High-energy diet enhances spermatogenic function and increases sperm midpiece length in fallow deer (Dama dama) yearlings

Nutrition is a major factor involved in the sexual development of livestock ruminants. In the male, a high-energy diet enhances the reproductive function, but its effects on the underlying processes such as spermatogenic efficiency are not yet defined. Moreover, the possible changes in sperm size due to a supplemented diet remain poorly investigated. The main goal of this study was to evaluate whether a high-energy diet affects the spermatogenic activity, epididymal sperm parameters (concentration, morphology, morphometry and acrosome integrity) and blood testosterone levels in fallow deer yearlings. For this purpose, 32 fallow deer were allocated into two groups according to their diet: control (pasture) and experimental (pasture and barley grain) groups. Fallow deer from the experimental group showed a significant increase in the Sertoli cell function and sperm midpiece length, together with a higher testicular mass, sperm concentration and percentage of normal spermatozoa than the control group (p < 0.05). We also found a tendency for higher blood testosterone levels in the animals fed with barley grain (p = 0.116). The better sperm quality found in the experimental group may be related to their higher efficiency of Sertoli cells and to an earlier onset of puberty. The results of the present work elucidate the mechanisms by which dietary supplementation enhances the male sexual development and might be useful for better practices of livestock management in seasonal breeders.

This study investigates an interesting and relatively understudied question. Many novel aspects of reproductive responses to diet have been explored in this study. My major concerns pertain to the use of energy as the primary indicator of nutrient quality without actually providing estimates for dietary energetic differences and the potentially strong alternative explanations driving the differences in response variables. The nutritive composition of the diet is really only cited in a single sentence (Lines 92-93), while later in the Discussion section only the protein and energetic content seems to be robustly considered. The protein content was slightly higher in the grass and the fat content was slightly higher in the barley grain. However, the dry matter content was over twice as great in the barley, while the fiber and lignin content were approximately three times as high in the grass. These factors might contribute to digestibility and assimilation differences/costs between the diets, which could be interacting with the response variables (in addition to energy intake). The study also seems to have been conducted on single population (divided into a control and treatment) during a single season, with most sample collection occurring at the terminus, which perhaps limits extrapolation of the findings.
Specific Comments: 1. Line 71-73: This statement seems to offer one of the strongest nutritive explanations for why supplementation with barley might increase fertility in male fallow deer. However, essential amino acids did not seem to be quantified in this study. Other nutritive explanations for the advantages of supplementation (digestibility, higher fat content, etc.) might give broader perspective to the study from the dietary data provided. 2. Lines 98-104: Was nutrient composition of barley grain determined using the same analyses as pasture or were other methods used to infer? 3. Lines 106-108: This seems like sufficient justification for using an alternative to randomized sample collection. 4. Lines 175-177: It looks like the deer were initially size-matched for the treatments, which I think was well recognized to be a necessary part of the experimental design. 5. Lines 189-193 and 215-217: These seemed to be the most significant experimental findings. Many of the other response variables did not seem to differ (e.g. testosterone, acrosomal status, and several aspects of sperm morphology). 6. Lines 230-253: The authors seem well versed in the function and production of male gametes, however, the link to diet is not yet well-elucidated. The only direct nutritional explanation is from the early reference to essential amino acids present (but not actually measured in this study) in the grains. 7. Lines 291-293: These lines provide a citation to the broader diversity of animals in the literature, which is relatively sparse up to this point in the paper. However, the animals in the current study seemed to be neither food restricted nor in poor body condition given the gain in mass and at least some equivalent aspects of fertility in both treatment and control. 8. Lines 300-302: It is difficult to assess this claim because Table 1 appears to show higher protein available for consumer intake in the grass compared to grain. 9. Line 498: At present, Table 1 only seems to be provided as background rather than as a supporting piece of evidence. This table is only cited once (within the Methods) and only protein and perhaps fat are alluded to. It is not yet clear why other nutrient analyses in the table were conducted and if these biochemicals might also contribute to the observed differences in the response variables. The arguments for higher-energy diet contributing to increased spermatogenic function and sperm midpiece length would also be strengthened if calorimetry findings or standard energy conversion estimates were to be included for the diets (e.g. protein=17 kJ/g, and fat=37 kJ/g; Raubenheimer and Rothman. 2013. Nutritional ecology of entomophagy in humans and other primates. Annu Rev Entomol).

