Phytochemical Composition and Effects of Aqueous Extracts from Moringa oleifera Leaves on In Vitro Ruminal Fermentation Parameters

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In the manuscript titled “Phytochemical composition and effects of aqueous extracts from Moringa oleifera leaves on in vitro ruminal fermentation parameters”, the authors describe a set of experiments designed to evaluated the use of different moringa extracts over ruminal fermentation.

In general, the study is interesting. However, it seems that the evaluation of different extraction methods has already been evaluated in other studies, as also mentioned by the authors (Ref. 12, Vongsak et al. 2013).  For this reason, the authors should point out why they expected a different result in their study and highlight the most important findings.

Also, I suggest some corrections to the manuscript:

Figures should be improved, and choose different point marks por each treatment.

Avoid using the same connector in consecutive paragraphs in the discussing part.

Methodology of protozoans count was not indicated in the text.

Author Response

REVISOR 1: No manuscrito intitulado “Composição fitoquímica e efeitos de extratos aquosos de folhas de Moringa oleifera em parâmetros de fermentação ruminal in vitro ”, os autores descrevem um conjunto de experimentos projetados para avaliar o uso de diferentes extratos de moringa durante a fermentação ruminal.

1) Em geral, o estudo é interessante. No entanto, parece que a avaliação de diferentes métodos de extração já foi avaliada em outros estudos, como também mencionado pelos autores (Ref. 12, Vongsak et al. 2013). Por esse motivo, os autores devem apontar por que esperavam um resultado diferente em seu estudo e destacar as descobertas mais importantes.

Resposta: Caro revisor, apesar das semelhanças, nosso estudo introduz métodos e aspectos inovadores do processamento de folhas para a produção de extrato aquoso. Em contraste, o trabalho de Vongsak et al. (2013) obteve outros tipos de extratos, especificamente extratos etanólicos.

2 ) Também sugiro algumas correções no manuscrito: as figuras devem ser melhoradas e escolher pontos de referência diferentes para cada tratamento.

Resposta: Caro revisor, optamos por usar cores diferentes devido ao limite de marcadores em nosso editor de texto.

3 ) Evite usar o mesmo conector em parágrafos consecutivos na parte de discussão.

Resposta: Obrigado pela sugestão. Revisamos o texto do artigo de acordo.

4 ) A metodologia de contagem de protozoários não foi indicada no texto.

Resposta: Obrigado pela sugestão. Adicionamos a metodologia ao texto do manuscrito

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript (ruminants-3403529) evaluated the phytochemical composition and antioxidant properties of aqueous extracts from Moringa oleifera leaves prepared using maceration, infusion, and decoction methods. It also investigated the effects of these extracts on in vitro ruminal fermentation. The manuscript has several promising aspects, but it requires revisions as several points need to be carefully revised before the next resubmission as follows:

1)      Abstract; The experimental design is not clearly described, and the results section lacks sufficient detail to represent the study comprehensively. Please add more information about the results obtained.

2)      L38-40; "Several strategies have been developed focusing on the manipulation of ruminal fermentation, to increase the efficiency of nutrient utilization and reduce energy losses, especially those related to methane formation and emission. " Please provide some examples or references to clarify these strategies and their relevance to the study.

3)      Are there statistics on the quantities produced from Moringa oleifera globally or in your country? Please add to the introduction section.

4)      Could the production of Moringa oleifera leaves meet the ruminant industry demand? 

5)      Several studies have investigated the effects of Moringa oleifera extracts both in vivo and in vitro. However, the gap of knowledge and specific hypothesis addressed by your study are not clearly stated. Please clarify these aspects in the introduction section.

6)      L152; Please provide a more detailed description of the bovine used for rumen fluid collection. Specify whether they were male or female, their average weight, and their feeding regimen. Additionally, clarify whether the rumen fluid was collected before or after feeding.

7)      L153-158; What is the extract dose for each treatment? Is it consistent across all extraction methods, and what was the rationale for selecting only one dose? Additionally, three replicates seem insufficient to provide a reliable recommendation.

8)      L161; What is the composition of the buffer solution used?

