Impact of Different Levels of Crude Protein on Production Performance and Meat Quality in Broiler Selected for Slow Growth

Round 1

Reviewer 1 Report (Previous Reviewer 1)

The manuscript is recommended for publication in present form.

Author Response

Honorable Reviewer,

Thank you for taking time in judging our manuscript and appreciating our work. We still have operated some adjustments in comparison with the latter form of the manuscript, basing on the other reviewers' suggestion.

Thank you indeed!

Cordially yours,

On behalf of all authors,

Razvan RADU-RUSU

Reviewer 2 Report (New Reviewer)

Regarding MS entitled ‘’ Impact of different levels of crude protein on production performance and meat quality in broiler selected for slow growth’’

Why did the authors choose a slow-growing chicken hybrid?

For different levels of CP, the study only has standard and treatment groups!

L21-22. It is logical to decrease CP in the finisher than in the grower, why did the authors increase CP in the finisher phase?

In the abstract add a p-value for significant findings.

L23, what about FCR, feed intake, and body weight gain?

L27. A conclusion sentence with recommendations should be added at the end of the abstract.

Introduction

Please combine short paragraphs. In the introduction, the authors should address why they increased CP content. In the poultry industry, nowadays the farmers are trying to decrease CP content because the production cost is markedly increasing. Why did the authors not think about increasing AA requirements instead of CP?

Materials and Methods

There are many short paragraphs, please try to combine some of them together.

What is the sex of the birds? Male or females or mixed sex? If mixed sex, how the authors randomly distributed them in groups.

A link for the production performance of the breed should be added within text.

Table 2 title, please rephrase. Please add ME/CP ratio.

The authors totally removed full-fat soybean in the treatment diets, which decrease EE content compared to the control. This may affect the performance of the broilers of the standard compared to treatment!

In conclusion, please add recommendations to the readers.

Author Response

Honorable Reviewer,

Thank you for taking time in judging our manuscript and appreciating our work.

We  have operated some adjustments in comparison with the latter form of the manuscript, basing on your recommendations on the other reviewers' suggestions.

Hereby you may find the explanation for the suggestions you have made us:

Question 1> Regarding MS entitled ‘’ Impact of different levels of crude protein on production performance and meat quality in broiler selected for slow growth’’ Why did the authors choose a slow-growing chicken hybrid?

Answer: we have chosen a slow-growing hybrid because this is the trend among large size and medium size poultry companies, adapting to the market demands and such studies are well received by them as examples of optimising the costs. Also, conventional broilers are well studied. Thank you indeed!

Question 2> For different levels of CP, the study only has standard and treatment groups!

Answer: we have a control (Standard) group (T1) and a Treatment group with increased CP levels throughout Grower and Finisher diets (T2). At the moment of the experimental protocol design, we did not want to approach higher levels of CP, in order to avoid increasing of dietary costs. Thank you indeed! 

Question 3> L21-22. It is logical to decrease CP in the finisher than in the grower, why did the authors increase CP in the finisher phase?

Answer: Crude protein conversion is better after the enzymes equipment is better adapted (after starter period). Also, costs were cut because we have withdrawn full fat soy and used  soybean meal and oil.

Question 4> In the abstract add a p-value for significant findings.

ANSWER: We added these, thank you!

Question 5> L23, what about FCR, feed intake, and body weight gain?

Answer: We added these, thank you!

Question 6 > L27. A conclusion sentence with recommendations should be added at the end of the abstract.

The last sentences in the abstract underline the efficacy of supplementing CP during Grower phase only,

Question 7> Introduction. Please combine short paragraphs. In the introduction, the authors should address why they increased CP content. In the poultry industry, nowadays the farmers are trying to decrease CP content because the production cost is markedly increasing. Why did the authors not think about increasing AA requirements instead of CP?

Answer: we have opt out for crude protein increase due to lowering cost reasons instead of  adding supplemental AA. It would be useful to optimize a mixture of essential AA using the ideal protein concept.

Question 8 > Materials and Methods. There are many short paragraphs, please try to combine some of them together.

We proceeded accordingly, thank you!