Review form: Reviewer 2
Is the manuscript scientifically sound in its present form? No

Do you have any ethical concerns with this paper? No
Have you any concerns about statistical analyses in this paper? No

Recommendation?
Major revision is needed (please make suggestions in comments)

Comments to the Author(s)
In this work, the authors elicit the influence of an energy-rich diet on the onset of sexual maturation of fallow deer (Dama dama). To achieve this, the authors randomly allocated 32 prepubertal fallow deer to two groups: one fed with pasture and one supplemented with barley. Six month later, the animals were sacrificed and biometric parameters, serum testosterone, sperm parameters, spermatogenic function and acrosomal status were accessed. Overall, the authors report improvements in sperm parameters and reproductive function, compatible to an earlier onset of sexual maturation in the group fed with pasture supplemented with barley. This paper presents some results that explore the potential commercial viability of fallow deer, based on an adequate methodology. The evaluated parameters are significant according to the aims of the work, and variables are properly defined. Besides, considering the amount of collected data and its further treatment, the reported results seem reliable. Nevertheless, there are some major concerns regarding the way the authors present their data, and some gaps that must be filled in order to draw more consistent conclusions. 1. The authors have not identified the most critical aim of their work. The most evident conclusion of this work is that a high-energy diet anticipates the puberty onset on fallow deer. The authors do not present any conclusive proof that it was diet, and not sexual development during puberty, the responsible for the improvement in sperm parameters. In fact, even the bibliography cited by the authors raise the idea that it was the maturation state, likely due to nutritional state, the main differentiating factor between the two groups of deer. Therefore, the authors must rewrite the paper around this aim, which will forcibly mean changes in all sections, including title. 2. The nomenclature of scientific terms is incoherent throughout the paper, rendering it unclear. As example, the nomenclature used for germ cell development phases is not coherent between Figure 1, Table 2 and the manuscript (the authors use round spermatocytes in text, then in table 2 they present counts for primary and secondary spermatocytes). Additionally, the authors should clearly define what are "sperm parameters", "spermatogenic function" and "acrosomal status". 3. Figure 1 is an essential figure in the context of the work but needs further refinement. It should show a wider field, or the authors could supplement it with an image of lower magnification, in order to show the seminiferous tubule structure and integrity. 4. During discussion, the authors overestimate their observations based upon inadequate bibliography. It is not comparable sperm mid-piece size of lizards with deer, particularly if no measurement of mitochondria number and fitness was performed. Besides, it is not acceptable to relate that eventual gain to a diet effect, based upon data draw from a study in rat liver. If the authors want to make this connection, they must present data on mitochondrial number and function in spermatozoa's midpiece. 5. Since the authors collected serum of the animals, they should quantify other hormones of the reproductive axis, in order to further characterize the maturation phase of the deer at sacrifice. 6. Although the study focuses on fallow deer, the authors must not disregard works published in other mammal species, notably rodents and humans, where it is stated that high-energy diets lead to impaired sperm parameters and poorer fertility outcomes. 7. I would not draw conclusions out of non-significant results as if there is a difference (e.g. testosterone levels).
Additionally, there are minor issues that should be addressed when rewriting the article. 1. Please, review the superscripts in the boxplots and state what "a" and "b" stand for. 2. I advise the authors to look at the following bibliography to improve their work, especially regarding germ cell -Sertoli cell cooperation: PMID 23495257, 29453570; ISBN 978-3-319-19791-3.

