Essential Oils as a Dietary Additive for Laying Hens: Performance, Egg Quality, Antioxidant Status, and Intestinal Morphology: A Meta-Analysis
Round 1
Reviewer 1 Report
This is a well written manuscript in which the authors examined a valuable meta-analysis regarding the effect of essential oils used as a dietary additive for laying hens on performance traits, egg quality parameters, as well as an antioxidant status indicators and intestinal morphology.
Introduction:
Overall, this section is prepared correctly and contains a typical background for the conducted topic, including, among others, a definition of EOs, their utilization in the nutrition of laying hens, as well as an explanation the reasons of why such a meta-analysis was performed by the authors.
However, by giving some detailed examples of inconsistent results found in several studies, it would be easier for the reader get the equal examples in this section. Therefore, it is worth show with numerical data, what parameters and in what directions have changed, as, for example, oppositional cases, if the selected literature data allows it.
Methods:
I do not have any comments to the methodological part of the manuscript, as the authors meticulously presented the scheme of the meta-analysis and justify each of performed steps justifying them with an adequate reference.
Results:
The description of the results is concise and adequate and it is worth to note that the authors have mentioned and justified the reason of use of the performed meta-regression technique only for selected parameters.
Discussion:
I don’t have any major comments for this section, since all considered issues are thoroughly discussed. The obtained results are briefly and synthetically discussed in relation to the cited literature, not only from a strictly statistical point of view, but also in terms of physiological mechanisms which improves the clarity the presented results of the performed meta-analysis.
Author Response
Response to Reviewer #1:
We would like to thank the reviewer for the careful and thorough reading of this manuscript and for the thoughtful comments and constructive suggestions, which help to improve the quality of this manuscript. Our response follows (the reviewer's comments are in italics).
Comments and Suggestions for Authors
This is a well written manuscript in which the authors examined a valuable meta-analysis regarding the effect of essential oils used as a dietary additive for laying hens on performance traits, egg quality parameters, as well as an antioxidant status indicators and intestinal morphology.
Comment 1. Introduction:
Overall, this section is prepared correctly and contains a typical background for the conducted topic, including, among others, a definition of EOs, their utilization in the nutrition of laying hens, as well as an explanation the reasons of why such a meta-analysis was performed by the authors.
However, by giving some detailed examples of inconsistent results found in several studies, it would be easier for the reader get the equal examples in this section. Therefore, it is worth show with numerical data, what parameters and in what directions have changed, as, for example, oppositional cases, if the selected literature data allows it.
Response: Thanks for the suggestion. The following information related to this comment has been added in the introduction section: “For example, some studies have reported up to 7% higher egg production, 15% better feed conversion, 81% higher total blood antioxidant capacity, and 24% longer intestinal villi in laying hens supplemented with EOs [10,18,19]. However, in other studies [22,23,24] using laying hens, less than 2% of benefits in egg production, feed conversion, and intestinal villi have been detected in response to EOs supplementation”. Lines 56-61.
Comment 2. Methods:
I do not have any comments to the methodological part of the manuscript, as the authors meticulously presented the scheme of the meta-analysis and justify each of performed steps justifying them with an adequate reference.
Response: Thanks.
Comment 3. Results:
The description of the results is concise and adequate and it is worth to note that the authors have mentioned and justified the reason of use of the performed meta-regression technique only for selected parameters.
Response: Thanks.
Comment 4. Discussion:
I don’t have any major comments for this section, since all considered issues are thoroughly discussed. The obtained results are briefly and synthetically discussed in relation to the cited literature, not only from a strictly statistical point of view, but also in terms of physiological mechanisms which improves the clarity the presented results of the performed meta-analysis.
Response: Thanks.
Author Response File: 
Author Response.docx
Reviewer 2 Report
The meta-analysis manuscript entitled (Essential Oils as a Dietary Additive for Laying Hens: Performance, Egg Quality, Antioxidant Status, and Intestinal Morphology: A Meta-Analysis) is good presented and written but I have some comments needs to be answered by the authors
Is the number of 32 used manuscript in the analysis is enough to produce valuable data for such manuscript
Secondly, the high differences in the doses used in used manuscripts may lead to high differences and false significance for obtained data.
Author Response
Response to Reviewer #2:
We would like to thank the reviewer for careful and thorough reading of this manuscript and for the thoughtful comments and constructive suggestions, which help to improve the quality of this manuscript. Our response follows (the reviewer's comments are in italics).
Comments and Suggestions for Authors
The meta-analysis manuscript entitled (Essential Oils as a Dietary Additive for Laying Hens: Performance, Egg Quality, Antioxidant Status, and Intestinal Morphology: A Meta-Analysis) is good presented and written but I have some comments needs to be answered by the authors
Comment 1. Is the number of 32 used manuscript in the analysis is enough to produce valuable data for such manuscript
Response: Dear reviewer, the database used included 38 manuscripts, not 32. This information was mentioned in the manuscript in Figure 1 and line 103.