9)      L161-162; “…………….0.4 mL of aqueous extract (corresponding to a daily dose of 80 mL per  animal per day).” How was the quantity of 80 mL per animal determined?

10)  Throughout the manuscript, It would be more accurate to use the term 'degradability' instead of 'digestibility' when referring to in vitro studies.

11)  In the Results section for Figures 1 to 5, the effect of time is indicated by significant letters, but the differences between treatments are not clearly presented. I suggest converting these figures into tables to clarify the effects of time, treatments, and their interactions. Additionally, it should clarify this in the Statistical Analysis section.

12)  The discussion section requires extensive revision. It relies heavily on speculation rather than solid scientific evidence. The discussion must be fortified with possible attributions supported by references.

Author Response

REVIEWER 2: The manuscript (ruminants-3403529) evaluated the phytochemical composition and antioxidant properties of aqueous extracts from Moringa oleifera leaves prepared using maceration, infusion, and decoction methods. It also investigated the effects of these extracts on in vitro ruminal fermentation. The manuscript has several promising aspects, but it requires revisions as several points need to be carefully revised before the next resubmission as follows:

1) Abstract; The experimental design is not clearly described, and the results section lacks sufficient detail to represent the study comprehensively. Please add more information about the results obtained.

Answer: Thank you for the suggestion. We have followed your recommendations in the article's abstract.

2) L38-40; "Several strategies have been developed focusing on the manipulation of ruminal fermentation, to increase the efficiency of nutrient utilization and reduce energy losses, especially those related to methane formation and emission. " Please provide some examples or references to clarify these strategies and their relevance to the study.

Answer: We have followed your recommendations in the article's introduction.

3) Are there statistics on the quantities produced from Moringa oleifera globally or in your country? Please add to the introduction section.

Answer: The Moringa production chain does not have a large scale of production, which limits the information regarding these production data.

4) Could the production of Moringa oleifera leaves meet the ruminant industry demand?

Answer: In the present study, the amount of moringa to be used is relatively low, as its extract is incorporated into the diet as a micro-ingredient, meaning in small quantities. Thus, the use of moringa as an additive in ruminant diets can be easily supported by the current availability of moringa in different global regions.

5) Several studies have investigated the effects of Moringa oleifera extracts both in vivo and in vitro. However, the gap of knowledge and specific hypothesis addressed by your study are not clearly stated. Please clarify these aspects in the introduction section.

Answer: We added the hypothesis to our study.

6) L152; Please provide a more detailed description of the bovine used for rumen fluid collection. Specify whether they were male or female, their average weight, and their feeding regimen. Additionally, clarify whether the rumen fluid was collected before or after feeding.

Answer: We have added these details to the article text. Regarding the donor animal, we used a crossbred adult male bovine raised in a continuous grazing system. The animal was taken to a restraining chute next to the paddock. Thus, at the time of collection, the cow had likely already partially fed.

7) L153-158; What is the extract dose for each treatment? Is it consistent across all extraction methods, and what was the rationale for selecting only one dose? Additionally, three replicates seem insufficient to provide a reliable recommendation.

Answer: Dear reviewer, in the text, we stated that we used 0.4 mL of each extract in the in vitro ruminal fermentation study. We chose to use the same dose of extracts to demonstrate that, at similar doses, the results can be influenced by the different concentrations of bioactive compounds.

8)      L161; What is the composition of the buffer solution used?

Answer: We have added the composition of the buffer solution to the text.

9) L161-162; “…………….0.4 mL of aqueous extract (corresponding to a daily dose of 80 mL per animal per day).” How was the quantity of 80 mL per animal determined?

Answer: Dear reviewer, the dose equivalent to 0.4 ml was based on the studies by Kholiff et al. (2018) and Pedraza Hernandez (2019). We have removed the information of equivalence to 80 ml in an animal. However, this equivalence is based on the following calculation: in 50 ml of fluid (ruminal liquid and buffer solution), we use 0.4 ml of aqueous Moringa extract. In 10,000 ml of ruminal fluid (adult sheep), 80 ml would be used.