15-Mar-2019
Dear Dr Pintus, The editors assigned to your paper ("High-energy diet enhances spermatogenic function and increases sperm midpiece length in fallow deer (Dama dama)") have now received comments from reviewers. We would like you to revise your paper in accordance with the referee and Associate Editor suggestions which can be found below (not including confidential reports to the Editor). Please note this decision does not guarantee eventual acceptance.
Please submit a copy of your revised paper before 07-Apr-2019. Please note that the revision deadline will expire at 00.00am on this date. If we do not hear from you within this time then it will be assumed that the paper has been withdrawn. In exceptional circumstances, extensions may be possible if agreed with the Editorial Office in advance. We do not allow multiple rounds of revision so we urge you to make every effort to fully address all of the comments at this stage. If deemed necessary by the Editors, your manuscript will be sent back to one or more of the original reviewers for assessment. If the original reviewers are not available, we may invite new reviewers.
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We suggest the following format: AB carried out the molecular lab work, participated in data analysis, carried out sequence alignments, participated in the design of the study and drafted the manuscript; CD carried out the statistical analyses; EF collected field data; GH conceived of the study, designed the study, coordinated the study and helped draft the manuscript. All authors gave final approval for publication.
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Once again, thank you for submitting your manuscript to Royal Society Open Science and I look forward to receiving your revision. If you have any questions at all, please do not hesitate to get in touch.
Kind regards, Royal Society Open Science Editorial Office Royal Society Open Science openscience@royalsociety.org on behalf of Dr Punidan Jeyasingh (Associate Editor) and Professor Kevin Padian (Subject Editor) openscience@royalsociety.org Associate Editor's comments (Dr Punidan Jeyasingh): This manuscript reporting effects of an experimental high-energy diet on sperm morphology and functionality in a livestock ruminant was evaluated by two experts. I apologize for the long delay because I had a hard time finding suitable reviewers. The experts liked the work, and clearly see its importance. Nevertheless, they have raised some serious issues. Of particular note is the lack of data on the nutritional parameters of the diet. This information must be more clearly furnished, and the results discussed in this light. Clearly, dietary changes are bound to have myriad physiological implications, making the links between diet and sperm traits, as claimed in this study, rather obscure. I believe the manuscript can be improved along these lines. Moreover, the authors appear to have ignored similar work in other taxa which can be leveraged to strengthen inferences. I felt the reviews were fair and constructive. With much gratitude to the expert reviewers, I wish the authors the best in addressing these comments and improving their manuscript.
Comments to Author: Reviewers' Comments to Author: Reviewer: 1 Comments to the Author(s) Comments to the Authors: The authors explore the role of diet in shaping the sexual development in male fallow deer. Supplemental feed and pasture composition were determined using several nutritional analyses. Moreover, metrics for growth, testis mass, spermatogenic activity, sperm traits, and testosterone levels were determined. The authors found that males grew more, had higher testicular mass, and also had higher sperm quality when provided supplemental grains.
This study investigates an interesting and relatively understudied question. Many novel aspects of reproductive responses to diet have been explored in this study. My major concerns pertain to the use of energy as the primary indicator of nutrient quality without actually providing estimates for dietary energetic differences and the potentially strong alternative explanations driving the differences in response variables. The nutritive composition of the diet is really only cited in a single sentence (Lines 92-93), while later in the Discussion section only the protein and energetic content seems to be robustly considered. The protein content was slightly higher in the grass and the fat content was slightly higher in the barley grain. However, the dry matter content was over twice as great in the barley, while the fiber and lignin content were approximately three times as high in the grass. These factors might contribute to digestibility and assimilation differences/costs between the diets, which could be interacting with the response variables (in addition to energy intake). The study also seems to have been conducted on single population (divided into a control and treatment) during a single season, with most sample collection occurring at the terminus, which perhaps limits extrapolation of the findings.
