Determination of the Requirements of Standardized Ileal Digestible Methionine Plus Cysteine and Lysine in Male Chicks of a Layer Breed (LSL Classic) During the Starter Period (1–21 d)
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsLines 36-41: There is only one reference. Add more references
Lines 64-69: Add reference
In introduction, highlight the gap in research field
Add p-value in table 3 and table 5
Lines 263-280: discuss the practical implications of this difference, such as avoiding over-supplementation, reducing feed costs, and improving environmental sustainability through lower nitrogen excretion
In conclusion, highlight the key findings and suggest future directions in research field
Author Response
Reviewer 1:
Lines 36-41: There is only one reference. Add more references (see line 41)
Response: Two additional references have been added (see line 41).
Lines 64-69: Add reference
Response: Two references have been added (see line 74).
In introduction, highlight the gap in research field
Response: The gap in the research field has been reported (see lines 66-69)
Add p-value in table 3 and table 5
Response: p values have been added in the Tables (see Tables 3 and 5)
Lines 263-280: discuss the practical implications of this difference, such as avoiding over-supplementation, reducing feed costs, and improving environmental sustainability through lower nitrogen excretion
Response: We added a paragraph to the discussion in which we specifically highlight the effects of an oversupply of protein and amino acids on sustainability, the environment, climate, and feed costs (see lines 334-371).
In conclusion, highlight the key findings and suggest future directions in research field
Response: We added a chapter “Conclusions”, which highlights the major findings of the study, and gives some suggestions for future studies (see lines 389-409).
Reviewer 2 Report
Comments and Suggestions for AuthorsDear Authors,
thank you very much for the nicely written manuscript.
I have a few comments/questions:
- line 111: According to the title and former information, the trial duration was day 1 - 21. Please double-check if the weight and feed intake was controlled on day 22 or 21 and adjust if necessary.
- Table 1 shows the basal diet composition. What is the reason for a rather high deviation in the Isoleucine (Ile) setting in the two trials (SID Ile trial 1: 6.66 vs trial 2: 8.14)? The usage of free Ile was also higher in trial 2, so it seems to be an intended difference.
- Line 160: I would add "...markedly below the assumed level required for..." because the whole purpose of the trial is that we don´t know exactly where the animals needs are.
- Line 166 - 168 and 195/196: Please clarify which criteria you used to decide which model fits the data best, e.g R2, AIC, AICC...
- Line 282 - 284: You calculated the corresponding Met+Cys ratio. Please clarify if this calculation is based on the Lys setting of trial 1 or the estimated Lys setting of trial 2.
- Discussion: In the discussion part, the estimated requirements from trial 1 and 2 are compared to those of high-performing meat type chicken, in particular Cobb 500 and Ross 308. However, as also correctly noted in the discussion part, the body composition of LSL birds and broilers are quite different. In this regard, wouldn´t it be better to compare the estimated requirements with dual-use bids of the LSL itself?
Best regards.
Author Response
Reviewer 2:
line 111: According to the title and former information, the trial duration was day 1 - 21. Please double-check if the weight and feed intake was controlled on day 22 or 21 and adjust if necessary.
Response: Thank you for this comment. Indeed, the feeding trials were rum from the morning of day 1 to the evening of day 21. Weights and feed refusals were than recorded in the morning of day 22 (see line 129).
Table 1 shows the basal diet composition. What is the reason for a rather high deviation in the Isoleucine (Ile) setting in the two trials (SID Ile trial 1: 6.66 vs trial 2: 8.14)? The usage of free Ile was also higher in trial 2, so it seems to be an intended difference.