Question 9 > What is the sex of the birds? Male or females or mixed sex? If mixed sex, how the authors randomly distributed them in groups. 

Answer: They were brooded as hatched, as is commonly done in most poultry farms. The results reflects the average of replicates (pens), where the birds were randomly allocated.

Question 10> A link for the production performance of the breed should be added within text.

ANSWER: added, thank you!

Question 11> Table 2 title, please rephrase. Please add ME/CP ratio.

Answer: We have proceeded accordingly, thank you!

Question 12> The authors totally removed full-fat soybean in the treatment diets, which decrease EE content compared to the control. This may affect the performance of the broilers of the standard compared to treatment!

Answer: we have withdrawn full fat soy in order to cut costs in T2 but we kept soymeal and oil to have the proper fat level and fatty acids levels. Thank you!

Question 13> In conclusion, please add recommendations to the readers.

Answer> conclusions were reformulated and the last 2 of them comprise recommendation. 

Thank you indeed!

Cordially yours,

On behalf of all authors,

Razvan RADU-RUSU

Reviewer 3 Report (New Reviewer)

Report

Introduction

very long, prolix and not very objective.

Authors should focus on the main research problem.

Abbreviation

CP, define. Line 85.

CP, line 98.

PC, define. Line 85.

V1 and V2, prefer: G for groups or T for treatments.

Define our experimental unity. Both: 50% males and 50% females? Unsexed? Make age clear at all stages of diet change.

Line 120- “equipment”: prefers systems, or apparatus…

Table 1. Experimental protocol Insert the age each phase. ie:

Starter diet - Y until X days.

Grower diet - Y until X days.

Finisher diet - Y until X days.

Line 109: were set up, then 100 day-old broilers (15 heads/m²). Prefer:  density- boiler or chicks/m².

Use a standard term: heads, chicks, broilers or birds.

Line 109 heads/m²

Line 114 600 chicks/treatment

Line 115 100 broilers/replicate

Body weight or Live weight.

pen or replicate.

Table 2. Structure and nutritional features of used diets - Requirements - amino acid levels: Basis: Total or SID?

Calculate and present in Table 2.

Total AAE essential amino acids (EAA) in basis CP or diet

Total NEAA non-essential amino acids (NEAA) in basis CP or diet.

Relation: EAA/NEAA in basis CP or diet.

 Statistical analyzes must consider the random effect of the association between repeated measurements taken in the same experimental unit.

In this research there is an evaluation considering the cumulative effect of the treatments on the growth of broilers.

Statistical analyzes must consider the random effect of the association between repeated measurements taken in the same experimental unit.

Therefore, it is an error to compare the phases considering that they are independent.

These results should be reanalyzed considering the procedure of repeated measurements in time.

Table 3. Live weight dynamics (n=50/treatment).

Table 4. Dynamics of daily weight gain (n=50/treatment).

Table 6. Feed intake and feed conversion rate

The others can be presented in the current form.

Especially the results of economic analysis must be calculated by replicate and must carry out the statistical analysis described in the item (2.6. Data treatment).

Table 7. European Production Efficiency Factor

Table 8. Economic efficacy indices (Expense, Incomes, Revenue, Rate of Profitability)

The mortality results should be performed a non-parametric statistical analysis. But first, it is advisable to check the assumptions for the normality of errors.

Table 5. Mortality dynamics

In general, the results can be presented in the same table when they are related to the same theme, for example, Table 3, 4 and 6 can be presented in the same table.

The same exercise can be performed on the other variables when there is similarity with the theme.

 Line 479- In Lingnan slow growing chicks’ or In Lingnan chicks’ slow growing?

The discussion should be more objective and faithful to the results of the statistical analyses.

It is recommended, after reanalyzing the data and having new p-values, to check the pertinence of some comparisons using percentage differences between groups.

Author Response

Suggestion 1: Introduction very long, prolix and not very objective.

Adjusted, thank you!

Suggestion 2: Authors should focus on the main research problem.

Abbreviation

CP, define. Line 85.

CP, line 98.

PC, define. Line 85.

We have tried to emphasise on the main research problem within the regional context of poultry production. Adjusted and used CP (crude protein) all over the place, thank you!