Specific Comments: 1. Line 71-73: This statement seems to offer one of the strongest nutritive explanations for why supplementation with barley might increase fertility in male fallow deer. However, essential amino acids did not seem to be quantified in this study. Other nutritive explanations for the advantages of supplementation (digestibility, higher fat content, etc.) might give broader perspective to the study from the dietary data provided. 2. Lines 98-104: Was nutrient composition of barley grain determined using the same analyses as pasture or were other methods used to infer? 3. Lines 106-108: This seems like sufficient justification for using an alternative to randomized sample collection. 4. Lines 175-177: It looks like the deer were initially size-matched for the treatments, which I think was well recognized to be a necessary part of the experimental design. 5. Lines 189-193 and 215-217: These seemed to be the most significant experimental findings. Many of the other response variables did not seem to differ (e.g. testosterone, acrosomal status, and several aspects of sperm morphology). 6. Lines 230-253: The authors seem well versed in the function and production of male gametes, however, the link to diet is not yet well-elucidated. The only direct nutritional explanation is from the early reference to essential amino acids present (but not actually measured in this study) in the grains. 7. Lines 291-293: These lines provide a citation to the broader diversity of animals in the literature, which is relatively sparse up to this point in the paper. However, the animals in the current study seemed to be neither food restricted nor in poor body condition given the gain in mass and at least some equivalent aspects of fertility in both treatment and control. 8. Lines 300-302: It is difficult to assess this claim because Table 1 appears to show higher protein available for consumer intake in the grass compared to grain. 9. Line 498: At present, Table 1 only seems to be provided as background rather than as a supporting piece of evidence. This table is only cited once (within the Methods) and only protein and perhaps fat are alluded to. It is not yet clear why other nutrient analyses in the table were conducted and if these biochemicals might also contribute to the observed differences in the response variables. The arguments for higher-energy diet contributing to increased spermatogenic function and sperm midpiece length would also be strengthened if calorimetry findings or standard energy conversion estimates were to be included for the diets (e.g. protein=17 kJ/g, and fat=37 kJ/g; Raubenheimer and Rothman. 2013. Nutritional ecology of entomophagy in humans and other primates. Annu Rev Entomol).
Comments to the Author(s) In this work, the authors elicit the influence of an energy-rich diet on the onset of sexual maturation of fallow deer (Dama dama). To achieve this, the authors randomly allocated 32 prepubertal fallow deer to two groups: one fed with pasture and one supplemented with barley. Six month later, the animals were sacrificed and biometric parameters, serum testosterone, sperm parameters, spermatogenic function and acrosomal status were accessed. Overall, the authors report improvements in sperm parameters and reproductive function, compatible to an earlier onset of sexual maturation in the group fed with pasture supplemented with barley. This paper presents some results that explore the potential commercial viability of fallow deer, based on an adequate methodology. The evaluated parameters are significant according to the aims of the work, and variables are properly defined. Besides, considering the amount of collected data and its further treatment, the reported results seem reliable. Nevertheless, there are some major concerns regarding the way the authors present their data, and some gaps that must be filled in order to draw more consistent conclusions. 1. The authors have not identified the most critical aim of their work. The most evident conclusion of this work is that a high-energy diet anticipates the puberty onset on fallow deer. The authors do not present any conclusive proof that it was diet, and not sexual development during puberty, the responsible for the improvement in sperm parameters. In fact, even the bibliography cited by the authors raise the idea that it was the maturation state, likely due to nutritional state, the main differentiating factor between the two groups of deer. Therefore, the authors must rewrite the paper around this aim, which will forcibly mean changes in all sections, including title. 2. The nomenclature of scientific terms is incoherent throughout the paper, rendering it unclear. As example, the nomenclature used for germ cell development phases is not coherent between Figure 1, Table 2 and the manuscript (the authors use round spermatocytes in text, then in table 2 they present counts for primary and secondary spermatocytes). Additionally, the authors should clearly define what are "sperm parameters", "spermatogenic function" and "acrosomal status". 3. Figure 1 is an essential figure in the context of the work but needs further refinement. It should show a wider field, or the authors could supplement it with an image of lower magnification, in order to show the seminiferous tubule structure and integrity. 4. During discussion, the authors overestimate their observations based upon inadequate bibliography. It is not comparable sperm mid-piece size of lizards with deer, particularly if no measurement of mitochondria number and fitness was performed. Besides, it is not acceptable to relate that eventual gain to a diet effect, based upon data draw from a study in rat liver. If the authors want to make this connection, they must present data on mitochondrial number and function in spermatozoa's midpiece. 5. Since the authors collected serum of the animals, they should quantify other hormones of the reproductive axis, in order to further characterize the maturation phase of the deer at sacrifice. 6. Although the study focuses on fallow deer, the authors must not disregard works published in other mammal species, notably rodents and humans, where it is stated that high-energy diets lead to impaired sperm parameters and poorer fertility outcomes. 7. I would not draw conclusions out of non-significant results as if there is a difference (e.g. testosterone levels).
Additionally, there are minor issues that should be addressed when rewriting the article. 1. Please, review the superscripts in the boxplots and state what "a" and "b" stand for. 2. I advise the authors to look at the following bibliography to improve their work, especially regarding germ cell -Sertoli cell cooperation: PMID 23495257, 29453570; ISBN 978-3-319-19791-3.

