Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Review of Fatemi and Peebles - Manuscript-poultry-3738536-6.27

Summary

The results, showing there is only a slow decline in of 25 hydroxy vitamin D₃ (25OHD) with reduction in intakes, is interesting.

The paper describes a feeding experiment in broiler chickens to compare the effects of Hy-D diets containing high concentrations of 25OHD (but lower that some reported references) on growth and commercial production parameters with those of control broilers fed diets containing recommended dietary concentrations of vitamin D₃. The concentration of vitamin D₃ was similar in the 3 control diets (starter, grower and finisher) and in the Hy-D diets: in the Hy-D diets the concentrations of 25OHD decreased from starter through grower to finisher (550 µg/kg diet, 276 µg/kg diet, 34.5 µg/kg diet): control diets contained no 25OHD.

Despite the decrease in 25OHD intake with age, body weights, all measures of production during starter, grower and finisher phases were significantly lower (except for FCR which was higher) in Hy-D broilers than controls. Mortality during grower and finisher stages was higher in Hy-D than controls. Serum 25OHD concentrations decreased with decreased dietary concentration but remained significantly higher than controls. The authors conclusions were that adding 25OHD at 8x recommended dietary concentrations to starter then 4x to grower and 2x to finisher diets resulted in a toxicity perhaps resulting from pathological changes in the starter phase that were not overcome with reduction of dietary 25OHD.

General concept

The objectives (lines 16 – 18) mention only the high 25OHD concentration in the starter diet – no mention is made of objectives for elevated circulating concentrations reduced with different dietary concentration as the broilers age.

Introduction

I felt the Introduction (particularly the first paragraph) would be improved by shortening it. The description of metabolism of vitamin D is not relevant to the experimental methodology or results. It would also be easier to read if the text was formed into shorter paragraphs each covering a specific topic or idea. The whole article would benefit from careful editing.

Line 66 reference to toxicity needed

L68 “interior” change to “internal”

L89 Is the half-life in serum?

L92 change “the safe” to “a safe”

L 96 change “has” to “have”

L97 meaning not clear

Materials and Methods

Again, shorter paragraphs would be helpful to the reader. The section is mostly clearly explained.

L129 – Are these measured at the end of each dietary phase that is  - 14, 28, 42 doa.

L133 add killed, muscles removed and weighed.

L138 after “… artery.”  Add allowed to clot and then centrifuged. Approximately 1 ml serum was ….

Table 1

delete “Pct” in all places

Add manufacturer and concentration of 25OHD solution used.

Table 2

Units would be better in µg/kg.

Results

For consistency it is better to use controls and Hy-D to name diets or treatment groups.

Table 3

Add a heading

In footnotes define abbreviations and units eg BWG per d

Table 4

Add a heading.

In footnote define % (percentage of what)

The information in Fig. 1 could easily be in text of results.

Discussion

Again, breaking up into smaller paragraphs would be helpful.

Some descriptions of, or comparisons with, published information on clinical effects of toxicity are not necessary because they were not investigated in this paper.

L195 – 196 this isn’t very clear. I am not sure what “renal classification” refers to.

L207 – 208 Needs a reference

L226 delete “even”

Line 227-228 delete sentence as it is not relevant.

Line 229 delete “highly”

L233 English expression could be improved with a bit of editing

L243 – 244 English expression could be improved with a bit of editing

L250 – 255 Perhaps this should be in the aims.

Conclusions

Some of the conclusions are a repetition of results and should be deleted.

Suggestions for future work should be a separate paragraph.

A reference I thought might be relevant is

Gabriela Gómez-Verduzco, Rene Morales-López and Ernesto Avila-Gozàlez Use of 25-hydroxycholecalciferol in Diets of Broiler Chickens: Effects on Growth Performance, Immunity and Bone Calcification J. Poult. Sci., 50: 60-64, 2013

Comments on the Quality of English Language

I strongly recommend that editing and English expression is improved.