Suggestion 3: V1 and V2, prefer: G for groups or T for treatments.

We have used T1 and T2 in the present form of the manuscript, thank you.

Suggestion 4: Define our experimental unity. Both: 50% males and 50% females? Unsexed? Make age clear at all stages of diet change.

Each treatment comprised  6 pens (replicates) x 100 day old chicks on brooding moment. They were populated as hatched (mixed males and females). Performances were recorded as mean of the pen. Thank you. We specified clearly the duration of each diet. Thank you!

Suggestion 5 Line 120- “equipment”: prefers systems, or apparatus…

Replaced equipments by ”technological systems”

Suggestion 6 Table 1. Experimental protocol Insert the age each phase. ie:

Starter diet - Y until X days.

Grower diet - Y until X days.

Finisher diet - Y until X days.

Adjusted, thank you! 

Line 109: were set up, then 100 day-old broilers (15 heads/m²). Prefer:  density- boiler or chicks/m².

Use a standard term: heads, chicks, broilers or birds.

Line 109 heads/m²

Line 114 600 chicks/treatment

Line 115 100 broilers/replicate

We decided to use ”heads”. Thank you!

Body weight or Live weight.

We decided to keep ”body weigh” everywhere

pen or replicate.

We used pen, specifying as well that 1 pen containing 100 heads = 1 replicate

Suggestion 7: Table 2. Structure and nutritional features of used diets - Requirements - amino acid levels: Basis: Total or SID?

Calculate and present in Table 2.

Total AAE essential amino acids (EAA) in basis CP or diet

Total NEAA non-essential amino acids (NEAA) in basis CP or diet.

Relation: EAA/NEAA in basis CP or diet.

We have operated the changes related to AA, EAA, NEAA, ratios. Also, we kept AA as per feed (total) and digestible AA content.

Suggestion 8: Statistical analyzes must consider the random effect of the association between repeated measurements taken in the same experimental unit.

In this research there is an evaluation considering the cumulative effect of the treatments on the growth of broilers.

Statistical analyzes must consider the random effect of the association between repeated measurements taken in the same experimental unit.

Therefore, it is an error to compare the phases considering that they are independent.

These results should be reanalyzed considering the procedure of repeated measurements in time.

Answer: we did not compare the phases, we have run comparisons between T1 and T2 in the same phase.

Suggestion 9

Table 3. Live weight dynamics (n=50/treatment).

Table 4. Dynamics of daily weight gain (n=50/treatment).

We merged data in tables 3, 4, 5 into one table

Table 6. Feed intake and feed conversion rate

we presented these data as means +- Std error of mean basing on the values per replicate (measured in each of the 6 pens per treatment)

The others can be presented in the current form.

Suggestion 10 Especially the results of economic analysis must be calculated by replicate and must carry out the statistical analysis described in the item (2.6. Data treatment).

Table 7. European Production Efficiency Factor

Table 8. Economic efficacy indices (Expense, Incomes, Revenue, Rate of Profitability)

We have presented the results per treatment (for the whole flock of each treatment that have reached each moment of diet switching and that have reached the slaughterhouse). In this case, we had almost 600 heads slaughtered for each treatment. Otherwise, the data would reflect the mean and statistics of 6 repetitions of almost 100 heads per treatment. Whole treatment is more realistic when comes to slaughtering.

Suggestion 11 Table 5. Mortality dynamics

The mortality results should be performed a non-parametric statistical analysis. But first, it is advisable to check the assumptions for the normality of errors.

The mortality was expressed as mean & SEM, resulted from 6 replications per treatment and it was introduced in t test, T1 vs T2, per each analysis moment.

Suggestion 12 - In general, the results can be presented in the same table when they are related to the same theme, for example, Table 3, 4 and 6 can be presented in the same table.

The same exercise can be performed on the other variables when there is similarity with the theme.

We have merged some tables with compatible variables, Thank you

 Line 479- In Lingnan slow growing chicks’ or In Lingnan chicks’ slow growing?

in ”Lignan” slow growing chickens is correct Lignan is the genotype, slow growing is functioning as an adjective. The apostrophe after chickens ' it is not required because it is not a genitive case. Thank you!