Author's Response to Decision Letter for (RSOS-181972.R0)
See Appendix A.

Comments to the Author(s)
Overall, I find that the authors have comprehensively addressed my concerns from the previous draft. The authors have provided additional clarification and seemed to effectively revise paragraph structure within the Methods. Inclusion of Table 2 (Lines 589-592) sufficiently reports the energetics of pasture versus the supplemental diet. This is exemplified through partitioning of the gross and digestible/metabolisable components, which seems to support a more robust series of explanations in the revised Discussion section. Further, the authors have integrated consideration of scope to their findings (Lines 242-243, 305-307) while also providing a more thorough consideration of the literature base (throughout Discussion) necessary to compel future study in this understudied research area.

Review form: Reviewer 2
Is the manuscript scientifically sound in its present form? Yes

Recommendation?
Accept with minor revision (please list in comments)

Comments to the Author(s)
In this work, the authors elicit the influence of an energy-rich diet on the onset of sexual maturation of fallow deer (Dama dama). To achieve this, the authors randomly allocated 32 prepubertal fallow deer to two groups: one fed with pasture and one supplemented with barley. Six month later, the animals were sacrificed and biometric parameters, serum testosterone, sperm parameters, spermatogenic function and acrosomal status were accessed. Overall, the authors report improvements in sperm parameters and reproductive function, compatible to an earlier onset of sexual maturation in the group fed with pasture supplemented with barley. After reading their revised paper, and considering all the changes in result of the reviewer's criticism, the outcome is a clearly an improved manuscript. I would like to particularly address the improvements in discussion, which now reflect a sounder rational and easier reading. I consider that my central criticism regarding this paper was addressed. As I pointed out before, one of the limitations of this work is the impossibility to separate developmental and dietary factors as the driving force of the male fertility parameters. This concern is addressed by the authors (lines 317-318), but a plethora of other methodologies could suit this problem in further works. Therefore, my advice is to restrict your conclusions to "young adult fallow deer", or a more suitable veterinarian term to describe the developmental phase of animals included in this study. This change should also be reflected in title. Lastly, I call the author's attention to a minor aspect. In pages 71 and 72, the significance marks are not clear. The authors should state what the lower-case letters "a" and "b" stand for in the image subtitle. I understand they denote significance, but does "a" and "b" stand for different levels of significance? Or does it stand for group-group comparisons?