Author Response

Reviewer 1

Review of Fatemi and Peebles - Manuscript-poultry-3738536-6.27

Summary

The results, showing there is only a slow decline in of 25 hydroxy vitamin D3 (25OHD) with reduction in intakes, is interesting.

The paper describes a feeding experiment in broiler chickens to compare the effects of Hy-D diets containing high concentrations of 25OHD (but lower that some reported references) on growth and commercial production parameters with those of control broilers fed diets containing recommended dietary concentrations of vitamin D3. The concentration of vitamin D3 was similar in the 3 control diets (starter, grower and finisher) and in the Hy-D diets: in the Hy-D diets the concentrations of 25OHD decreased from starter through grower to finisher (550 µg/kg diet, 276 µg/kg diet, 34.5 µg/kg diet): control diets contained no 25OHD.

Despite the decrease in 25OHD intake with age, body weights, all measures of production during starter, grower and finisher phases were significantly lower (except for FCR which was higher) in Hy-D broilers than controls. Mortality during grower and finisher stages was higher in Hy-D than controls. Serum 25OHD concentrations decreased with decreased dietary concentration but remained significantly higher than controls. The authors conclusions were that adding 25OHD at 8x recommended dietary concentrations to starter then 4x to grower and 2x to finisher diets resulted in a toxicity perhaps resulting from pathological changes in the starter phase that were not overcome with reduction of dietary 25OHD.

Answer:

We appreciate the time spent on readying meticulously our manuscript and we will try to properly address your comments

General concept

1. The objectives (lines 16 – 18) mention only the high 25OHD concentration in the starter diet – no mention is made of objectives for elevated circulating concentrations reduced with different dietary concentration as the broilers age.

Answer:

Thank you for the comments. The elevated circulating concentration is in response to the  dietary concentration provided. Therefore, we did not aim to raise the blood stream level of 25OHD₃, as this just happened because of the high dietary level of 25OHD₃. At certain levels, this rise did not occur because the bird’s body was to catabolize it. The whole objective was  to demonstrate that the elevated level of circulating 25OHD₃ can be a good indicator in determining the of toxicity of vitamin D sources.

Introduction

2. I felt the Introduction (particularly the first paragraph) would be improved by shortening it. The description of metabolism of vitamin D is not relevant to the experimental methodology or results. It would also be easier to read if the text was formed into shorter paragraphs each covering a specific topic or idea. The whole article would benefit from careful editing.

Answer:

Thank you for the suggestion, however, the necessity of providing basic information on vitamin D metabolism is for obtaining a better understanding of the mechanisms by which vitamin D sources are observed to become actively functional, and what could interfere with this process. Also, knowing the function of vitamin D is also needed to show why this vitamin is important as a supplemental material, and  what dietary levels of vitamin D are expected to exhibit positive effects. After the inclusion of that information, the terminology of vitamin D toxicity (or hypervitaminosis D) and its relevant consequences in both broiler and layers were introduced. It is also important because of the differences that high levels of vitamin D may have different effects on the tolerance and on the BW and growth rate of different aged broilers.

3. Line 66 reference to toxicity needed

Answer:

The relevant correction was applied.

4. L68 “interior” change to “internal”

Answer:

The relevant correction was applied.

5. L89 Is the half-life in serum?

Answer:

Yes, the serum level was measured. The relevant correction was applied.

6. L92 change “the safe” to “a safe”

Answer:

The relevant correction was applied. 

7. L 96 change “has” to “have”

Answer:

The grammatical correction was made.

8. L97 meaning not clear

Answer:

The relevant correction was applied, and now it reads as

“indicating that a 10-fold use of supplemental 25OHD₃ may not be safe in new generation broilers.”

Materials and Methods

9. Again, shorter paragraphs would be helpful to the reader. The section is mostly clearly explained.

Answer:

Thank you for the suggestion; however, this section is approximately one page and an effort was made to cite those methodologies that were similar to previous work in order to shorten this section.

10. L129 – Are these measured at the end of each dietary phase that is - 14, 28, 42 doa.

Answer:

Yes, the relevant correction was added so as to more clearly showthis statement.

