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Cited by 9Animal Behavior and Welfare StudiesAnimal testing and alternativesVeterinary Pharmacology and Anesthesia

Ultrasonic Vocalization Analysis as a Novel Metric to Assess Cage Enrichment in Rats

Open access
Bigelow, Logan J, Cohen, Andrew J, Pimm, Robyn, Knight, Jennifer B, Bernard, Paul B·2022·Journal of the American Association for Laboratory Animal Science
DOI 10.30802/aalas-jaalas-21-000024PMC PMC8956216
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Abstract

Laboratory rodent housing conditions vary significantly across laboratories and facilities. Variation in housing can be associated with animal stress leading to study variability and the subsequent inability to replicate experimental findings. Optimization and standardization of animal housing are necessary to promote animal welfare and data consistency, thereby reducing the number of animals necessary to detect treatment effects. While interest in environmental enrichment is increasing, many studies do not examine the behavior of animals in the home cage, neglecting important aspects of enrichment. To determine how increased vertical home cage area affects animal welfare, double-decker cages (enriched), which allow rats to upright stand, were compared with standard single-level cages, which impede the ability to upright stand. Home cage welfare was assessed by analyzing ultrasonic vocalizations, fecal corticosterone, upright standing, and fighting. Ultrasonic vocalization was further explored by analyses of call type as defined by a 14 call-type schematic. Rats housed in enriched cages spent more time fighting, produced fewer 50 kHz calls, and had higher levels of fecal corticosterone. Rats in standard cages attempted to upright stand more often but remained upright for a shorter amount of time due to the height limitation imposed by standard cages. In addition, standard cages restrict some naturalistic behaviors such as upright standing and reduce fighting, which may be attributable to their single-tier organization and floor space. Enriched cages permit rats to engage in normal ethological behavior but also increase fighting. This study demonstrates that housing conditions have a meaningful impact on multiple measures of animal affect. When considering study design, researchers should be aware of how housing conditions affect animal subjects.

AI snapshot

Written from the paper’s full text.

TL;DR

Male CD rats housed in double-decker enriched cages showed fewer 50-kHz calls, more 22-kHz calls, higher fecal corticosterone, and longer upright standing than rats in standard single-level cages. The authors conclude that housing conditions meaningfully affect multiple welfare/affect measures and that USVs may help assess home-cage welfare, though the enriched cage also appeared to increase negative indicators.

Key findings

  • Enriched-cage rats produced fewer 50-kHz calls than standard-cage rats on day 18 (t(6) = 2.66, P = 0.0378), and more 22-kHz calls overall (F(1, 6) = 9.84, P = 0.0202).
  • Enriched-cage rats had higher fecal corticosterone than standard-cage rats (t(11) = 3.70, P = 0.00350).
  • Standard-cage rats attempted upright standing more frequently (F(1, 6) = 28.8, P = 0.00170), but enriched-cage rats stood upright for longer total duration (F(1, 6) = 34.7, P = 0.00110).
  • The abstract states enriched rats spent more time fighting, but the Results report no significant difference in total fighting duration between enriched and standard cages (t(6) = 2.13, P = 0.0775); fighting duration was related to 22-kHz call number (F(1,6) = 23.3, P = 0.00290, R² = 0.795). Complex call analysis found condition effects for flat-trill (P = 0.00590), inverted-U (P = 0.0435), multi-step (P = 0.048), and trill (P = 0.0308).

Methods

  • Male CD rats (Charles River) were allocated to standard (4 cages) or enriched double-decker (4 cages) housing, 3 rats/cage, 12 rats/treatment group; rats were approximately 80 d old at allocation and remained in conditions for 33 d.
  • USV and video recordings were made in the home cage every 3 d for 18 d and 15 d, respectively, with a single 30-min USV/video recording on day 33; upright standing was scored from 1-min video intervals on days 1, 3, 6, 9, 12, and 15, and fighting was scored from the day-33 30-min video.
  • Fecal corticosterone was measured by commercial ELISA on day 33; analyses used unpaired 2-tailed Student t tests, 2-way ANOVA with day and condition, and linear regression, with α = 0.05.