Suggestion 13- The discussion should be more objective and faithful to the results of the statistical analyses.

It is recommended, after reanalyzing the data and having new p-values, to check the pertinence of some comparisons using percentage differences between groups.

We reported the differences now as percentage differences and in relation with t test p values. Thank you!

Thank you indeed for your precious suggestions!

Cordially yours, on behalf of all authors,

Razvan RADU-RUSU

Reviewer 4 Report (New Reviewer)

The Manuscript deals with an interesting topic and the research brings results that can be applied in practice. There are some inconsistencies and certain shortcomings in the work that should be fixed.

Introduction: In line 67, you state that there are many studies that investigated the same topic, but by the end of the Introduction, you state only one. There are some in the previous part of the Introduction, but after such a statement I expect several references in this or at least the next sentence.

Material and methods: You very precisely describe the Methods you used in your research, but again you do not provide a single Reference. I understand that it is not necessary for everything, but perhaps a Reference for the protocol of sensory research, as you have ISO standards for chemical analysis.

Results and Discussion: I have no objections in this part, maybe only that you repeat the results you have shown in the tables, but nothing significant.

However, I would like an explanation in the Discussion, such as that the treatment with additional more protein was cheaper per unit mass. It is known that protein feeds are the most expensive, and your treatment with them turned out to be cheaper. Is this due to the specifics of the local market, own production, ..., but not globally, so please explain how you came to this in your research.

Conclusion: This part of the Manuscript is problematic for me. You conclude that additional protein feeding improves growth parameters, meat production and other properties compared to standard feeding. Maybe soften these conclusions a bit, since you didn't get such a significant difference between the treatments in the results, that is, you got only a few of them. In the results, you got only a few statistically significant differences, but not enough for such a conclusion. Therefore, I propose to correct the Conclusion according to the obtained Results.

After accepted comments and corrections, we will gladly propose the manuscript for publication in the Journal.

Author Response

Honorable reviewer,

Thank you indeed for taking time in helping us improving our manuscript.

Hereby are our answers to your suggestion!

The Manuscript deals with an interesting topic and the research brings results that can be applied in practice. There are some inconsistencies and certain shortcomings in the work that should be fixed.

Suggestion 1

Introduction: In line 67, you state that there are many studies that investigated the same topic, but by the end of the Introduction, you state only one. There are some in the previous part of the Introduction, but after such a statement I expect several references in this or at least the next sentence.

Answer> we have improved the citations and all Introduction to bring it closer to the main topic.

Suggestion 2

Material and methods: You very precisely describe the Methods you used in your research, but again you do not provide a single Reference. I understand that it is not necessary for everything, but perhaps a Reference for the protocol of sensory research, as you have ISO standards for chemical analysis.

Answer: we added references for the analytical standards. Thank you indeed!

Suggestion 3

Results and Discussion: I have no objections in this part, maybe only that you repeat the results you have shown in the tables, but nothing significant.

However, I would like an explanation in the Discussion, such as that the treatment with additional more protein was cheaper per unit mass. It is known that protein feeds are the most expensive, and your treatment with them turned out to be cheaper. Is this due to the specifics of the local market, own production, ..., but not globally, so please explain how you came to this in your research.

answer> The cost was cut because we withdrawn the full fat soybean and sunflower meal in T2 and the cost per kg feed was a bit lower. Also, overall costs were lower due to lower feed intake in T2 and to better gain in T2. Therefore, feed conversion ratio was better in T2 and the European Production Efficiency factor was better in T2 (lower mortality, lower FCR, higher body weight at slaughter).

Suggestion 4 Conclusion: This part of the Manuscript is problematic for me. You conclude that additional protein feeding improves growth parameters, meat production and other properties compared to standard feeding. Maybe soften these conclusions a bit, since you didn't get such a significant difference between the treatments in the results, that is, you got only a few of them. In the results, you got only a few statistically significant differences, but not enough for such a conclusion. Therefore, I propose to correct the Conclusion according to the obtained Results.

we have modified the conclusions, thank you!

After accepted comments and corrections, we will gladly propose the manuscript for publication in the Journal.

Thank you indeed!