On the other hand, the answer is yes. According to some authors (Orzuna-Orzuna et al., 2023a; Orzuna-Orzuna et al., 2023b; Sierra-Galicia et al., 2023), valuable and reliable results can be obtained when the analyzed response variables were reported in at least three studies. Only response variables reported in at least three studies were included in this manuscript, as indicated in lines 120-122.
Orzuna-Orzuna, J.F.; Dorantes-Iturbide, G.; Lara-Bueno, A.; Chay-Canul, A.J.; Miranda-Romero, L.A.; Mendoza-Martínez, G.D. Meta-analysis of flavonoids use into beef and dairy cattle diet: Performance, antioxidant status, ruminal fermentation, meat quality, and milk composition. Front. Vet. Sci. 2023a, 10, 1134925. https://doi.org/10.3389/fvets.2023.1134925
Orzuna-Orzuna, J.F.; Lara-Bueno, A.; Mendoza-Martínez, G.D.; Miranda-Romero, L.A. Meta-analysis of hydroxycinnamic acids into finishing lambs’ diet: Growth performance, antioxidant status, and meat quality. Small Rumin. Res. 2023b, 223, 106963. https://doi.org/10.1016/j.smallrumres.2023.106963
Sierra-Galicia, M.I.; Rodríguez-de Lara, R.; Orzuna-Orzuna, J.F.; Lara-Bueno, A.; Ramírez-Valverde, R.; Fallas-López, M. Effects of supplementation with bee pollen and propolis on growth performance and serum metabolites of rabbits: A meta-analysis. Animals 2023, 13, 439. https://doi.org/10.3390/ani13030439
Comment 2. Secondly, the high differences in the doses used in used manuscripts may lead to high differences and false significance for obtained data.
Response: Being a compilation of several studies published in different research centers, the variability in doses and other management characteristics is obvious. However, as indicated in lines 65-67 of the present manuscript, “Meta-analysis is a statistical method with high analytical power (Viechtbauer, 2010), which uses rigorous procedures that allow statistically combining and analyzing data sets from various experiments to obtain objective evidence on the effect of a treatment (Orzuna-Orzuna et al., 2023a; Orzuna-Orzuna et al., 2023b)”. In other words, no matter the variability of the experimental conditions between studies, meta-analytic methods make it possible to eliminate this variability and obtain reliable results (the possibility of false significance is eliminated). In addition, a complement used to measure variability is the analysis of heterogeneity. In the present meta-analysis, heterogeneity was not statistically significant for the covariate "essential oil dose," indicating that the dose range used does not significantly influence the expected response.
Viechtbauer, W. Conducting meta-analysis in R with the metaphor package. J. Stat. Softw. 2010, 36, 1–48. https://doi.org/10.18637/jss.v036.i03
Orzuna-Orzuna, J.F.; Dorantes-Iturbide, G.; Lara-Bueno, A.; Chay-Canul, A.J.; Miranda-Romero, L.A.; Mendoza-Martínez, G.D. Meta-analysis of flavonoids use into beef and dairy cattle diet: Performance, antioxidant status, ruminal fermentation, meat quality, and milk composition. Front. Vet. Sci. 2023, 10, 1134925. https://doi.org/10.3389/fvets.2023.1134925
Orzuna-Orzuna, J.F.; Lara-Bueno, A.; Mendoza-Martínez, G.D.; Miranda-Romero, L.A. Meta-analysis of hydroxycinnamic acids into finishing lambs’ diet: Growth performance, antioxidant status, and meat quality. Small Rumin. Res. 2023, 223, 106963. https://doi.org/10.1016/j.smallrumres.2023.106963
Last but not least, there are several recent meta-analyses published in MDPI journals and other prestigious publishers in which it can be observed that the variability of the doses of the evaluated additives does not represent a limitation or lead to false significance of the results obtained. Some of these meta-analyses are listed below:
Orzuna-Orzuna, J.F.; Dorantes-Iturbide, G.; Lara-Bueno, A.; Miranda-Romero, L.A.; Mendoza-Martínez, G.D.; Santiago-Figueroa, I. A Meta-Analysis of Essential Oils Use for Beef Cattle Feed: Rumen Fermentation, Blood Metabolites, Meat Quality, Performance and, Environmental and Economic Impact. Fermentation 2022, 8, 254. https://doi.org/10.3390/fermentation8060254
Darmawan, A.; Hermana, W.; Suci, D.M.; Mutia, R.; Sumiati; Jayanegara, A.; Ozturk, E. Dietary Phytogenic Extracts Favorably Influence Productivity, Egg Quality, Blood Constituents, Antioxidant and Immunological Parameters of Laying Hens: A Meta-Analysis. Animals 2022, 12, 2278. https://doi.org/10.3390/ani12172278
Dorantes-Iturbide, G.; Orzuna-Orzuna, J.F.; Lara-Bueno, A.; Mendoza-Martínez, G.D.; Miranda-Romero, L.A.; Lee-Rangel, H.A. Essential Oils as a Dietary Additive for Small Ruminants: A Meta-Analysis on Performance, Rumen Parameters, Serum Metabolites, and Product Quality. Vet. Sci. 2022, 9, 475. https://doi.org/10.3390/vetsci9090475
Orzuna-Orzuna, J.F.; Dorantes-Iturbide, G.; Lara-Bueno, A.; Mendoza-Martínez, G.D.; Miranda-Romero, L.A.; Hernández-García, P.A. Effects of Dietary Tannins’ Supplementation on Growth Performance, Rumen Fermentation, and Enteric Methane Emissions in Beef Cattle: A Meta-Analysis. Sustainability 2021, 13, 7410. https://doi.org/10.3390/su13137410
Author Response File: Author Response.docx
Reviewer 3 Report
This is a valuable meta-analysis of some of the main effects of essential oil supplements in the feed of laying hens. The authors rightly define essential oils as plant derived substances, however, in several of the legends of their tables, they indicate that only essential oils from micro algae were included, which was, however, not indicated as such in the M&M section. Subtitle 2.6. is identical to subtitle 2.5., which obviously is a mistake.