07-May-2019
Dear Dr Pintus: On behalf of the Editors, I am pleased to inform you that your Manuscript RSOS-181972.R1 entitled "High-energy diet enhances spermatogenic function and increases sperm midpiece length in fallow deer (Dama dama)" has been accepted for publication in Royal Society Open Science subject to minor revision in accordance with the referee suggestions. Please find the referees' comments at the end of this email.
The reviewers and Subject Editor have recommended publication, but also suggest some minor revisions to your manuscript. Therefore, I invite you to respond to the comments and revise your manuscript.
• Ethics statement If your study uses humans or animals please include details of the ethical approval received, including the name of the committee that granted approval. For human studies please also detail whether informed consent was obtained. For field studies on animals please include details of all permissions, licences and/or approvals granted to carry out the fieldwork.
• Data accessibility It is a condition of publication that all supporting data are made available either as supplementary information or preferably in a suitable permanent repository. The data accessibility section should state where the article's supporting data can be accessed. This section should also include details, where possible of where to access other relevant research materials such as statistical tools, protocols, software etc can be accessed. If the data has been deposited in an external repository this section should list the database, accession number and link to the DOI for all data from the article that has been made publicly available. Data sets that have been deposited in an external repository and have a DOI should also be appropriately cited in the manuscript and included in the reference list.
If you wish to submit your supporting data or code to Dryad (http://datadryad.org/), or modify your current submission to dryad, please use the following link: http://datadryad.org/submit?journalID=RSOS&manu=RSOS-181972.R1 • Competing interests Please declare any financial or non-financial competing interests, or state that you have no competing interests.
• Authors' contributions All submissions, other than those with a single author, must include an Authors' Contributions section which individually lists the specific contribution of each author. The list of Authors should meet all of the following criteria; 1) substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data; 2) drafting the article or revising it critically for important intellectual content; and 3) final approval of the version to be published.
All contributors who do not meet all of these criteria should be included in the acknowledgements.
We suggest the following format: AB carried out the molecular lab work, participated in data analysis, carried out sequence alignments, participated in the design of the study and drafted the manuscript; CD carried out the statistical analyses; EF collected field data; GH conceived of the study, designed the study, coordinated the study and helped draft the manuscript. All authors gave final approval for publication.
• Acknowledgements Please acknowledge anyone who contributed to the study but did not meet the authorship criteria.
• Funding statement Please list the source of funding for each author.
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Because the schedule for publication is very tight, it is a condition of publication that you submit the revised version of your manuscript before 16-May-2019. Please note that the revision deadline will expire at 00.00am on this date. If you do not think you will be able to meet this date please let me know immediately.
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Supplementary files will be published alongside the paper on the journal website and posted on the online figshare repository (https://figshare.com). The heading and legend provided for each supplementary file during the submission process will be used to create the figshare page, so please ensure these are accurate and informative so that your files can be found in searches. Files on figshare will be made available approximately one week before the accompanying article so that the supplementary material can be attributed a unique DOI. I thank the authors for a thoroughly revised version of the manuscript. This revision was assessed by the original reviewers, and both are clearly happy with it. With much gratitude to the reviewers, I am happy to recommend this manuscript for publication. One of the reviewers picked up a couple of important issues that should be addressed before the manuscript can be accepted for publication.
Reviewer comments to Author: Reviewer: 2 Comments to the Author(s) In this work, the authors elicit the influence of an energy-rich diet on the onset of sexual maturation of fallow deer (Dama dama). To achieve this, the authors randomly allocated 32 prepubertal fallow deer to two groups: one fed with pasture and one supplemented with barley. Six month later, the animals were sacrificed and biometric parameters, serum testosterone, sperm parameters, spermatogenic function and acrosomal status were accessed. Overall, the authors report improvements in sperm parameters and reproductive function, compatible to an earlier onset of sexual maturation in the group fed with pasture supplemented with barley. After reading their revised paper, and considering all the changes in result of the reviewer's criticism, the outcome is a clearly an improved manuscript. I would like to particularly address the improvements in discussion, which now reflect a sounder rational and easier reading. I consider that my central criticism regarding this paper was addressed. As I pointed out before, one of the limitations of this work is the impossibility to separate developmental and dietary factors as the driving force of the male fertility parameters. This concern is addressed by the authors (lines 317-318), but a plethora of other methodologies could suit this problem in further works. Therefore, my advice is to restrict your conclusions to "young adult fallow deer", or a more suitable veterinarian term to describe the developmental phase of animals included in this study. This change should also be reflected in title. Lastly, I call the author's attention to a minor aspect. In pages 71 and 72, the significance marks are not clear. The authors should state what the lower-case letters "a" and "b" stand for in the image subtitle. I understand they denote significance, but does "a" and "b" stand for different levels of significance? Or does it stand for group-group comparisons?
Reviewer: 1 Comments to the Author(s) Overall, I find that the authors have comprehensively addressed my concerns from the previous draft. The authors have provided additional clarification and seemed to effectively revise paragraph structure within the Methods. Inclusion of Table 2 (Lines 589-592) sufficiently reports the energetics of pasture versus the supplemental diet. This is exemplified through partitioning of the gross and digestible/metabolisable components, which seems to support a more robust series of explanations in the revised Discussion section. Further, the authors have integrated consideration of scope to their findings (Lines 242-243, 305-307) while also providing a more thorough consideration of the literature base (throughout Discussion) necessary to compel future study in this understudied research area.

13-May-2019
Dear Dr Pintus, I am pleased to inform you that your manuscript entitled "High-energy diet enhances spermatogenic function and increases sperm midpiece length in fallow deer (Dama dama) yearlings" is now accepted for publication in Royal Society Open Science.
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