Review Reports - Context-Dependent Effects of Maternal Behaviour on Lamb Growth in Tibetan Sheep

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Requires Major Revision

The Methods section requires major revision to fully explain to the readers how the behavioral tests were conducted. There are also no references to previous research on behavioral testing. Studying the behavior of sheep and productivity is an important area of research. In some situations, the tests you are using may be measuring separation stress instead of exploration. Sheep sometimes react strongly by vocalizing when they are separated from other sheep. This is due to separation distress, and it is not exploration.

Line 13 – Explain how behavioral responsiveness was measured.

Line 32 – Explain how maternal activity was measured.

Section 2.4.1 – Provide references for all the behavioral tests.

Line 176 – Latency to enter is a good measure for boldness.

Line 189 – There are two types of distances traveled. They are calm walking and exploration, which would measure boldness. The other type is wild frantic running around the arena. This would be a measure of separation distress, and it is not boldness. Did your sheep walk around calmly, or did they run around wildly and vocalize? Please explain.

Line 200 – What were the novel objects? Please describe them. Most animals are often more willing to approach a stationary object. They will also have a startle reaction if an object suddenly moves.

Line 214 – How did you measure minimal agitation? You need to have a scoring ethogram. Another factor is familiarity with the resting space. An animal may be calmer in a familiar space.

Line 223 – Describe method of restraint for collecting samples.

Line 236 – Please describe the standardized handling procedure. You also need to explain how you quantified struggle rate. Did you have a numbering system?

Line 252 – From my experience with behavioral testing of animals and productivity, I predict that vocalization and struggling by the ewes will be associated with lamb weights. Ewes that vocalize more when isolated or have greater struggling may have lighter lambs. These two measures may be a more accurate measure of docility. Another test you can conduct is a ewe’s willingness to approach a familiar caretaker. Docile animals are more willing to approach familiar people who are associated with good experiences, such as feeding.

Line 672 – Need to add references for behavior testing methods.

Final comment – I encourage you to make revisions in your paper. Animal behavior is an important area of research, and you need to have simple accurate methods to assess it.

Author Response

Comment 1:

Response 1:

Thank you for this important and constructive comment. We agree that the original Methods section did not provide sufficient detail on the behavioural testing procedure or adequate methodological references. In the revised manuscript, we have substantially expanded the description of the behavioural protocol, including the arena structure, test sequence, trial duration, behavioural recording procedure, and operational definitions of the measured variables. We have also added relevant references on sheep behavioural testing, livestock fear/temperament assessment, and open-field and novel-object paradigms.

We also agree that, in sheep, some responses expressed during individual testing may reflect temporary social isolation or separation-related distress rather than exploration alone. Accordingly, we have revised the manuscript to distinguish locomotor exploration from vocal responses more clearly. In the revised version, vocalisation is interpreted more cautiously as an indicator of emotional reactivity under temporary isolation rather than as a direct measure of exploration. We have also added a corresponding clarification in the Discussion.

Comment 2:

Line 13 – Explain how behavioral responsiveness was measured.

Response 2:

Thank you for this helpful comment. We agree that the term “behavioural responsiveness” was too broad and did not clearly indicate how it was measured. In the revised manuscript, we have replaced this general expression with the specific behavioural variables that were actually recorded in this study, including latency-based responses, locomotor activity in the open-field zone, vocalisation during individual testing, novelty response to an unfamiliar object, and docility-related settling behaviour in the resting zone. We have also revised the corresponding text in the Methods to ensure that each behavioural variable is explicitly defined and consistently referred to throughout the manuscript. These revisions clarify how maternal behavioural responses were quantified and improve the precision of the manuscript.

Comment 3:

Line 32 – Explain how maternal activity was measured.

Response 3:

Thank you for this helpful comment. We have revised the manuscript to define maternal activity explicitly as locomotor movement recorded during the open-field phase of the behavioural test. We have also ensured that this definition is used consistently in the Methods, Results, and related Discussion text.