Cordially yours,

On behalf of all team of authors,

Razvan RADU-RUSU

Round 2

Reviewer 2 Report (New Reviewer)

Thank you for your revisions. Following your revisions, I have minor comments.

1. Short paragraphs in introduction and conclusion sections should be combined, for example, paragraphs L45 and L49. It is not usual in scientific research.

2. Please go through your manuscript and correct any grammar mistakes.

Author Response

Honorable reviewer,

We have considered your recommendations and merged some of the small paragraphs into larger ones, in order to facilitate fluent reading.

Also, we have run an English check for the whole manuscripts.

Thank you indeed for all of your professional and valuable suggestion and support in improving our manuscript.

Cordially yours,

Marius USTUROI, main author

Reviewer 3 Report (New Reviewer)

The authors accepted the suggestions.

Author Response

Honorable reviewer,

We have considered your recommendations and we have run an English check for the whole manuscripts.

Thank you indeed for all of your professional and valuable suggestion and support in improving our manuscript.

Cordially yours,

Marius USTUROI, main author

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Line 62.  It is also well known that approximately 75-85% of poultry meat production costs are 62 generated by feeding.

Needs to be rewritten with figures 70-75%.

Causalities in the whole manuscript be replaced by Mortality.

Line 310. Dressing percentage data needs rechecking and appropriate changes be incorporated.

Author Response

Coverletter for Reviewer 1

Former manuscript Agriculture-2115629, Resubmitted as Agriculture-2184331

Honorable reviewer,

Thank you for your suggestions. We have complied with them and adjusted the manuscript accordingly.

Related to your punctual suggestions, here are our answers:

62. Line 62.  It is also well known that approximately 75-85%of poultry meat production costs are 62 generated by feeding.

Needs to be rewritten with figures 70-75%.

Answer: We put in text 70-75%, thank you!

1. Causalities in the whole manuscript be replaced by Mortality.

Answer: We have replaced casualties by Mortality, thank you! 

Line 310. Dressing percentage data needs rechecking and appropriate changes be incorporated.

Answer: We have recalculated the dressed yield values, data were not right, indeed. Thank you!

Thank you indeed and please rest assured of our kind consideration!

On behalf of all authors,

Razvan Mihail RADU-RUSU

Reviewer 2 Report

This article has shortage in the methodology. For repeatability of experiment all components of all diets which was used in the experiment must be exact quantified. For example the proportion of soybean meal, or soybean oil, or essential amino acids in the diets is unclear. Right this are factors affecting efficiency of broiler meat production. This was feeding experiment, therefore entire information about diet (feed mixtures) composition must be listed in the methodology. In addition, results of meat quality parameters and sensory analysis is missing.

Author Response

Coverletter for Reviewer 2

Former manuscript Agriculture-2115629, Resubmited as Agriculture-2184331

Honorable reviewer,

Thank you for your suggestions. We have complied with them and adjusted the manuscript accordingly.

Related to your punctual suggestions, here are our answers:

This article has shortage in the methodology. For repeatability of experiment all components of all diets which was used in the experiment must be exact quantified. For example the proportion of soybean meal, or soybean oil, or essential amino acids in the diets is unclear. Right this are factors affecting efficiency of broiler meat production. This was feeding experiment, therefore entire information about diet (feed mixtures) composition must be listed in the methodology. In addition, results of meat quality parameters and sensory analysis is missing.

Answers:

• We simplified the methodology by reorganising the experimental protocol and leaving one single genotype, in order to compare the standard diet with the one enriched in crude protein. Also, we extracted data from the main experimental database, in order to present it as it was collected, as 6 replicates per each treatment.
• We have updated the manuscript by providing detailed diet specifications, both as feedstuffs proportion and nutritional value. We discussed the effects of crude protein and essential amino acids supplementations.
• We added data related to meat proximate composition and with sensory scoring.
• All data was recalculated statistically to achieve new descriptors and analysis of variance p values, in accordance with the new arranged data.

Thank you indeed and please rest assured of our kind consideration!

On behalf of all authors,

Razvan Mihail RADU-RUSU

Reviewer 3 Report

The manuscript is unclear on experimental design and statistical analysis.