In the discussion, the authors should also envisage the undectable publication bias that perhaps only experiments which yielded positive results may have been written up for publication. The range of dosages used in the studies included in the meta analysis should be reported in the results section, as ranges are used from literature in the discussion section.
Author Response
Response to Reviewer #3:
We would like to thank the reviewer for careful and thorough reading of this manuscript and for the thoughtful comments and constructive suggestions, which help to improve the quality of this manuscript. Our response follows (the reviewer's comments are in italics).
Comments and Suggestions for Authors
This is a valuable meta-analysis of some of the main effects of essential oil supplements in the feed of laying hens. The authors rightly define essential oils as plant derived substances, however, in several of the legends of their tables, they indicate that only essential oils from micro algae were included, which was, however, not indicated as such in the M&M section. Subtitle 2.6. is identical to subtitle 2.5., which obviously is a mistake.
Response: Dear reviewer, thank you for the comments. We apologize that the footnotes in Tables 2, 3, 4 and 5 were incorrect. In fact, all kinds of essential oils were included in the present manuscript, not just essential oils from microalgae. The footnotes of Tables 2, 3, 4 and 5 have been corrected and now read: “N: represents the number of studies; NC: represents the number of comparisons between essential oils treatment and control treatment”.
Also, subheading 2.6 has been corrected and now reads: “Meta-Regression and Subgroup Analysis”.
Comment 2. In the discussion, the authors should also envisage the undectable publication bias that perhaps only experiments which yielded positive results may have been written up for publication. The range of dosages used in the studies included in the meta analysis should be reported in the results section, as ranges are used from literature in the discussion section.
Response: Dear reviewer, we, the authors, disagree with the first comment because, theoretically, the reasoning that has been established in the literature on meta-analytic methods is in the opposite direction. The absence of publication bias suggests that all the literature related to the topic has been published and is available. In contrast, the presence of publication bias suggests that there were likely studies that were not published or that only studies with positive effects were published (Littell et al., 2008). Therefore, it is not necessary to discuss an effect that was not significant.
Littell, J.H.; Corcoran, J.; Pillai, V. Systematic reviews and meta-analysis, 1st ed.; Oxford University Press: Oxford, UK, 2008; pp. 111–132.
Last but not least, below we list some recent articles published in prestigious journals in which there was no publication bias and consequently nothing was discussed about it:
Sierra-Galicia, M. I., Rodríguez-de Lara, R., Orzuna-Orzuna, J. F., Lara-Bueno, A., Ramírez-Valverde, R., & Fallas-López, M. (2023). Effects of Supplementation with Bee Pollen and Propolis on Growth Performance and Serum Metabolites of Rabbits: A Meta-Analysis. Animals, 13(3), 439.
Orzuna-Orzuna, J. F., Dorantes-Iturbide, G., Lara-Bueno, A., Chay-Canul, A. J., Miranda-Romero, L. A., & Mendoza-Martínez, G. D. (2023). Meta-analysis of flavonoids use into beef and dairy cattle diet: Performance, antioxidant status, ruminal fermentation, meat quality, and milk composition. Frontiers in Veterinary Science, 10, 142.
de Nazaré Santos Torres, R., Bertoco, J. P. A., de Arruda, M. C. G., de Melo Coelho, L., Paschoaloto, J. R., Neto, O. R. M., ... & Baldassini, W. A. (2023). Effects of molasses supplementation on animal performance and carcass parameters of beef cattle: a meta-analysis. Tropical Animal Health and Production, 55(3), 1-13.
Orzuna-Orzuna, J. F., Lara-Bueno, A., Mendoza-Martínez, G. D., & Miranda-Romero, L. A. (2023). Meta-analysis of hydroxycinnamic acids into finishing lambs’ diet: Growth performance, antioxidant status, and meat quality. Small Ruminant Research, 223, 106963.
On the other hand, the range of doses used in the studies included in the meta-analysis was reported in the Results section 3.1. To complement this section, the age ranges of the laying hens and the supplementation periods were reported. Likewise, the main breeds/strains of laying hens and primary bioactive compounds of the EOs were described. Lines 169-180.
Author Response File: Author Response.docx
Round 2
Reviewer 2 Report
Thanks for authors efforts