Comment 4:

Section 2.4.1 – Provide references for all the behavioral tests.

Response 4:

Thank you for this helpful comment. We agree that the original manuscript did not provide sufficient methodological references for the behavioural tests used in this study. In the revised manuscript, we have added appropriate references for the major behavioural assays and their interpretation, including the open-field test, the novel-object test, and livestock temperament/fear-testing approaches. Specifically, we now cite established reviews of fear and behavioural reactivity tests in domestic animals and sheep, as well as our previous Tibetan sheep study that used a related behavioural testing framework. These additions have been incorporated into the relevant parts of the Methods section to ensure that each behavioural test is properly supported by the literature.

The relative references are as followings:

Forkman, B.; Boissy, A.; Meunier-Salaün, M.-C.; Canali, E.; Jones, R.B. A critical review of fear tests used on cattle, pigs, sheep, poultry and horses. Physiology & Behavior 92(3): 340–374.

Dodd, C.L.; Pitchford, W.S.; Hocking Edwards, J.E.; Hazel, S.J. Measures of behavioural reactivity and their relationships with production traits in sheep: A review. Applied Animal Behaviour Science 140(1–2): 1–15.

Yu, Y.; Wang, Y.; Zhong, L.; Zhu, H.; Qu, J. Variations in Behavioral and Physiological Traits in Yearling Tibetan Sheep (Ovis aries). Animals 11(6): 1676.

Stubsjøen, S.M.; Bohlin, J.; Dahl, E.; Knappe-Poindecker, M.; Fjetland, O.; Lepschy, M.; et al. 2018. Effect of short-term seclusion of sheep on their welfare indicators. Journal of Veterinary Behavior 28: 1–7.

Comment 5:

Line 176 – Latency to enter is a good measure for boldness.

Response 5:

Thank you for this helpful comment. We agree that latency to enter is an appropriate measure of boldness in this context. In the revised manuscript, we now state explicitly that latency to enter the open-field zone was used as the operational measure of boldness, with shorter latencies indicating greater boldness and longer latencies indicating greater caution or shyness. The corresponding changes have been made in the Methods and related text.

Comment 6:

Response 6:

Thank you for this important and helpful comment. We agree that total distance travelled alone cannot distinguish calm exploratory movement from agitation-driven movement associated with separation-related distress, and therefore should not be interpreted directly as boldness. In the revised manuscript, we have clarified that boldness was represented by latency to enter the open-field zone, whereas movement after entry was interpreted more cautiously as locomotor activity/exploration rather than as boldness per se. Because the present study did not include a separate behavioural classification of calm walking versus frantic running, total distance travelled was interpreted conservatively, and the Discussion now acknowledges that some locomotor responses during individual testing may reflect temporary social isolation or separation-related emotional reactivity. The corresponding changes have been made in the Methods, Results, and Discussion sections to ensure consistency across the manuscript.

Comment 7:

Response 7:

Thank you for this helpful comment. We agree that the novel-object stimulus should be described more clearly. In the revised manuscript, we have added a specific description of the novel objects used in the novelty assay. The novel stimuli were an orange-and-white parking cone, a wet-floor sign, and a yellow man-shaped “danger” sign, which were used at different testing stages. We have also clarified that all objects were placed in a fixed position before the focal animal entered the novelty zone and remained stationary throughout the trial. Therefore, the assay measured the ewe’s response to an unfamiliar stationary object, rather than to sudden object movement or a startle-inducing stimulus. The corresponding changes have been made in the Methods text.

Comment 8:

Line 214 – How did you measure minimal agitation? You need to have a scoring ethogram. Another factor is familiarity with the resting space. An animal may be calmer in a familiar space.