11. L133 add killed, muscles removed and weighed.

Answer:

The relevant correction was applied. 

12. L138 after “… artery.” Add allowed to clot and then centrifuged. Approximately 1 ml serum was ….

Answer:

The relevant correction was applied. 

Table 1

delete “Pct” in all places

Answer:

Thank you for the suggestion; however, this is common practice in showing the percentage of ingredients in diets, and we used the same format in our earlier publications as was previously recommended.

13. Add manufacturer and concentration of 25OHD solution used.

Answer:

The relevant information was added. 

Table 2

14. Units would be better in µg/kg.

Answer:

Thank you for the suggestion; however, both IU/Kg and µg/kg are acceptable units for the expression of all vitamin D sources. Also, because IU/Kg is a more common expression for Vitamin D₃ level, use of this term for both vitamin D₃ and 25OHD₃ was decided upon.

Results

15. For consistency it is better to use controls and Hy-D to name diets or treatment groups.

Answer:

On lines 113-114, definition of the term “Hy-D diet” was provided (“Hy-D diets that consisted of 552 (8x) μg/kg of 25OHD₃ (water-soluble source) for the starter, 276 (4x) μg/kg of 25OHD₃ for the grower, and 34.5 (0.5 x) μg/kg of 25OHD₃ for the finisher dietary phases”). The terms “Controls and Hy-D” were subsequently used to designate those specific diets.

Table 3

16. Add a heading

Answer:

The relevant correction was applied. 

17. In footnotes define abbreviations and units eg BWG per d

Answer:

Units are available in the Table and all performance variables are defined in the footnotes.

Table 4

18. Add a heading.

Answer:

The relevant correction was applied. 

19. In footnote define % (percentage of what)

Answer:

The relevant information was added to the heading.

20. The information in Fig. 1 could easily be in text of results.

Answer:

Thank you, that information is also in the text of the results.

Discussion

21. Again, breaking up into smaller paragraphs would be helpful.

Answer:

We would like to minimize the length, but for a better understanding the findings we needed to comprehensively discuss all relevant concepts that may be involved with or linked to our results. It is worth-mentioning that the overall length of the discussion is 3 to 4 pages, which is in the acceptable range.

22. Some descriptions of, or comparisons with, published information on clinical effects of toxicity are not necessary because they were not investigated in this paper.

Answer:

Thank you for the suggestion. The relevant corrections was applied.

23. L195 – 196 this isn’t very clear. I am not sure what “renal classification” refers to.

Answer:

The relevant correction was made to this sentence and now it reads as:

“In contrast to that of VitD_3, renal calcification was observed in broilers when dietary 25OHD₃ was supplemented at levels that were 5 to 10 times greater than the recommended level [33].”

24. L207 – 208 Needs a reference

Answer:

The relevant reference was added.

25. L226 delete “even”

Answer:

The relevant correction was applied.

26. Line 227-228 delete sentence as it is not relevant.

Answer:

Please see added and rewritten statements encompassing lines 177-218 of the R1 version that replaced lines 207-250 in the submitted version.

27. Line 229 delete “highly”

Answer:

Please see added and rewritten statements encompassing lines 177-218 of the R1 version that replaced lines 207-250 in the submitted version.

28. L233 English expression could be improved with a bit of editing

Answer:

Please see added and rewritten statements encompassing lines 177-218 of the R1 version that replaced lines 207-250 in the submitted version.

29. L243 – 244 English expression could be improved with a bit of editing

Answer:

Please see added and rewritten statements encompassing lines 177-218 of the R1 version that replaced lines 207-250 in the submitted version.

30. L250 – 255 Perhaps this should be in the aims.

Answer:

This information is in the conclusions of the discussion. Nevertheless,, the goal of this research was to show that a 10 fold increase in the dietary source of 25OHD₃ is not safe and that further investigation is required to determine actual safer doses.