Limitations

  • Stated: experimenters could not be blind to condition for in-cage behavior analysis, and a single analyst scored in-cage behaviors, so IRR was not performed; complex call classification had IRR of 67% based on 231 calls, with discrepancies especially for modulated pitch calls.
  • Stated: fecal corticosterone alone cannot assign a physiologic state, because increased arousal may not be negative; the authors also note future work is needed to determine whether the corticosterone elevation affects affect or memory tests.
  • Evident: the study used only male rats, small group sizes (4 cages/group), and the enriched cage differed by both a shelf and less floor space, which the authors hypothesize may confound fighting, 22-kHz calls, and corticosterone.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.30802/aalas-jaalas-21-000024

Cites

47 of 48 references verified0 citation errors · 1 not checkable

Statistics

60 tests recomputed · 0 with the reported and recomputed p on opposite sides of .05

Claims the paper makes

11 of 45 cited sources checked · 5 supported · 1 flagged · accuracy not yet measured

  • Premisecitation
    § Introduction

    Rat ultrasonic vocalizations can be categorized into two main groups: positive/"happy" 50 kHz USVs and negative/"distress" 22 kHz USVs.

    “USVs in rats can be categorized into 2 main groups: positive/“happy” 50 kHz USVs and negative/“distress” 22 kHz USVs.7-9,24,33,38,39,41”
    Quote in the paperSubstantial claim2 of 8 sources checked
    • cites [7] Brudzynski S. 2015. Pharmacology of ultrasonic vocalizations in adult rats: significance, call classification and neural substrate. CN 13:180–192. 10.2174/1570159X13999150210141444 [DOI] [PMC free article] [PubMed] [Google Scholar] source
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    • cites [9] Brudzynski SM. 2013. Ethotransmission: communication of emotional states through ultrasonic vocalization in rats. Curr Opin Neurobiol 23:310–317. 10.1016/j.conb.2013.01.014. [DOI] [PubMed] [Google Scholar] source
    • cites [24] Kisko TM, Wöhr M, Pellis VC, Pellis SM. 2015. From play to aggression: high-frequency 50-kHz ultrasonic vocalizations as play and appeasement signals in rats, p 91–108. In: Wöhr M Krach Seditors. Social behavior from rodents to humans. vol. 30. Cham: Springer International Publishing. [DOI] [PubMed] [Google Scholar] source
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      The Introduction identifies 50-kHz calls with appetitive behavior and 22-kHz calls with aversion, supporting the positive/negative distinction. But it explicitly defines three classes—40-, 22-, and 50-kHz calls—not the two main groups stated in the claim. (LLM, conf=0.92)

      Experimental — accuracy not yet measured
  • Premisecitation
    § Discussion

    Rats generally do not emit USVs when rearing.

    “A comparison of USV data between groups with apparent differences in upright standing is complicated in that rats generally do not emit USVs when rearing.14”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [14] Burke CJ, Kisko TM, Swiftwolfe H, Pellis SM, Euston DR. 2017. Specific 50-kHz vocalizations are tightly linked to particular types of behavior in juvenile rats anticipating play. Cooper BG, editor. PLoS ONE 12:e0175841. 10.1371/journal.pone.0175841 [DOI] [PMC free article] [PubMed] [Google Scholar] source

      The Results ('Specific correspondences between vocalizations and behaviors'; page not supplied) associate rearing with a marked reduction in the probability of calls, supporting the gist of the claim. But the study examined 50-kHz calls in juvenile rats anticipating play; it does not establish that rats generally do not emit USVs while rearing. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
  • Findingown data
    § Abstract

    Rats in standard cages attempted to upright stand more often than rats in enriched cages.