Response 8:

Thank you for this helpful comment. We agree that the original wording “minimal agitation” was imprecise and did not clearly indicate how this behaviour was assessed. In the revised manuscript, we have therefore removed this qualitative expression and replaced it with a more explicit description of the measured behaviour, based on latency to enter and settle in the resting zone under the standardized post-test condition. We now state that individuals entering the resting zone promptly and settling quickly were considered more behaviourally calm in this context. Because the present study did not apply a separate ethogram specifically designed to score agitation intensity, we have avoided interpreting this variable as a formal agitation score and instead describe it more conservatively as a settling- or recovery-related behavioural response. We have also revised the Discussion to acknowledge more explicitly that behaviour in the resting zone may reflect not only recovery after novelty exposure, but also the animal’s perception of that compartment as relatively sheltered or less aversive. The corresponding changes have been made in the Methods.

Comment 9:

Line 223 – Describe method of restraint for collecting samples.

Response 9:

Thank you for this helpful comment. We have revised the Methods section to describe the restraint procedure more clearly. Specifically, we now state that physiological measurements and sample collection were performed under brief, standardized light manual restraint by trained personnel, with care taken to minimize stress and ensure animal safety.

Comment 10:

Line 236 – Please describe the standardized handling procedure. You also need to explain how you quantified struggle rate. Did you have a numbering system?

Response 10:

Thank you for this helpful comment. We agree that the original manuscript did not describe the handling procedure and struggle-related behaviour with sufficient clarity. In the revised manuscript, we have added a clearer description of the standardized handling phase, during which each animal was subjected to brief, light manual restraint by trained personnel for physiological measurement and sample collection. We have also clarified that struggle-related behaviour was quantified during this handling period, based on the animal’s escape-related movements under restraint, and that the description of this variable has been kept strictly consistent with the original scoring procedure. Because no separate formal numbering or ordinal scoring system was used beyond the original handling-based recording procedure, we have avoided overstating this measure in the revised manuscript.

Comment 11:

Response 11:

Thank you for this valuable suggestion. We have revised the Discussion at the relevant location to note more explicitly that, in sheep, vocalization during individual testing and struggle-related behaviour during handling may reflect more immediate forms of behavioural reactivity linked to temporary isolation and restraint. We have also added a statement that approach to a familiar caretaker would be a useful complementary direction for future studies of docility in sheep.

Comment 12:

Line 672 – Need to add references for behavior testing methods.

Response 12:

Thank you. We understand that this comment refers to the need for references supporting the behavioural testing methods themselves. Accordingly, in the revised manuscript, we have added relevant methodological references at the corresponding locations in the Methods section, including the descriptions of the open-field test, novel-object test, and sheep behavioural reactivity measures. The references already cited in the Discussion were retained because they support the broader theoretical interpretation rather than the test procedures themselves.

Comment 13:

Final comment – I encourage you to make revisions in your paper. Animal behavior is an important area of research, and you need to have simple accurate methods to assess it.

Response 13:

Thank you for this constructive comment. We agree that behavioural assessment in sheep should rely on simple, clear, and biologically meaningful methods. In response to the reviewer’s suggestions, we have substantially revised the manuscript, especially the Methods and related Discussion, to improve clarity, refine the behavioural definitions, add appropriate methodological references, and reduce overinterpretation where necessary. We believe these revisions have substantially improved the manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

The article addresses the important issue of assessing the factors that determine lambs' growth and development in relation to their mothers' characteristics under specific regional/production conditions.

In the last paragraph of the Introduction, the authors describe their research study. In my opinion, it would be more valuable to state the research study's purpose at this point. One could write: "The purpose of the research was..." Regardless of the general purpose of the research study, it would also be worthwhile to describe the cognitive (scientific) and utilitarian (practical) goals of the research.

The Introduction discusses sheep and their behavior extensively, taking into account the relationships between different generations of animals. In my opinion, this valuable information can be used to formulate (identify) a research problem. This problem can, for example, be formulated as a single research question or multiple research questions. It would also be worthwhile to identify a gap in current knowledge related to the research problem developed in the study.

Alternatively, at the end of the Introduction chapter, you could present (write) a brief outline of the research tasks within the study.