Response: Thank you for this comment, which is absolutely correct. In the first trial, the concentrations of SID isoleucine, valine, and threonine were approximately aligned with the breeder’s recommendations for LOHMANN LSL-CLASSIC pullets and layers. Although we assumed these levels might be sufficient, we decided to slightly increase the concentrations of these amino acids in the second trial for safety reasons. However, it became evident that, with an adequate supply of the amino acids under investigation (SID Met + Cys, SID Lys), bird performance was comparable in both trials. Thus, we can conclude that the first trial also provided sufficient levels of these amino acids. In the second trial, the concentrations of these amino acids were clearly above the requirement, but this had no negative effects on the birds. Therefore, in our opinion, this point does not require further discussion.
Line 160: I would add "...markedly below the assumed level required for..." because the whole purpose of the trial is that we don´t know exactly where the animals needs are.
Response: This comment has been addressed (see lines 123-127).
Line 166 - 168 and 195/196: Please clarify which criteria you used to decide which model fits the data best, e.g R2, AIC, AICC...
Response: We considered R2 to decide which model gives an adequate information of the requirement (see lines 184-187, 279-283, 301-303).
Line 282 - 284: You calculated the corresponding Met+Cys ratio. Please clarify if this calculation is based on the Lys setting of trial 1 or the estimated Lys setting of trial 2.
Response: To calculate the optimum ratio of Met+Cys/Lys we used the data of trial 1 for optimum Met + Cys concentration and the data of trial 2 for optimum Lys concentration. In the revised version of the manuscript, we used SID Met + Cys/MJ AMEn and SID Lys/MJ AMEn as a base for calculation of the optimum ratio, as AMEn levels differed in both trials (see lines 313-323).
Discussion: In the discussion part, the estimated requirements from trial 1 and 2 are compared to those of high-performing meat type chicken, in particular Cobb 500 and Ross 308. However, as also correctly noted in the discussion part, the body composition of LSL birds and broilers are quite different. In this regard, wouldn´t it be better to compare the estimated requirements with dual-use bids of the LSL itself?
Response: thank you for this valid comment. Unfortunately, there are less data in the scientific literature regarding the optimum concentrations of amino acids in dual-use birds. Recommendations for these birds are largely coming from practical observations than from scientific studies. Therefore, we compared the optimum concentrations of SID Met + Cys and SID Lys with recommndations given for LSL classic pullets (see lines 295-297, 36-308).
Reviewer 3 Report
Comments and Suggestions for AuthorsThis study determined the optimal levels of standardized ileal digestible methionine + cysteine (0.58%) and lysine (0.74%), as well as their 78:100 ratio, in 1- to 21-day-old male LSL Classic layer chicks using a dose-response approach, providing a scientific basis for diet formulation under Germany’s relevant rearing ban. The manuscript itself has several shortcomings that need revision.
1 L40-41 Please cite literature comparing growth rate and feed conversion ratio differences between male LSL Classic chicks and those of other mainstream layer breeds (e.g., ISA Brown) to justify selecting this breed as the experimental subject.
2 L55-57 Please clarify how the ban affects chick-source purity (to avoid misidentified embryos being reared), the determination of sample size, and how the experimental design aligns with the new regulation, thereby strengthening the link between the study and policy.
3 L103-104 Please clarify whether the database values are derived from studies on the same or closely related chick strains, or provide preliminary trial data to validate the accuracy of the digestibility coefficients used in this experiment.
4 L130 Please add the key parameters for amino acid detection under the VDLUFA method to ensure reproducibility.
5 L257-259 Please discuss the interaction effect of the AMEâ‚™ difference between Trial 1 (13.97 MJ/kg DM) and Trial 2 (13.16 MJ/kg DM) on amino acid requirement estimation.
6 L287-290 The underlying physiological mechanism for this ratio difference is not elaborated; the discussion only links it to muscle-to-feather ratios and lacks metabolic-level evidence. Please supplement with data on metabolic-pathway differences between slow- and fast-growing chickens regarding Met + Cys versus Lys (e.g., gene expression related to feather synthesis, efficiency of muscular protein deposition), citing relevant gene-expression or enzyme-activity studies to deepen the mechanistic discussion.
