questions you raised, we have taken them into account during the selection of the core growth model, Basis for model selection: Although most studies suggest that the Gompertz model describes animal growth more accurately, our data calculation showed that the predicted mature weight of the Gompertz model was much lower than the actual value (1980 g at 70 weeks of age from the manual). Its prediction accuracy could not meet the needs of growth characteristic analysis in this study. Reason for choosing the Von Bertalanffy model: After comprehensive comparison, the fitting result of the Von Bertalanffy model was more consistent with the actual data characteristics of this study. Therefore, we finally selected this model as the core growth model. Explanation of limitations: We clearly recognize that due to the inherent characteristics of the model itself, there will indeed be a certain error in the weight prediction results for the later growth stage of laying hens (beyond the 84-day-old sampling range of this study). At present, the limitations of this issue have been detailed in the discussion section of the paper (specific location: Lines 744–755), and this part of the content has been highlighted with gray shading for your easy reference.

 

Comments13: L_800-801: “Proteins are digested in the diet to release...” 

                   Suggestion: “Dietary proteins are digested to release...”

Response: Thank you very much for your suggestion. I have made revisions to the article. Change the position to highlight in gray fill on line 839.

Comments14: L_864: “In general, the model predictions generally align with previous studies.”

              Suggestion: “Overall, the model predictions are in line with previous studies.”

Response: Thank you very much for your suggestion. I have made revisions to the article. Modify the position to highlight with gray filling on line 905

We believe that the manuscript has been significantly improved after addressing the reviewers’ comments. We would like to express our sincere gratitude to you and the reviewers for the constructive comments and the opportunity to revise our work. We hope that the revised manuscript is now acceptable for publication in Animals.

Thank you for your time and consideration.

Sincerely,
Jiatong Li
Henan Agricultural University
18836947816@163.com

Reviewer 3 Report

Comments and Suggestions for Authors

Please recheck the text; there are still a lot of spaces missing after words.

missing space: line 30, after „protein”; line 59, after „body weight”; line 78-80, line 91, line 96, etc.

 

The citation is incorrect: line 56: reference number 2 is about lactating show and not hens. Reference number 8 is about pigs, not small mammals.

Line 284: AIA was already explained in line 238.

Table 2 and 3: the explanations of AIC and BIC are missing.

Line 605: the table legend should be above the table.

Line 845-846: please use the abbreviations of amino acids.

Author Response

Dear Reviewer,

Thank you very much for giving us the opportunity to revise our manuscript “[[Establishment of an amino acid nutrition prediction model for laying hens during the brooding and early-growing period]” (Manuscript ID: [animals-3905240]). We highly appreciate the reviewers’ time and their positive and constructive comments, which have helped us to improve the quality of our work. We have incorporated all the suggestions into the revised manuscript, and our point-by-point responses are detailed below.

 

Comments1: Please recheck the text; there are still a lot of spaces missing after words.

missing space: line 30, after „protein”; line 59, after „body weight”; line 78-80, line 91, line 96, etc.

Response: Regarding the issue of missing spaces you pointed out (for instance, after "protein," "body weight," and in lines 78-80, 91, 96, etc.), we have carefully revised the manuscript. We have added the necessary spaces and removed any erroneous ones at the specified locations. Furthermore, we have performed an additional check of the entire document's formatting using Microsoft Word's spelling and grammar check feature to ensure consistency and correctness throughout the text. We sincerely appreciate your meticulous work in helping us improve the manuscript. Please do not hesitate to let us know if any further revisions are required.

 

 

Comments2: The citation is incorrect: line 56: reference number 2 is about lactating show and not hens. Reference number 8 is about pigs, not small mammals.

Response: Thank you for your meticulous attention to detail and for identifying the incorrect citations in our manuscript. You are absolutely correct. The previously cited references (number 2 regarding lactating sows and number 8 regarding pigs) were inappropriately used in the context of hens and small mammals, respectively. This was an oversight on our part. We have now carefully reviewed and replaced these references with the most appropriate and relevant literature that directly supports our statements concerning hens and small mammals. The specific changes have been made at Line 81 in the revised manuscript. To make it easy for you to locate this correction, we have highlighted the changes with purple shading.

 

Comments3: Line 284: AIA was already explained in line 238.

Response: We thank the reviewer for pointing out the redundant definition. The repeated explanation of "AIA" has been deleted to improve the manuscript's conciseness. This modification is located around line 290 and is marked with purple fill for easy identification.

Comments4: Table 2 and 3: the explanations of AIC and BIC are missing.

Response:  As requested, we have added the definitions of AIC and BIC to the footnotes of Tables 2 and 3. The new text is highlighted with gray fill for easy identification.

Comments5: Line 605: the table legend should be above the table.

Response: We have corrected the formatting error noted by the reviewer. The legend is now correctly positioned above the table in line with standard practice.

Comments6: Line 845-846: please use the abbreviations of amino acids

Response: The full amino acid names in the original lines have been replaced with their standard abbreviations. This change is reflected at line 867 and is highlighted with purple fill. In addition, I also checked other parts of the text to ensure that all abbreviations were used after the first use of amino acids

Once again, we thank the reviewers for their thoughtful comments which have made our paper stronger. We are very pleased with the improvements and hope that the revised version now meets the high standards of Animals. We look forward to hearing from you.

With best regards,
Jiatong Li
Henan Agricultural University
18836947816@163.com