Evaluating the Impact of Synthesised Scent Enrichment on Behavioural and Endocrine Responses in Captive Alaotran Gentle Lemurs (Hapalemur alaotrensis)
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsConsidering the critically endangered status of the Alaotran gentle lemur as per the IUCN Red List, and the limited breeding success of this species found in ex situ institutions in only one region (EAZA), this study to determine an olfactory enrichment paradigm to effect behavioral and endocrine responses that may increase breeding success in the ex situ population was well worth investigating. While the small sample size of 3 non-breeding pairs in 3 zoos may have been a limiting factor in interpreting results, this study serves as an initial investigation to perfect methods for further studies. Although the results show that olfactory enrichment may not be fully successful in terms of enhancing breeding, there was potential for further studies to investigate enhancement of welfare and breeding using olfactory cues for this species of lemur and other threatened primates.
The manuscript would be enhanced by identifying the status of breeding management for this species in EAZA, identifying the ages of the paired animals and their breeding history – had they never bred, or hadn’t bred with this pairing? Is there a difference in the anogenital secretions of a female when in estrus or not in estrus? Otherwise, this study paves the way for additional studies to enhance the reproductive potential of this population of gentle lemurs and other lemur species.
With some revision, this manuscript will be ready for publication.
Specific comments:
Edits and additions
Line 59. and Critically Endangered on the IUCN Red List: https://www.iucnredlist.org/species/9676/182236363#population
59. Identify what EEP stands for and define what it is. Mention that there is an international studbook through WAZA that tracks and facilitates breeding management.
61. Mention that there were only 7 births from 5 pairs within the last year and cite ZIMS Species Holding report. This will support the statement that there are only a few active breeding pairs in the population of 56 adults.
69. Does the chemical profile change when the female is in estrus vs when she has given birth? Was the analysis done when the female was soliciting breeding? Did you analyze the anogenital secretions from a non-breeding female as a control?
86-87. In looking at ZIMS, I found possible individuals that may have been used in the study: the Mulhouse pair may consist of a 10-year old male and 15 year-old female; the ZSL pair may be a 17 year-old male and 5 year-old female (who recently produced 0.1.2 young that DNS in 2023 and 2024 after the study was done); at Jersey there were a number of possibilities but one male, born in 2005 had never bred and another, born in 2008 had lots of offspring. For the females, one 19 year-old had never bred. In any case, it would be important to include ages of the animals in the pairs and indicate if they had ever bred before. Saying "non-breeding pairs" may mean they had never bred with this pairing, or each one had never bred successfully, which is two different things.
133. Was there work done to determine marking behavior by females to inform where to best place the samples?
149. both male and female study subjects
Please see attached manuscript with comments and additions.
Comments for author File: 
Comments.pdf
Author Response
Please see attached point-to-point reply to Reviewer 1's comments (with our responses highlighted in blue). Thanks!
Author Response File: Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsA helpful little study, on an important lemur.
Abstract
L20 Two additional compounds – additional to what? Perhaps include more information about the relevant compounds and how the two mixture differed in L14 where the two synthetic mixtures are first mentioned?
Introduction
L74 ‘between the two mixtures’ – remove as its clear from the start of the sentence that you are comparing the effects of your two mixtures.
Methods
L93 Were baseline / control data collected to compare to the enrichment days?
L121 When was enrichment added and how long after were behavioural data collected?
L140 What was the rationale for within 12cm being the criterion for proximity?
L182 The description of the two way ANOVA could make it clearer that the factors were sex and mixture.
L188 Wilcoxon test is described last yet it’s the first test in the results – reorder this section of the methods to better match the order of analyses presented in the results? As pairs were analysed separately is day or observation the unit of analysis?
Results
L209 What is the rationale for analysing each pair of lemurs separately? Need to state the unit of analysis e.g. proximity / day or hour, and provide the N.
L212 It would help to reiterate here that the test was a two-way ANOVA, what the unit of analysis was and the N.
L217 States Tab 6 is Tukey test results, its not, its ANOVA.
L222 Why no Tukey test results for olfactory behaviours?
Fig 2 For clarity can the sexes be labelled on the X axis rathe than using a legend? Such low values, especially at ZSL, mean there’s no colour visible in the bar. The Y axis needs detail in terms of the units, behavioural frequency per what time unit?
L243 Need to state what the stats test was (ANOVA?) and provide sample sizes.
Discussion
L265 Remove reference to abnormal and aggressive behaviours as they were too infrequent to be analysed, or include a statement to that effect?
L270-275 this needs consideration of the findings otherwise it’s more suited to the introduction.
L293 scientific names missing
L296 Without baseline cortisol it is difficult to talk about a reduction in stress.
Author Response
Please see attached point-to-point reply to the Reviewer 2's comments (with our responses highlighted in blue).
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsSee review report.
Comments for author File: Comments.pdf
See review report.
Author Response
Please see attached point-to-point reply to Reviewer 3's comments (with our responses highlighted in blue).
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThank you for these changes! Everything looks great! Please see the comments below and in the manuscript.
See lines 70 and 73 for suggested changes. For line 73 "the latest ZIMS Species Holding report" - please identify ZIMS with "the latest Species Holdings report for Alaotran gentle lemur in Species360 Zoological Information Management System (ZIMS), which shows only seven births..."
Because you received this information rather than looking it up yourself (only Species360 members have access to ZIMS), the reference should have the format: ZIMS Species Holdings, (date data was received). Species360 Zoological Information Management System. http://zims.species360.org.
Otherwise, readers would try to access this report and would be unable to do so.
Otherwise, this manuscript is ready for publication. Well done!
Comments for author File: Comments.pdf
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsMy earlier comments have been dealt with.
Author Response
I thank you very much, once again, for your comments and suggestions which have helped us prepare a, we believe, much improved report.
Sincerely yours,
Dr. Stefano Vaglio
(on behalf of all authors)
Reviewer 3 Report
Comments and Suggestions for AuthorsSummary: I commend the authors for the significant improvements made to the manuscript and for an interesting study on a rare species. I find all responses to my first round of edits to be acceptable and thorough. Most significantly, pre-enrichment data is now presented, which allows for much stronger and interesting conclusions. Authors may consider implementing a few more suggested changes for enhanced readability, but overall this manuscript should now make an excellent addition to the literature.
Additional suggestions:
Line 19: better to say ….”female fertility on the behavior of three…"
Throughout the manuscript, I feel that it would greatly enhance readability if the baseline vs enrichment condition methods or data was presented FIRST, followed by the comparison across the two enrichment conditions. For instance, beginning with the abstract, it seems cleaner if the order of the sentences in lines 19-24 was reversed. And in line 207, I wouldn’t say that the “analysis was extended” to include the baseline period; I would say that was the crux of the experiment!
I believe that you could completely eliminate the unscented strips from the enrichment periods and still have an acceptable experiment (really would have been nice to have the unscented strips in the “pre” condition, to make sure the presence of the strips alone didn’t have an impact, but I won’t press the issue). Therefore, referring to the unscented strips as a control seems a bit confusing to me when the real control is your baseline data in the “pre” condition. Consider eliminating that terminology. I see it as a nice additional measure to show that the lemurs took interest in the provided scent, but not as a “control” per say.
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
