Counteracting the Toxic Hidroarsenicism Effects upon Reproductive-Physiological Outcomes in Male Goats: The Selenium and Vitamin-E Mitigation Approach

Round 1

Reviewer 1 Report

In this manuscript, the authors describe an experiment in which treatment with selenium and vitamin E, was shown to prevent loss of sperm quality in goats caused by sodium arsenite.
 In my opinion, the chapters are written in a clear and logical manner, and the results of the study are followed by appropriate discussion. 

However, I have three comments on the results presented:

1. In Table 1 and Figure 2 there are results on 'Latency' shown, but there is no mention of this parameter in the main text of the manuscript (in Materials and Methods or Results). If this was a parameter measured in the study, it should also be described and the results commented on in the text.

2. Line 153-155: the sentence is unclear: does the variable 'color' at week 8 refer to SG?

3. Line 91-92: What does the abbreviation 's.v.' stand for?

Correction of the text is required: there is incorrect punctuation in some places and some sentences are unclear, e.g.:  Line 107, lines 183-187, lines 233 -235, lines 251-254.
Line 220: "Selenium" should be in lowercase.

Author Response

 

May 17, 2023

 

RE: Counteracting the toxic hidroarsenicism effects upon reproductive-physiological outcomes in male goats: the Selenium and Vitamin-E mitigation approach (Animals-2396891).

 

Dear Reviewer 1: 

 

We appreciate your feedback and valuable comments on our manuscript and the opportunity to address your comments and improve our manuscript. Below are our responses to the comments. 

1.- For comment 1, a paragraph has been added in the materials and methods section (in red), as well as the corresponding bibliography. Also in the results section, a paragraph was added (in red) that describes the results corresponding to latency, prior to which mention is made of table and figure 1.

Added bibliography:

Delgadillo, J. A., Leboeuf, B., & Chemineau, P. (1991). Decrease in the seasonality of sexual behavior and sperm production in bucks by exposure to short photoperiodic cycles. Theriogenology, 36(5), 755–770. doi:10.1016/0093-691x(91)90341-a 

2.- For comment 2. Yes indeed, the 'color' variable at week 8 refers to SG, which is the selenium group. It was reiterated to make a change adding that it is in the SG (in red).

3.- For comment 3. The abbreviation "s.v." by subcutaneous (SC) injection (in red).

4.- For comment 4, the punctuation and diction to the English language in the lines indicated have been revised and improved. The word selenium and others have been corrected. 

Thank you for considering our revised paper. 

Sincerely, 

 

Javier Morán Martínez, PhD 

Department Head 

Department of Cell Biology and Ultrastructure 

Medicine Faculty

Autonomous University of Coahuila, Torreón 

Email: [email protected]

Author Response File: Author Response.pdf

Reviewer 2 Report

Ortega-Morales and collogues have evaluated the detoxification effects of selenium and vitamin-E on the toxicity of hidroarsenicism upon reproductive-physiological outcomes in male goats. The topic is interesting. The study is compactly and well written, the figures and tables are well-presented. This reviewer, however, has a few suggestions that would improve the manuscript for readers.

 

1.     L28, not sure how to give the selenite 6 mg + vitamin-E 420 I.U. every 14 days? Every day during the 14 days experiment period?

2.     In the abstract, no need to add the “± SD/SE” information in the abstract.

3.     P value need to be written in italic.

4.     Lines 62-68, introduction need to write more information about the function of the selenium; and why selenium has been selected. Too much selenium also not good for the sperm quality. Please see and cite the latest references, such as: 1) Both Selenium deficiency and excess impair male reproductive system via inducing oxidative stress-activated PI3K/AKT-mediated apoptosis and cell proliferation signaling in testis of mice. Free Radical Biology and Medicine. 2023; 2) Selenium deficiency-induced multiple tissue damage with dysregulation of immune and redox homeostasis in broiler chicks under heat stress. SCIENCE CHINA-Life Sciences. 2023; 3) Dietary selenium excess affected spermatogenesis via DNA damage and telomere-related cell senescence and apoptosis in mice. 2023. etc.

5.     The sample size could be improved in the future study.

6.     Please add the replicates n=?, mean±SE/SD etc. information in the figure legends and footnote.

7.     Please check the table and make sure they followed the right style of the journal.

8.     Please add space before and after “<”.

9.     Please check the reference and make sure all of them followed the right form of the journal style.

Author Response

may 17, 2023 

 

RE: Counteracting the toxic hidroarsenicism effects upon reproductive-physiological outcomes in male goats: the Selenium and Vitamin-E mitigation approach (Animals-2396891).

 

Dear Reviewer 2: 

 

We appreciate your feedback and valuable comments on our manuscript and the opportunity to address your comments and improve our manuscript. Below are our responses to the comments. 