In the Materials and Methods section, the authors mentioned animal welfare twice. In my opinion, it would be beneficial to elaborate on the issue of maintaining animal welfare requirements during the study. If an experiment is planned, it is worth providing information on whether the dimensions of the technical equipment, including the testing areas, were designed to allow the animals to feel comfortable and have sufficient space.

It is worth describing how the data from the recorded material observations were transferred to the research forms. Were the forms created in an Excel spreadsheet? Perhaps it would be worthwhile to show a fragment (part) of such a form? It would also be worthwhile to provide more details, including a summary of specific animal behavioral events in the ethogram.

It would be helpful to provide more information about the animals/herd that participated in the study. Were there any guidelines for selecting animals (sheep) for the experiment, taking into account age, lactation rate, etc.? Were the animals previously trained (before the experiment), or was this their first exposure to the experimental equipment? It's worth expanding on these details in the research description. In this section (Materials and Methods), it would also be helpful to provide more information about the animals. How many were there, what was their condition at the beginning and end of the experiment, what was the average weight of the animals, and what was their average age?

The authors have described the location of the experiment and its details. It would be helpful to include a diagram of the arena and surrounding infrastructure used in the experiment. A photo of the location where the experiment was conducted could also be added to the article. This would greatly facilitate the reader's understanding of the experimental procedure and situational analysis.

If the animals and their behavior were recorded during the experiment, it may be worthwhile to provide more details about the camera and recording software. The camera model and the name of the recording software can be provided. Was it a black-and-white or color camera? (I used only a black-and-white camera in my research because it was easier to identify the animals). The comment about providing details (models, manufacturers) in the article also applies to the devices used to measure (analyze) the struggle rate, respiration rate, and heart rate. If this information could be supplemented, it would benefit the presentation of the technical equipment used in the research (experiment).

I think it would be worthwhile to verify the notes (in the text) regarding tables and figures. The notes in the text include, for example, Figure S2a (line 468) and Figure S2b (line 469). Figure 2 is in the article, but I was unable to identify parts a and b in that figure. Are you referring to supplementary materials? Perhaps it should be stated more precisely (emphatically) that this refers to supplementary materials.

In the final section of the article, you could write about any limitations that need to be considered when planning this type of research.

The final section of the article also discusses how farmers can use the research results in practice. The practical application of these observations can provide valuable input for improving livestock (sheep) production on farms.

Author Response

Comment 1:

Response 1:

We sincerely thank the reviewer for this positive and encouraging comment. We appreciate the recognition of the scientific and practical relevance of this study. We agree that understanding how maternal characteristics relate to lamb growth and development under specific regional and production conditions is an important topic, particularly in Tibetan sheep managed under semi-captive conditions on the Qinghai–Tibetan Plateau.

Comment 2:

Response 2:

Thank you for this valuable suggestion. We agree that the final paragraph of the Introduction should present the purpose of the study more explicitly and should distinguish more clearly between its scientific and practical aims. In the revised manuscript, we have therefore rewritten the closing part of the Introduction to state the purpose of the study directly and to clarify both the scientific objective, namely to examine the relationships among maternal behavioural traits, offspring behavioural traits, and early growth under semi-captive livestock conditions, and the practical objective, namely to assess the potential relevance of these behavioural differences for Tibetan sheep management and breeding.

Comment 3:

Response 3:

Thank you for this thoughtful comment. We agree that the Introduction should define the research problem and the associated knowledge gap more clearly. In the revised manuscript, we have strengthened the end of the Introduction to make this point more explicit. In particular, we now state more clearly that, although maternal behaviour, offspring behaviour, and growth performance have each been studied previously, they have rarely been examined together within a unified framework under livestock production conditions. We have linked this gap directly to the questions addressed in the present study.

Comment 4:

Alternatively, at the end of the Introduction chapter, you could present (write) a brief outline of the research tasks within the study.