    “Rats in standard cages attempted to upright stand more often but remained upright for a shorter amount of time due to the height limitation imposed by standard cages.”
    Quote in the paperNot a substantial claim
  • Findingown data
    § Results

    For 22 kHz calls, there was a significant treatment effect (F(1,6)=9.84, P=0.0202).

    “For 22 kHz calls, a 2-way ANOVA revealed a significant treatment effect (F (1, 6) = 9.84, P = 0.0202), a nonsignificant day effect, F (6, 36) = 0.767, P = 0.601, and a nonsignificant day x treatment interaction, F (6, 36) = 0.690, P = 0.659).”
    Quote in the paperSubstantial claim
  • Findingreasoning
    § Abstract

    Rats in standard cages remained upright for a shorter amount of time than rats in enriched cages.

    “Rats in standard cages attempted to upright stand more often but remained upright for a shorter amount of time due to the height limitation imposed by standard cages.”
    Quote in the paperNot a substantial claim
  • May combine two claims. 18
  • Classified as probably not a substantial claim. 16

  • May combine two claims.

    Premisecitation
    § Introduction

    Previous Canadian Council on Animal Care guidelines mandated a minimum cage height that did not allow upright standing for most rats; current guidelines recommend a minimal height equivalent to the nose-to-tail-base distance plus 4 cm, which would allow upright standing.

    “Previous guidelines from the Canadian Council on Animal Care (CCAC) mandated a minimum cage height that did not allow upright standing for most rats; however, the current guidelines recommend a minimal height equivalent to the nose-to-tail-base distance plus 4 cm, which would allow upright standing.16,28”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [16] Canadian Council on Animal Care. 2020. CCAC Guidelines: Rats. Ottawa, Ontario: Canadian Council on Animal Care. [Google Scholar]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [28] Makowska IJ, Weary DM. 2016. The importance of burrowing, climbing and standing upright for laboratory rats. R Soc Open Sci 3:160136. 10.1098/rsos.160136. [DOI] [PMC free article] [PubMed] [Google Scholar] source

      Split into 2 atoms — 1 confirmed, 0 partially supported, 1 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation
    § Introduction

    Play fighting between rats, as well as play between a rat and a human, are known to elicit intense 50 kHz calling, while threatening scenarios, such as an intruder or exposure to a predator, are more commonly associated with 22 kHz calls.

    “Play fighting between rats, as well as play between a rat and a human, are known to elicit intense 50 kHz calling,20,25 while threatening scenarios, such as an intruder or exposure to a predator is more commonly associated with 22 kHz calls.46”
    Quote in the paperSubstantial claim1 of 3 sources checked
    • cites [20] Himmler BT, Kisko TM, Euston DR, Kolb B, Pellis SM. 2014. Are 50-kHz calls used as play signals in the playful interactions of rats? I. Evidence from the timing and context of their use. Behav Processes 106:60–66. 10.1016/j.beproc.2014.04.014. [DOI] [PubMed] [Google Scholar] source
    • cites [25] LaFollette MR, O’Haire ME, Cloutier S, Blankenberger WB, Gaskill BN. 2017. Rat tickling: a systematic review of applications, outcomes, and moderators. Pellis S, editor. PLoS ONE 12:e0175320. 10.1371/journal.pone.0175320 [DOI] [PMC free article] [PubMed] [Google Scholar] source
    • cites [46] Thomas DA, Takahashi LK, Barfield RJ. 1983. Analysis of ultrasonic vocalizations emitted by intruders during aggressive encounters among rats (Rattus norvegicus). J Comp Psychol 97:201–206. 10.1037/0735-7036.97.3.201. [DOI] [PubMed] [Google Scholar] source

  • Classified as probably not a substantial claim.

    Premisecitation
    § Introduction

    Exploration of distinct ultrasonic vocalizations has revealed a linkage between certain call types and behaviors.