Conclusions

31. Some of the conclusions are a repetition of results and should be deleted.

Answer:

Part of the conclusion section is to revisit the objectives of the study and to briefly describe the main results of study; therefore the first few sentences of the conclusion were allocated for that information.

32. Suggestions for future work should be a separate paragraph.

Answer:

The relevant changes were applied.

33. A reference I thought might be relevant is

Gabriela Gómez-Verduzco, Rene Morales-López and Ernesto Avila-Gozàlez Use of 25-hydroxycholecalciferol in Diets of Broiler Chickens: Effects on Growth Performance, Immunity and Bone Calcification J. Poult. Sci., 50: 60-64, 2013

Answer:

The recommended reference is added and is used in the Introduction section.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

General comments:

The present study aims to evaluate the effects of disproportionally high levels of 25OHD3 on broiler performance and meat yield. While the topic is of interest, especially given the growing attention on vitamin D metabolites in poultry nutrition, several aspects of the experimental design and reporting need further clarification to strengthen the scientific rigor and reproducibility of the work.

The rationale behind the use of such a high dosage (8 times the recommended level) of 25OHD3 is not clearly explained. Moreover, it is unclear why lower levels were adopted during the grower and finisher phases, and whether this was based on previous evidence, safety considerations, or other factors. A more detailed justification of the dosage strategy across the different growth phases would enhance the clarity and scientific basis of the study.

In addition, the manuscript focuses primarily on performance and meat yield outcomes. While these are important, the inclusion of data on health-related parameters (e.g., blood 25OHD3 levels, and potential effects on key organs such as the liver and kidneys) would provide a more comprehensive understanding of the biological implications of such high vitamin D metabolite levels.

Specific comments:

• Several important details on housing and management conditions are missing (e.g., pen dimensions, litter type, environmental temperature, lighting program). These are essential for evaluating the reproducibility and external validity of the study.

• The diet composition is reported in three separate tables, although the only difference between groups is the inclusion level of 25OHD3. Consolidating this information into a single table or describing the differences in the text would improve readability and reduce redundancy.

• The vitamin premix used in the diet already contains cholecalciferol. This should be explicitly addressed in the discussion to better interpret the interactions between vitamin D3 sources.

• In the finisher phase, there appears to be a notable difference in actual vitamin D3 content between control and experimental diets, which warrants further explanation.

• Key methodological details regarding animal slaughter and sample collection are missing and should be added for transparency and reproducibility.

• Lines 153–154 mention an in ovo treatment, which does not apply to this study and should be corrected.

• In the results section, some references to tables are missing. These should be added to guide the reader through the data.

• Table 1 does not include the initial body weight of the animals. Including this information would allow readers to assess whether groups were comparable at baseline.

• The presentation of both FI and ADFI may be redundant. Additionally, feed intake data should be interpreted with caution if the number of animals per group changed due to mortality. Clarifying this aspect would improve data interpretation.

Author Response

Reviewer 2

General comments:

1. The present study aims to evaluate the effects of disproportionally high levels of 25OHD3 on broiler performance and meat yield. While the topic is of interest, especially given the growing attention on vitamin D metabolites in poultry nutrition, several aspects of the experimental design and reporting need further clarification to strengthen the scientific rigor and reproducibility of the work.

Answer:

The relevant responses are provided below to all of the specific comments.

2. The rationale behind the use of such a high dosage (8 times the recommended level) of 25OHD₃ is not clearly explained. Moreover, it is unclear why lower levels were adopted during the grower and finisher phases, and whether this was based on previous evidence, safety considerations, or other factors. A more detailed justification of the dosage strategy across the different growth phases would enhance the clarity and scientific basis of the study.