 

1.- For comment 1, the paragraph "for 12 weeks" was added, lines 28 and 30 (in red).

2.- For comment 2, the suggestion to eliminate the information in the abstract of the ± DS/ES description was addressed.

3.- For comment 3, the value of P was corrected to p, as indicated by the journal (in red).

4.- We added more information to the introduction section. For example, we add a paragraph and more references on different animal species treated with selenium and its effects on the reproductive system, mainly sperm motility (in red). The literature suggested by the reviewer (for which we are grateful) has been integrated into the manuscript.

Added bibliography:

Xu ZJ, Liu M, Niu QJ, Huang YX, Zhao L, Lei XG, Sun LH. Both selenium deficiency and excess impair male reproductive system via inducing oxidative stress-activated PI3K/AKT-mediated apoptosis and cell proliferation signaling in testis of mice. Free Radic Biol Med. 2023 Mar;197:15-22. doi: 10.1016/j.freeradbiomed.2023.01.024. Epub 2023 Jan 31. PMID: 36731804.

 

Zhao L, Liu M, Sun H, Yang JC, Huang YX, Huang JQ, Lei X, Sun LH. Selenium deficiency-induced multiple tissue damage with dysregulation of immune and redox homeostasis in broiler chicks under heat stress. Sci China Life Sci. 2023 Feb 10. doi: 10.1007/s11427-022-2226-1. Epub ahead of print. PMID: 36795182.

 

Zhang Y, Liu J, Li X, Zhou G, Sang Y, Zhang M, Gao L, Xue J, Zhao M, Yu H, Zhou X. Dietary selenium excess affected spermatogenesis via DNA damage and telomere-related cell senescence and apoptosis in mice. Food Chem Toxicol. 2023 Jan;171:113556. doi: 10.1016/j.fct.2022.113556. Epub 2022 Dec 9. PMID: 36502996.

 

5.- For comment 5, it will indeed be considered to increase the "n" in future investigations. In this work, this sample size was used due to the existence of goats that met the homogeneity criteria in terms of previous exposure time, age, weight, background, and for economic reasons.

 

6.- For comment 6, the number of animals per group was added, in table 1, as well as the number of animals per group and the DE/ES in the figure legends and footnote (in red)

7.- For comment 7, all the tables have been adjusted to the style of the Journal.

 

8.- For comment 8, all the spaces before the “<” symbols have been corrected.

 

9.- For comment 9, the style and form of all the references were verified according to the standards of the Journal.

 

 

Thank you for considering our revised paper. 

Sincerely, 

Javier Morán Martínez, PhD 

Department Head 

Department of Cell Biology and Ultrastructure 

Autonomous University of Coahuila, Torreón 

Email: [email protected]

 

Author Response File: Author Response.pdf

Reviewer 3 Report

I have only a few comments and questions for the authors:

Line 112: ¿Why did you use a scale from 1 to 3, if the correct scale to evaluate sperm motility is from 0-5? scale 0: represent completely immotile sperm.

Lines 120-121: The color (scale 1-3). Insert reference.

Introduction section is too short, it´s recommend  add more information about how arsenic can affect odor intensity, heart rate, respiratory rate, rumen movement, body temperatura in goats.

Line 132: Body condition score (BCS, scale from 1 to 3). Why did you use a scale from 1 to 3, if the correct scale is from 0 to 5?. Insert reference.

Lines: 96,106,200: remove the word spermatic. The correct word is sperm (sperm quality).

Lines 118, 226, 227, 238 and Figure 2: Remove the word spermatic. The correct word is sperm (sperm concentration).

At the moment of taking the samples, the animals get stressed ¿does this not affect the Physiological constant? Insert references.

Line 209: ¿Is 2.5 (scale) a good motility?

In conclusión section, suthors demonstrated that the treatment with selenium and vitamin-E, increases odor. However, this result it is not described in the abstract.

Lines 204-207, the autor mention: Arsenic-mediated toxicity in male reproduction is considered to be related to spermatotoxicity, caused by the production of reactive oxygen species (ROS), excessive levels of ROS can negatively affect sperm quality by decreasing motility while increasing DNA damage and lipid peroxidation of the cellular membrane. ¿Why lipoperoxidation (TBARS technique or Bodipy-511 fluorescence) or reactive oxigen species (CellRox-Green or DCHF-DA Fluorescence technique) levels were not measured in semen samples?