Response 4:

Thank you for this helpful suggestion. In the revised manuscript, we have improved the final paragraph of the Introduction so that the aims of the study are presented more directly and in a more structured manner. We now state the main objectives of the study clearly and explicitly, including the two specific questions tested, which together provide a concise outline of the principal research tasks.

Comment 5:

Response 5:

Thank you for this important suggestion. We agree that the description of animal welfare safeguards during the study should be more detailed. In the revised manuscript, we have expanded the Materials and Methods section to clarify that the behavioural tests and handling procedures were designed to minimize stress and to avoid unnecessary disturbance or prolonged restraint. We have also added a clearer statement indicating that the testing arena and associated setup were arranged to provide sufficient space for controlled movement during the behavioural assays while maintaining a standardized testing environment.

Comment 6:

Response 6:

Thank you for this constructive comment. We agree that the transition from recorded observations to analytical data should be described more clearly. In the revised manuscript, we have clarified that the video recordings were reviewed using predefined behavioural categories and time-stamped event logging, and that the scored observations were subsequently entered into structured data sheets for statistical analysis. We have also revised the text to describe the ethogram-based coding framework more explicitly, so that the behavioural variables extracted from the recordings are more clearly understood. At the present stage, we chose to improve the methodological description in the text rather than include a separate example of the scoring form.

Comment 7:

Response 7:

Thank you for this helpful suggestion. We agree that the description of the study animals should be more complete. In the revised manuscript, we have expanded the relevant part of the Materials and Methods section to clarify the study population more fully, including the number of focal animals and the general basis for their inclusion in the study. We have also improved the description of the testing context with respect to the animals’ participation in the experiment. Where this information was available from the original records, we have supplemented the manuscript with additional descriptive information on the focal animals and herd.

Comment 8:

Response 8:

Thank you for this valuable suggestion. We agree that a clearer visual presentation of the experimental setting would help readers better understand the testing procedure. In the revised manuscript, we have further clarified the arena structure and the sequence of the test zones in the Materials and Methods section. In addition, we have added a schematic diagram of the arena to facilitate understanding of the experimental procedure. We appreciate the reviewer’s suggestion that additional visual material could further improve the clarity of the study setting.

Comment 9:

Response 9:

Thank you for this useful suggestion. We agree that additional technical detail would improve the transparency of the Materials and Methods section. In the revised manuscript, we have clarified the role of the video recording system in behavioural data collection and, where available from the original records, we have supplemented the text with additional information on the recording and physiological measurement procedures. We also agree that clearer reporting of the technical setup benefits the presentation of the study and improves methodological transparency.

Comment 10:

Response 10:

Thank you for this careful observation. We agree that the references to the supplementary figures should be stated more explicitly. In the revised manuscript, we have checked the figure citations throughout the text and revised them so that references such as Figure S1a/S1b and Figure S2a/S2b are now clearly identified as referring to the Supplementary Materials. We believe this correction improves consistency and avoids possible confusion for the reader.

Comment 11:

In the final section of the article, you could write about any limitations that need to be considered when planning this type of research.

Response 11:

Thank you for this valuable suggestion. We agree that the limitations of the present study should be stated more explicitly. In the revised manuscript, we have strengthened the final section by noting that some behavioural measures may be context-dependent and that certain interpretations should therefore remain cautious. We have also indicated that further validation under additional sheep production systems would be important when designing and interpreting this type of research.

Comment 12:

Response 12:

Thank you for this helpful comment. We agree that the practical implications of the study should be presented more clearly. In the revised manuscript, we have strengthened the concluding section to indicate that the present findings may provide a useful basis for future management, selection, and breeding considerations in Tibetan sheep under semi-captive production conditions. At the same time, we emphasize that further validation will be necessary before direct on-farm application.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have made extensive revisions on manuscript ID Animals 4268266 - Context-Dependent Effects of Maternal Behavior on Lamb Growth in Tibetan Sheep. The paper should be accepted for publication. The authors added additional information on handling methods and a description of the novel objects.