Answer:

Thank you for the comment. Relevant explanations regarding each of the concerns were given in the manuscript, and to be more clear, the specific sections that correspond to each of the concerns are provided below:

1. Rational behind 8x.

Previous research regarding vitamin D toxicity showed a margin of safety up to 10x recommended levels. However, that previous research was conducted in the late 80s or early 90s using on broilers or layers that were not as fast growing as today’s birds. Furthermore, their Ca and P absorption capabilities were not as efficient as today’s modern birds. Therefore, they had a greater capacity to tolerate higher levels of 25OHD₃. Previously, broilers have been able to tolerate up 100x the level of dietary vitamin D₃ without interfering with their performance. However, currently, they cannot tolerate 35x the level of dietary vitamin D₃, in which negative effects are observed in their live performance variables and associated internal organ damages. (This information is provided in the Introduction and Discussion sections).

Secondly, this study was founded by USDA, which  requires that mortality rates don’t exceed 20% throughout the growing phase in all trials. [This information (given below) is now in the Introduction sections on lines 98 to 100].

“It should also be noted that this study was funded by the United States Department of Agriculture, which mandates that trials be terminated when a 20% mortality rate is reached..”

1. lower level was adopted

We disagree that the lower level was adopted in the grower and finisher phases, because the damages due to the 8x level was detrimental in the starter phase and did not allow birds to recover in the rest of the growing phase. Also, the mortality rate continued as the birds developed.

3. In addition, the manuscript focuses primarily on performance and meat yield outcomes. While these are important, the inclusion of data on health-related parameters (e.g., blood 25OHD₃ levels, and potential effects on key organs such as the liver and kidneys) would provide a more comprehensive understanding of the biological implications of such high vitamin D metabolite levels.

Answer:

We agree that the major concept of this manuscript is focused on the bird’s performance; however, there is also a close association between high serum concentrations of 25OHD₃ and vitamin D toxicity. Although we did not look at the organ morphological changes as a result of hypervitaminosis D, we suggested that further research is needed to determine the possible organ damages that may have been incurred in response to high doses of 25OHD₃. In addition, no studies have been conducted on modern broilers to determine safe levels of dietary 25OHD₃, and our results just showed that even 8x the recommended level of 25OHD₃ caused birds to suffer. As is indicated, further research is needed to determine safe dietary 25OHD₃ levels. This requires that more dietary treatments be tested, and that research should not only be focused on bird performance, but also on any physiological damages at the cellular level. We modified the Discussion section in order to be more focused on the aforementioned topics.

Specific comments:

4. Several important details on housing and management conditions are missing (e.g., pen dimensions, litter type, environmental temperature, lighting program). These are essential for evaluating the reproducibility and external validity of the study.

Answer:

Thank you for the comment. The relevant information was added on lines 119 to 122, and now reads as:

“Floor pens contained used litter top dressed with fresh wood shavings and were 1.22 m × 0.914 m (1.12 m²) in dimension, which allowed for a 0.062 m2/bird stocking density. Birds had ad libitum access to water and feed and were brooded according to the Ross 708 guidelines throughout the 42 doa grow out period [42].”

5. The diet composition is reported in three separate tables, although the only difference between groups is the inclusion level of 25OHD3. Consolidating this information into a single table or describing the differences in the text would improve readability and reduce redundancy.

Answer:

Thank you for the suggestion, however, it is common practice in nutrition based studies to provide the whole diet composition in order to allow the reader to have comprehensive information about diet formulation throughout all 3 growing phases. Additionally, that information is more readily readable when provided in the tables than in short incomplete sentences in the text.

6. The vitamin premix used in the diet already contains cholecalciferol. This should be explicitly addressed in the discussion to better interpret the interactions between vitamin D3 sources.

Answer:

We already mentioned in the Introduction on lines 60 to 64 that it is necessary that vitamin D be included at a level used in control diets in order to maintain normal production.

7. In the finisher phase, there appears to be a notable difference in actual vitamin D3 content between control and experimental diets, which warrants further explanation.

Answer:

Thank you for the comments. The relevant correction regarding this matter was applied to the value in Table 2.

8. Key methodological details regarding animal slaughter and sample collection are missing and should be added for transparency and reproducibility.