    “Exploration of distinct USVs has revealed a linkage between certain call types and behaviors.14”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [14] Burke CJ, Kisko TM, Swiftwolfe H, Pellis SM, Euston DR. 2017. Specific 50-kHz vocalizations are tightly linked to particular types of behavior in juvenile rats anticipating play. Cooper BG, editor. PLoS ONE 12:e0175841. 10.1371/journal.pone.0175841 [DOI] [PMC free article] [PubMed] [Google Scholar] source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation
    § Introduction

    Excess agonistic engagement between cage mates may be harmful to wellbeing while playful fighting may be beneficial.

    “Excess agonistic engagement between cage mates may be harmful to wellbeing while playful fighting may be beneficial.36,37”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [36] Pellis SM, Pellis VC. 2004. Play and fighting, p 298–306. The behavior of the laboratory rat: a handbook with tests. [place unknown]: Oxford University Press. [Google Scholar]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [37] Pellis SM, Pellis VC. 2017. What is play fighting and what is it good for? Learn Behav 45:355–366. 10.3758/s13420-017-0264-3. [DOI] [PubMed] [Google Scholar] source

      The Abstract (page not supplied) says play fighting may build psychological resilience, supporting the possibility that it is beneficial. The functions of play fighting section (page not supplied) says excessive force can cause play to escalate into serious fighting, but does not establish that excess agonistic engagement between cage mates harms wellbeing. (LLM, conf=0.84)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation
    § Introduction

    Rats that have the opportunity will stand upright; when unable to do so, they will stretch laterally more frequently, suggesting that the ability to extend the body is important.

    “Consistent with the implied benefit of increased vertical space, rats that have the opportunity will stand upright; when unable to do so, they will stretch laterally more frequently, suggesting that the ability to extend the body is important.”
    • Also stated in: § Introduction · hedged
      “Rats may lateral stretch more frequently in circumstances where general mobility is restricted.28”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [28] Makowska IJ, Weary DM. 2016. The importance of burrowing, climbing and standing upright for laboratory rats. R Soc Open Sci 3:160136. 10.1098/rsos.160136. [DOI] [PMC free article] [PubMed] [Google Scholar] source

  • May combine two claims.

    Premisecitation
    § Introduction

    Ultrasonic vocalizations are a sensitive method of assessing rodent communication and affective state.

    “Ultrasonic vocalizations (USVs) are a sensitive method of assessing rodent communication and affective state9,33”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [9] Brudzynski SM. 2013. Ethotransmission: communication of emotional states through ultrasonic vocalization in rats. Curr Opin Neurobiol 23:310–317. 10.1016/j.conb.2013.01.014. [DOI] [PubMed] [Google Scholar] source
    • cites [33] Parsana AJ, Li N, Brown TH. 2012. Positive and negative ultrasonic social signals elicit opposing firing patterns in rat amygdala. Behav Brain Res 226:77–86. 10.1016/j.bbr.2011.08.040. [DOI] [PMC free article] [PubMed] [Google Scholar] source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § Discussion

    The observed behavior of increased upright standing is consistent with another study that also observed an increase in upright standing in a permissive environment.

    “The observed behavior is consistent with another study that also observed an increase in upright standing in a permissive environment.28”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [28] Makowska IJ, Weary DM. 2016. The importance of burrowing, climbing and standing upright for laboratory rats. R Soc Open Sci 3:160136. 10.1098/rsos.160136. [DOI] [PMC free article] [PubMed] [Google Scholar] source

      The Results (Main study; page unavailable) report frequent upright standing in semi-naturalistic cages, and the Conclusion says standard cages prevented rats from standing upright. The paper does not report an increase in upright standing after a change to a permissive environment; it reports that standing declined as the rats aged. (LLM, conf=0.82)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation
    § Discussion

    Fecal corticosterone may reflect an increased state of arousal, but the arousal may not be negative.