10)  Throughout the manuscript, It would be more accurate to use the term 'degradability' instead of 'digestibility' when referring to in vitro studies.

Answer: Thank you for the suggestion. We have made the changes throughout the manuscript.

11)  In the Results section for Figures 1 to 5, the effect of time is indicated by significant letters, but the differences between treatments are not clearly presented. I suggest converting these figures into tables to clarify the effects of time, treatments, and their interactions. Additionally, it should clarify this in the Statistical Analysis section.

Answer: Dear reviewer, in the results section, we highlighted that there was no statistical difference between treatments at the different incubation times. Thus, the letters in the graphs indicate the differences between incubation times. Due to the individual effect of time, we preferred to use graphs to visually demonstrate this behavior.

12)  The discussion section requires extensive revision. It relies heavily on speculation rather than solid scientific evidence. The discussion must be fortified with possible attributions supported by references.

Answer: Dear reviewer, we have revised the discussion and removed the speculations. We acknowledge a limitation in this study regarding microbiological analysis, which constrains some explanations. However, based on widely recognized literature, our findings suggest some interpretations that could be confirmed through such an analysis. If further concerns arise, we kindly request more specific comments.

Reviewer 3 Report

Comments and Suggestions for Authors

It is an interesting and well-done study demonstrating the high nutritional value moringa might have for ruminants. However, it is necessary to clarify some issues to improve the article. 

 

Comments for author File: Comments.pdf

Author Response

REVISOR 3: É um estudo interessante e bem feito que demonstra o alto valor nutricional que a moringa pode ter para ruminantes. No entanto, é necessário esclarecer algumas questões para melhorar o artigo.

1) Como essas extrações foram armazenadas após o processamento?

Resposta: Obrigado pela sugestão. Adicionamos essa informação ao texto do manuscrito.

2) Você está se referindo às diferenças nos valores de digestibilidade da MS medidos em vários momentos?

Resposta: Exatamente, refere-se à degradabilidade da matéria seca (MS) nos tempos avaliados. Ressaltamos que os tratamentos não diferiram conforme os tempos de incubação, nem houve interação entre os fatores. Melhoramos a legenda da figura no artigo.

3) Explicar cada variável mais próxima de seu valor correspondente seria mais eficaz para facilitar o entendimento.

Resposta: Entendemos sua preocupação, caro revisor. No entanto, as diretrizes exigem que as figuras sejam anexadas imediatamente após sua primeira menção.

4) Você está se referindo às diferenças nos valores de digestibilidade do FDN medidos em vários momentos?

Resposta: Exatamente, refere-se à degradabilidade da fibra em detergente neutro (FDN) nos tempos avaliados. Ressaltamos que os tratamentos não diferiram conforme os tempos de incubação, nem houve interação entre os fatores. Melhoramos a legenda da figura no artigo.

5) Você está se referindo às diferenças nos valores de pH medidos em vários momentos?

Resposta: Exatamente! Melhoramos a legenda da figura no artigo.

6) Você está se referindo às diferenças nos valores amoniacais medidos em vários momentos?

Resposta: Exatamente! Melhoramos a legenda da figura no artigo.

7) Você está se referindo às diferenças na população de protozoários medidas em vários momentos?

Resposta: Exatamente! Melhoramos a legenda da figura no artigo.

8) Infelizmente, você não analisou a população microbiana do rúmen, o que significa que você não sabia a quantidade de bactérias gram-positivas no líquido ruminal necessária para uma avaliação adequada.

Resposta: Exatamente. Estamos cientes dessa limitação e, portanto, no manuscrito, fizemos algumas sugestões quanto às justificativas para os resultados obtidos. Essas hipóteses são baseadas em literatura mundialmente reconhecida.

9) O nome do periódico não deve ser abreviado.

Resposta: De acordo com o modelo Ruminants, o nome do periódico deve ser abreviado.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have thoroughly revised the manuscript and addressed all my comments comprehensively. One minor change is still recommended: in Figures 1 and 2, please revise the Y-axis label by replacing 'digestibility' with 'degradability'.