Answer:

The relevant information was added in the Material and Method section on lines 138 to 144. That information now reads as:

“At 14, 28, and 39 doa, one bird from each of the 10 replicate pens per dietary treatment were randomly selected (20 total birds), and were bled by venipuncture of the wing brachial artery. After collection, blood samples were allowed to clot and were then centrifuged. An approximate 1 ml volume of serum was subsequently extracted for determination of 25OHD₃ concentration according to an RIA assay procedure described by Hollis et al. [43]. Later, the same sample of birds were individually weighed and euthanized by CO₂ for meat yield determinations that included percentage pectoralis major (P. major), and pectoralis minor (P. minor) weights. Total breast yield percentage was also calculated by adding the values of the P. major and P. minor muscles.”

9. Lines 153–154 mention an in ovo treatment, which does not apply to this study and should be corrected.

Answer:

The relevant correction was applied.

10. In the results section, some references to tables are missing. These should be added to guide the reader through the data.

Answer:

Thank you for the comments. Some heading titles were missed in the original draft, but are now included, and the corresponding abbreviations are now split out in either the heading or foot notes of each table.

11. Table 1 does not include the initial body weight of the animals. Including this information would allow readers to assess whether groups were comparable at baseline.

Answer:

The relevant information was added to Table 3 where the live performance variables were reported. Table 1 belongs to feed composition.

12. The presentation of both FI and ADFI may be redundant. Additionally, feed intake data should be interpreted with caution if the number of animals per group changed due to mortality. Clarifying this aspect would improve data interpretation.

Answer:

Thank you for the comment. FI refers to feed intake within each dietary phase, but it is also important to report daily feed intake or daily gain. It is common practice to demonstrate not only the changes in each dietary phase but also to provide some estimation about daily changes, because it may be beneficial if daily changes are noteworthy.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This paper aims to investigate the effects of high dietary levels of 25-hydroxycholecalciferol (25OHD₃) on serum 25OHD₃ concentration, production performance, breast meat yield, and other indicators of Ross 708 broilers. The experimental results show that excessive intake of dietary 25OHD has adverse effects on broiler production performance and meat yield, which may be related to vitamin D toxicity. However, the manuscript has several major issues:

1. The experimental results data are insufficient to support the article's conclusions.

2. There are problems in the experimental design. Firstly, from the experimental results in the manuscript, the 8-fold commercial standard concentration has extremely strong negative effects, but the authors did not provide results of other concentrations (such as 4-fold) for comparison. Secondly, the manuscript lacks data on serum calcium ion concentration, histopathological examination of the liver and kidney, or other physiological effects of 25OHD₃, making it impossible to explain the metabolic process, tissue accumulation, or toxicological mechanism of 25OHD₃ in broilers. Thirdly, general tolerance tests on broilers require maintaining a fixed treatment concentration throughout the cycle, and it is clear that such an experimental design does not serve this purpose. Therefore, the dose settings of 8-fold in the starter phase, 4-fold in the grower phase, and 0.5-fold in the finisher phase lack clear significance.

3. Other issues: The control group uses vitamin D₃ as the source, while the treatment group uses 25OHD₃; the equivalence between the two needs to be clarified. The production performance data should use the 39-day-old data to match the 39-day slaughter performance measurement. The mortality statistics should use the t-test.

Author Response

Reviewer 3

this paper aims to investigate the effects of high dietary levels of 25-hydroxycholecalciferol (25OHD₃) on serum 25OHD₃ concentration, production performance, breast meat yield, and other indicators of Ross 708 broilers. The experimental results show that excessive intake of dietary 25OHD has adverse effects on broiler production performance and meat yield, which may be related to vitamin D toxicity. However, the manuscript has several major issues:

1. The experimental results data are insufficient to support the article's conclusions.

Answer:

Thank you for the comments. However, in nutrition-based studies, it is not mandatory that serological and morphological findings be provided to justify reporting the results of the research project. There is no doubt that having more data is helpful, but it is not necessary. Nevertheless, we have included serum 25OHD₃ concentrations, which are better indicators than that of organs of vitamin D toxicity. Moreover, we cited numerous previous studies showing that severe vitamin D toxicity will result in an impairment in live performance and ultimately death, whereas birds are able to maintain normal production at lower levels. This indicates that when severe negative impacts are observed, cellular and morphological damages have occurred beforehand. Additionally, we suggested that future research on other variables should be determined when various doses of dietary 25OHD₃ are tested.