Line 244, the authors mention: Ours results confirm that fact because the highest heart rate occurred in the AG group, the reason for this may be the oxidative stress caused by arsenic. It is well known that arsenic induced oxidative stress. Prolonged arsenic ingestion leads to its accumulation in liver, kidneys, heart, and lungs and in smaller amounts in muscle, nervous system, gastrointesti- nal tract, and spleen because these organs are rich in ox- idative enzyme systems. Some studies have pointed out that oxidative damage to hemoglobin has been shown to cause changes in its structure    and    function    resulting    in    denaturation, precipitation, and methemoglobin formation inside erythrocytes. Furthermore, oxidative stress in red blood cells (RBCs) is an indicator of overall oxidative stress and RBC related disorders. Keshavarsy et al. (2015) reported that there is a significant relationship between arsenic levels in drinking water and blood in sheep. Likewise, concluded that increased levels of arsenic in drinking water leads to increased deposition of arsenic in blood samples. ¿Why arsenic or rective oxugen species (ROS) levels were not measured in the blood?

Author Response

May 17, 2023

 

RE: Counteracting the toxic hidroarsenicism effects upon reproductive-physiological outcomes in male goats: the Selenium and Vitamin-E mitigation approach (Animals-2396891).

Dear Reviewer 3: 

We appreciate your feedback and valuable comments on our manuscript and the opportunity to address your comments and improve our manuscript. Below are our responses to the comments. 

1.- For the questions and comments 1, 2, and 3 on the variables motility, color, body weight, rumen movement, the arbitrary scale (1-3, with the corresponding explanation for each variable) was used, which is the one in the protocol established for reproductive evaluation and physiology in goats at the National Institute for Agricultural and Livestock Forestry Research (INIFAP) (experimental campus), in Matamoros Coahuila, Mexico. A paragraph has been added in this regard in the materials and methods section of this standardized technique in the INIFAP experimental field (in red), reference is added.

4.- For comment 4, in the introduction section, a paragraph has been added related to the effect of arsenic with some physiological variables according to this indication (in red). As well as the corresponding bibliography has been attached.

a.- Balarastaghi S, Barangi S, Hosseinzadeh H, Imenshahidi M, Moosavi Z, Razavi BM, Karimi G. Melatonin improves arsenic-induced hypertension through the inactivation of the Sirt1/autophagy pathway in rat. Biomed Pharmacother. 2022 Jul;151:113135. Doi: 10.1016/j.biopha.2022.113135. Epub 2022 May 19. PMID: 35598369.

 

b.- Rana R, Mehmood MH, Shaukat B, Shahid S, Malik A, Murtaza B. Evaluation of the Cardiovascular Effects of Coriandrum sativumand Citrus limon to Treat Arsenic-Induced Endothelial Damage and Hypertension in Rats. Life (Basel). 2022 Nov 10;12(11):1842. doi: 10.3390/life12111842. PMID: 36362997; PMCID: PMC9695286.

 

c.- Flora SJ, Pachauri V, Mittal M, Kumar D. Interactive effect of arsenic and fluoride on cardio-respiratory disorders in male rats: possible role of reactive oxygen species. Biometals. 2011 Aug;24(4):615-28. Doi: 10.1007/s10534-011-9412-y. Epub 2011 Jan 18. PMID: 21243404.

 

d.- Calidad Seminal del Macho Cabrío de la Comarca Lagunera. Centro de Investigación Regional Norte Centro. Campo Experimental la laguna. Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (INIFAP). Matamoros, Coahuila, México. Diciembre 2017. Folleto técnico No. 34. ISBN: 978-607-37-0832-6.

 

4.- For the comment 4, the word spermatic has been changed by the word sperm in the indicated lines (in red).

5.- For comment 5, we appreciate this comment. For the animals included in this study, management, facilities (corrals, drinking fountains, drinking, etc.) and feeding were as adequate as possible. The handlers of the animals as well as the researchers tried to give the best handling to the animals, with the aim of not generating alterations due to stress in the animals. Efforts were made to minimize these factors on sampling days as much as possible so as not to alter the measured variables.

 

6.- For comment 6, Yes, according to the scale used in this protocol, a good sperm motility is considered.

 

7.- For comment 7, the result of the odor variable was added to the abstract in the conclusion section, as suggested.

8 and 9.- For comments 8 and 9, ¿Why lipoperoxidation (TBARS technique or Bodipy-511 fluorescence) or reactive oxigen species (CellRox-Green or DCHF-DA Fluorescence technique) levels were not measured in semen samples? and ¿Why arsenic or rective oxugen species (ROS) levels were not measured in the blood?.

In this study the main objective was to study the supplementation of selenium and vitamin E, and to evaluate their effects on physiological and reproductive aspects in male goats. We plan to carry out other studies that consider these questions about reactive oxygen species, lipoperoxidation that are asked by the reviewer.

 

Thank you for considering our revised paper. 

Sincerely, 

 

Javier Morán Martínez, PhD 

Department Head 

Department of Cell Biology and Ultrastructure 

Medicine Faculty

Autonomous University of Coahuila, Torreón 

Email: [email protected]

 

Author Response File: Author Response.pdf