7 L298-299 Please go beyond the general statement that “rearing male layer chicks is problematic economically and ecologically” by using the optimal amino acid levels identified here to quantify the economic savings (e.g., percentage reduction in feed cost) and environmental benefits (e.g., decrease in nitrogen excretion).
8 Please revise the reference list to comply with the journal’s formatting requirements.
Author Response
Reviewer 3:
This study determined the optimal levels of standardized ileal digestible methionine + cysteine (0.58%) and lysine (0.74%), as well as their 78:100 ratio, in 1- to 21-day-old male LSL Classic layer chicks using a dose-response approach, providing a scientific basis for diet formulation under Germany’s relevant rearing ban. The manuscript itself has several shortcomings that need revision.
1 L40-41 Please cite literature comparing growth rate and feed conversion ratio differences between male LSL Classic chicks and those of other mainstream layer breeds (e.g., ISA Brown) to justify selecting this breed as the experimental subject.
Response: we used the LSL classic strain as female counterparts are the most widely used for commercial egg production in Germany. Nevertheless, we compared the performance of LSL Classic males with those of the ISA Brown strain (see lines 376-383).
2 L55-57 Please clarify how the ban affects chick-source purity (to avoid misidentified embryos being reared), the determination of sample size, and how the experimental design aligns with the new regulation, thereby strengthening the link between the study and policy.
Response: Thank you for this valid comment. We added a short paragraph on in-ovo sexing techniques (see lines 51-63).
3 L103-104 Please clarify whether the database values are derived from studies on the same or closely related chick strains, or provide preliminary trial data to validate the accuracy of the digestibility coefficients used in this experiment.
Response: We added a sentence on this comment. Digestibility data in the AMINODAT tables are coming from digestibility trials in chicken (see lines 118-123).
4 L130 Please add the key parameters for amino acid detection under the VDLUFA method to ensure reproducibility.
Response: we added a description of the amino acid analysis (see lines 149-154)
5 L257-259 Please discuss the interaction effect of the AMEâ‚™ difference between Trial 1 (13.97 MJ/kg DM) and Trial 2 (13.16 MJ/kg DM) on amino acid requirement estimation.
Response: Thank you for this very valid comment. As poultry is able to adapt feed intake to energy density of the diets, it is correct to relate amino acid concentrations to AMEn of the diets. We have done so and recalculated the optimum Met + Cys/Lys ratio based on the optmium concentrations of these amino acids per MJ AMEn (see lines 313-323).
6 L287-290 The underlying physiological mechanism for this ratio difference is not elaborated; the discussion only links it to muscle-to-feather ratios and lacks metabolic-level evidence. Please supplement with data on metabolic-pathway differences between slow- and fast-growing chickens regarding Met + Cys versus Lys (e.g., gene expression related to feather synthesis, efficiency of muscular protein deposition), citing relevant gene-expression or enzyme-activity studies to deepen the mechanistic discussion.
Response: Thank you for this comment. When recalculating the Met + Cys/Lys ratio based on the optimum concentrations of these amino acids per MJ AMEn, the ratio is 74:100 (instead of 78:100 based on concentrations of these amino acids per kg diet). The ratio of 74:100 is very consistent with many broiler studies, including studies with conventional broilers. Therefore, it is no longer required to discuss potential differences in this ratio between the present study and other broiler studies.
7 L298-299 Please go beyond the general statement that “rearing male layer chicks is problematic economically and ecologically” by using the optimal amino acid levels identified here to quantify the economic savings (e.g., percentage reduction in feed cost) and environmental benefits (e.g., decrease in nitrogen excretion).
Response: Thank you for this valid comment. We added a paragraph to the discussion in which we specifically highlight the effects of an oversupply of protein and amino acids on sustainability, the environment, climate, and feed costs (see lines 334-371).
8 Please revise the reference list to comply with the journal’s formatting requirements.
Response: has been done.
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors have addressed the revisions.
Reviewer 3 Report
Comments and Suggestions for AuthorsThe author has revised or responded to the questions or concerns I raised.