2. There are problems in the experimental design. Firstly, from the experimental results in the manuscript, the 8-fold commercial standard concentration has extremely strong negative effects, but the authors did not provide results of other concentrations (such as 4-fold) for comparison. Secondly, the manuscript lacks data on serum calcium ion concentration, histopathological examination of the liver and kidney, or other physiological effects of 25OHD₃, making it impossible to explain the metabolic process, tissue accumulation, or toxicological mechanism of 25OHD₃ in broilers. Thirdly, general tolerance tests on broilers require maintaining a fixed treatment concentration throughout the cycle, and it is clear that such an experimental design does not serve this purpose. Therefore, the dose settings of 8-fold in the starter phase, 4-fold in the grower phase, and 0.5-fold in the finisher phase lack clear significance.

Answer:

First, the comparison between Hy-D diet and control diet were compared in the results section, and relevant discussion was given in the Discussion section. The treatments are Hy-D vs control, and not each Hy-D class compared to each other. However, the only difference between these Hy-D doses was their effects on serum 25OHD₃ level, which is indicated in the results followed by relevant justification in the Discussion section.

Second, this manuscript, as indicated in the title, is focused on live performance and meat yield production in response to supplemental dietary 25OHD₃ up to 8x the recommended level. We also measured the appropriate serological indicator for vitamin D toxicity, which is serum 25OHD₃. The comprehensive relationships concerning the aforementioned items has been recently added to the R1 draft in the Discussion section on lines 222 to 250, and now reads as:

“It is well observed that renal calcification is initiated when dietary levels reach 100 µg/kg, and that renal Ca retention increases by 5-fold when dietary 25OHD₃ is supplemented at 1000 µg/kg (40,000 IU/kg) [29]. In addition, an excessive production of the active form of vitamin D (1,25 (OH)₂ D₃), in response to high levels of serum 25OHD₃ has been shown to result in an increase in the incidence of kidney calcification in mice [49]. Renal calcification in birds has also been observed when they are supplemented with 5x the recommended level of 25OHD₃ [19], which is associated with renal tubular, and less commonly, with arterial mineralization [50] in response to elevated serum 25OHD₃ concentration [19]. In humans, severe occurrences of hypervitaminosis D were observed when serum 25OHD₃ levels reached between 88 [51] and 150 [52] ng/mLConversely, serum 25OHD₃ concentrations at significantly lower levels have been associated with hypervitaminosis D in chickens [19]. An increase in serum 25OHD₃ is directly linked to an increase in Ca serum levels or hypercalcemia [1, 49-52]. An elevated level of circulating Ca is damaging, leading to alterations in cell membrane permeability linked to changes in Ca pump activity that can ultimately lead to cellular necrosis [49,50]. With increasing toxici levels of 25OHD₃, cardiac arrhythmias and death in response to both renal and cardiac failure have been reported [51]. It is well observed that normal 25OHD₃ serum concentrations result when birds are supplemented with between 10 and 20 ng/mL of the water-soluble form of 25OHD₃ [19, 52,53]. In this study, 8x, 4x, 1/2x levels of dietary 25OHD₃ resulted respectively in serum 25OHD₃ concentrations of 115, 50, and 37 ng/mL, indicating that not only was the 8x level damaging, but also that birds were still subject to some level of damage when doses were gradually lowered. In addition, hatchling chicks are more susceptible to toxic supplementation due to their immature digestive and immune systems [54]. Moreover, any significant damage that may be incurred in the internal organs of a bird can be unrecoverable as they develop. Our findings showed that chicks fed 8x the recommended level of 25OHD₃ failed to recover from the negative effects that hypervitaminosis D caused on their meat yield and performance despite the gradual reduction in supplementation throughout the growing phase.”