(1) The authors did not provide clear information about experimental design, what was the experimental unit (EU) and how many EUs were in this experiment? Moreover, the authors did not provide information about the number of replications and the number of animals in each replication, which is well known that the number of replications is the key to making the repeatability of experimental results.

(2) As far as I can understand, this experiment had 4 experimental groups without replication (n=1), so it was impossible to analyze the statistical results. The authors did not clarify what statistic that use for the significant test between the treatments. Even if the authors use ANOVA, when n=1 it is impossible to calculate the F-test.

(3) The authors may misunderstand that 50 birds/house was 50 replications (n=50), the word ‘replication’ is that 1 replication must be equal to 1 EU, meaning that the animals in each EU must be independent of each other. However, the 50 chickens were taken from the same house that they had social hierarchy which influence to the measured observations, so it cannot be concluded that the results are derived from the effect of the treatment alone. Consequently, the interpretation of the experimental results may be incorrect.

(4) The analysis of the significant test must do an ANOVA and F-test first, with only one P-value, and after significant findings, multiple comparisons will be performed to compare the differences between treatments.

From the reason stated above, I am unable to ensure that the results of this study are reliable or unreliable.

Author Response

Coverletter for Reviewer 3

Former manuscript Agriculture-2115629, Resubmited as Agriculture-2184331

Honorable reviewer,

Thank you for your suggestions. We have complied with them and adjusted the manuscript accordingly.

Related to your punctual suggestions, here are our answers:

1) The authors did not provide clear information about experimental design, what was the experimental unit (EU) and how many EUs were in this experiment? Moreover, the authors did not provide information about the number of replications and the number of animals in each replication, which is well known that the number of replications is the key to making the repeatability of experimental results.

 Answer:

• We clarified the methodology by reorganising the experimental protocol. We collected data from two treatments (V1 and V2), each of it having 6 replications (6 pens x 100 individuals). So the EU = 100 individuals, isolated in 6 pens from the rest of the hall. Thank you!

(2) As far as I can understand, this experiment had 4 experimental groups without replication (n=1), so it was impossible to analyze the statistical results. The authors did not clarify what statistic that use for the significant test between the treatments. Even if the authors use ANOVA, when n=1 it is impossible to calculate the F-test.

 Answer:

• We re-processed the experimental data and left the results of a single genotype, in order to compare the standard diet (V1 treatment) with the one enriched in crude protein (V2 treatment). Every treatment had 6 replicates (6 pens x 100 day old chicks at experiment onset). The data was reanalysed using the t test because only 2 treatments were compared (not multiple comparisons). Thank you!

(3) The authors may misunderstand that 50 birds/house was 50 replications (n=50), the word ‘replication’ is that 1 replication must be equal to 1 EU, meaning that the animals in each EU must be independent of each other. However, the 50 chickens were taken from the same house that they had social hierarchy which influence to the measured observations, so it cannot be concluded that the results are derived from the effect of the treatment alone. Consequently, the interpretation of the experimental results may be incorrect.

Answer:

• The number of replications = 6 / treatment, in each replicate we had 100 individuals. All statistics and analysis of variance were calculated for the newly reorganised protocol. Thank you!

(4) The analysis of the significant test must do an ANOVA and F-test first, with only one P-value, and after significant findings, multiple comparisons will be performed to compare the differences between treatments.

Answer:

• You are right, in the former manuscript, we have used a multiple ANOVA comparison. In this revised version, we did not use Fisher test, followed by a Post-Hoc, suh as Tukey, because we have decided to compare the single effect of the different dietary protein level applied on a single genotype (instead of 2 genotypes in the former version of the manuscript). Par consequence, we remained only with two treatments to compare and we prefered to run anTwo tailed Unpaired t Test with Welch correction (built in GraphPad Prism 9.4.1. statistical analysis software), assuming the standard deviations were not equal. Thank you!

Also, supplemental data was included in the manuscript (such as detailed diets – feedstuffs proportions and nutritional value), results on meat proximate composition and sensory analysis.

Thank you indeed and please rest assured of our kind consideration!

On behalf of all authors,

Razvan Mihail RADU-RUSU