    “Although fecal corticosterone may reflect an increased state of arousal, the arousal may not be negative.29”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [29] Mendl M, Burman OHP, Paul ES. 2010. An integrative and functional framework for the study of animal emotion and mood. Proc Biol Sci 277:2895–2904. 10.1098/rspb.2010.0303. [DOI] [PMC free article] [PubMed] [Google Scholar] source

      Section 4, “Implications for the Measurement of Animal Emotion” (page unavailable), says physiological measures, including hypothalamic-pituitary-adrenal activity, may reflect arousal without distinguishing positive from negative states. The paper does not specifically discuss fecal corticosterone, so it supports the general caution but not its application to that measure. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data
    § Results

    Rats in standard cages stood upright more frequently than did rats in enriched cages, with a significant treatment (cage type) effect (F(1,6)=28.8, P=0.00170).

    “A 2-way ANOVA revealed a significant treatment (cage type) effect for upright standing (F (1, 6) = 28.8, P = 0.00170), a nonsignificant day effect (F (5, 30) = 0.592, P = 0.706), and a nonsignificant treatment x day interaction (F (5, 30) = 0.496, P = 0.777). Rats in standard cages stood upright more frequently than did rats in enriched cages (Figure 7A).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data
    § Results

    Rats in enriched cages stood upright for a longer amount of time than rats in standard cages, with a significant treatment effect (F(1,6)=34.7, P=0.00110).

    “The total duration of standing upright showed a significant treatment effect (F (1, 6) = 34.7, P = 0.00110), a nonsignificant day effect (F (5, 30) = 1.15, P = 0.358), and a nonsignificant day x treatment interaction (F (5, 30) = 0.911, P = 0.487). Rats in enriched cages stood upright for a longer amount of time than rats in standard cages (Figure 7B).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingreasoning
    § Discussion

    The ability to stand upright did not result in an increase in 50 kHz calls or a decrease in 22 kHz calls.

    “However, the ability to stand upright did not result in an increase in 50 kHz calls or a decrease in 22 kHz calls.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductreasoning
    § Materials and Methods

    A single analyst scored the in-cage behaviors; therefore, interrater reliability was not performed.

    “A single analyst scored the in-cage behaviors; therefore, IRR was not performed.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data
    § Discussion

    Rats in enriched cages stood upright for longer durations compared with standard housing, suggesting improved ethologically relevant welfare.

    “Rats in enriched cages stood upright for longer durations as compared with rats in standard housing, suggesting improved ethologically relevant welfare.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Longer upright standing in enriched cages indicates improved ethologically relevant welfare.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given
    § Materials and Methods

    Cages in both conditions were randomly distributed on the rack at the time of cage changing.

    “Cages in both conditions were randomly distributed on the rack at the time of cage changing.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Study design and conductown data
    § Materials and Methods

    Analysts were consistent in scoring call duration and in recognizing that a call had occurred (IRR = 89%), but demonstrated discrepancies in classifying the call (IRR = 69%).

    “Analysts were consistent in scoring the call duration and in recognizing that a call had occurred (IRR = 89%), but demonstrated some discrepancies in classifying the call (IRR = 69%).”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductown data
    § Materials and Methods

    Rats were randomly allocated to standard or enriched cages.

    “After acclimation and handling, rats were randomly allocated to standard (508 × 406 × 216 mm) (Ancare Corporation, Bellmore, NY) or enriched (GR1800 Double Decker; 381 × 305 × 394 mm) (Tecniplast, Montreal, Quebec) cages”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given
    § Materials and Methods

    Ultrasonic vocalization and fecal corticosterone analysis data were assessed by investigators who were blind to experimental conditions.

    “Ultrasonic vocalization analysis and fecal corticosterone analysis data were assessed by investigators who were blind to experimental conditions.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § Introduction

    A previous study differentiated the two types of fight behavior based on the target and type of contact.

    “A previous study differentiated the 2 types of fight behavior based on the target and type of contact.34”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [34] Pellis SM, Pellis VC. 1987. Play‐fighting differs from serious fighting in both target of attack and tactics of fighting in the laboratory rat (Rattus norvegicus ). Aggress Behav 13:227–242. 10.1002/1098-2337(1987)13:4<227::AID-AB2480130406>3.0.CO;2-C. [DOI] [Google Scholar] source

  • May combine two claims.