As is discussed, an increase in circulating Ca serum level is highly associated with serum 25OHD_3, and the damaging effects on live performance are observed when internal organ as well as cellular damages occur. Therefore, when severe impairment of live performance is observed in broilers, those aforementioned damages have already occurred. In addition, the purpose of this study was not to define safe doses of dietary 25OHD₃ in broiler diets through the determination of various organ morphological changes, but rather through the examination of various bird performance variables. We suggested that further study is needed to measure various internal morphological variables when various excess amounts of dietary 25OHD₃ are employed.

Thirdly, previous research regarding vitamin D toxicity showed a margin of safety up to 10x recommended levels. However, that previous research was conducted in the late 80s or early 90s using on broilers or layers that were not as fast growing as today’s birds. Furthermore, their Ca and P absorption capabilities were not as efficient as today’s modern birds. Therefore, they had a greater capacity to tolerate higher levels of 25OHD₃. Previously, broilers have been able to tolerate up 100x the level of dietary vitamin D₃ without interfering with their performance. However, currently, they cannot tolerate 35x the level of dietary vitamin D₃, in which negative effects are observed in their live performance variables and associated internal organ damages. (This information is provided in the Introduction and Discussion sections).

Secondly, this study was founded by USDA, which requires that mortality rates don’t exceed 20% throughout the growing phase in all trials. [This information (given below) is now in the Introduction sections on lines 98 to 100].

“It should also be noted that this study was funded by the United States Department of Agriculture, which mandates that trials be terminated when a 20% mortality rate is reached..”

3. Other issues: The control group uses vitamin D₃ as the source, while the treatment group uses 25OHD₃; the equivalence between the two needs to be clarified. The production performance data should use the 39-day-old data to match the 39-day slaughter performance measurement. The mortality statistics should use the t-test.

Answer:

As we discussed, the control group does not contain 25OHD₃, because according to Ross 708 recommendations and the NRC, the minimum recommendation for vitamin D is based on vitamin D_3, and as we discussed, the level of vitamin D₃ needed to maintain normal production is 250 IU/kg. In addition, the aim of this research was to determine if up to 8x the recommended level is damaging. It was confirmed that it is detrimental. Furthermore, despite lowering the level of 25OHD₃ in subsequent dietary phases, the damaging effects were so severe in the starter phase that the birds were not able to overcome the imposed negative effects. With regards to the units, IU/kg is the common unit for vitamin D₃, while the µg/kg unit is more common for 25OHD₃. However, both units are comparable and can be used for both sources. In the text we clearly explained the equivalent value for each unit.

The times that some sample birds were selected for blood collection and meat yield evaluation were at days 14 and 39 post hatch. However, birds were kept until day 42 for live performance variable determinations. None of the birds were processed at the end of experiment. It is common practice to not use the day at the end of experiment as a sampling day.

The t-test is a very weak test that can be used for only 2 by 2 pairwise comparisons. Although the data could have been analyzed using the t-test, the ANOVA is a more robust method that allowed us to define the error term in order to improve the reliability and accuracy of the analysis.

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

Since mortality rate is binomially distributed data, it is still recommended to modify the statistical method for mortality rate, using the Generalized Linear Mixed Model (GLMM) for calculation. That is, the relationship between mortality probability and independent variables is established through the logit link function.

Author Response

Reviewer 3

Since mortality rate is binomially distributed data, it is still recommended to modify the statistical method for mortality rate, using the Generalized Linear Mixed Model (GLMM) for calculation. That is, the relationship between mortality probability and independent variables is established through the logit link function.

Answer:

Thank you for the comments. However, firstly, the mortality data is not binomially distributed because the observed percentage of mortality in each dietary phase was calculated by dividing the number of dead birds by the total number in each pen. Secondly, logit proc is used when there are categorical variables; however, all variables including the mortality rate variable were numerical, indicating that One way ANOVA using PROC GLIMMIX is a suitable method for analyzing the above-mentioned data.

Author Response File: Author Response.pdf