    Premisecitation
    § Introduction

    Ultrasonic vocalizations can reflect affective state or stress status continuously and noninvasively, making them preferable to other measures of stress or anxiety, such as fecal corticosterone, in rats.

    “One of the greatest benefits of USVs is their ability to reflect affective state or stress status continuously and noninvasively, making USVs preferable to other measures of stress or anxiety, such as fecal corticosterone, in rats.21,26,27,30”
    Quote in the paperSubstantial claim0 of 4 sources checked
    • cites [21] Hunt C, Hambly C. 2006. Faecal corticosterone concentrations indicate that separately housed male mice are not more stressed than group housed males. Physiol Behav 87:519–526. 10.1016/j.physbeh.2005.11.013. [DOI] [PubMed] [Google Scholar] source
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  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Study design and conductnone given
    § Materials and Methods

    Experimenters could not be blind in the analysis of in-cage behaviors.

    “Experimenters could not be blind in the analysis of in-cage behaviors.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation
    § Introduction

    Both 50 and 22 kHz calls are critical in guiding the nature of interactions between rats, suggesting an important communicative role for ultrasonic vocalizations in guiding social interactions.

    “Both 50 and 22 kHz calls are critical in guiding the nature of interactions between rats, suggesting an important communicative role for USV in guiding social interactions.12,13”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [12] Burke CJ, Euston DR, Pellis SM. 2020. What do you hear, what do you say? ultrasonic calls as signals during play fighting in rats. Int J Play 9:92–107. 10.1080/21594937.2020.1720126. [DOI] [Google Scholar] source
    • cites [13] Burke CJ, Kisko TM, Pellis SM, Euston DR. 2017. Avoiding escalation from play to aggression in adult male rats: The role of ultrasonic calls. Behav Processes 144:72–81. 10.1016/j.beproc.2017.09.014. [DOI] [PubMed] [Google Scholar] source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § Introduction

    Some authors have suggested as many as 14 distinct 50 kHz ultrasonic vocalizations in rats.

    “In recent years, further delineation of 50 kHz calls has occurred, with some authors suggesting as many as 14 distinct USVs.49”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [49] Wright JM, Gourdon JC, Clarke PBS. 2010. Identification of multiple call categories within the rich repertoire of adult rat 50-kHz ultrasonic vocalizations: effects of amphetamine and social context. Psychopharmacology (Berl) 211:1–13. 10.1007/s00213-010-1859-y. [DOI] [PubMed] [Google Scholar] source

  • May combine two claims.

    Study design and conductown data
    § Materials and Methods

    The automatic ultrasonic vocalization scoring program was 89% accurate for 50 kHz calls and 100% accurate for 22 kHz calls.

    “The program was 89% accurate for 50 kHz calls, and 100% accurate for 22 kHz calls.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § Discussion

    A previous study observed that 22 kHz vocalizations occurred after the first bite during resident-intruder scenarios that resulted in submission, suggesting an association between 22 kHz calls and non-play fighting.

    “A previous study observed that 22 kHz vocalizations occurred after the first bite during resident-intruder scenarios that resulted in submission, suggesting an association between 22 kHz calls and non-play fighting.32”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [32] Panksepp J, Burgdorf J, Beinfeld MC, Kroes RA, Moskal JR. 2004. Regional brain cholecystokinin changes as a function of friendly and aggressive social interactions in rats. Brain Res 1025:75–84. 10.1016/j.brainres.2004.07.076. [DOI] [PubMed] [Google Scholar] source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § Discussion

    Play-fighting or rough and tumble play is more closely associated with the emittance of 50 kHz calls.

    “Conversely, play-fighting or “rough and tumble play” is more closely associated with the emittance of 50 kHz calls.5”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [5] Brenes JC, Lackinger M, Höglinger GU, Schratt G, Schwarting RKW, Wöhr M. 2016. Differential effects of social and physical environmental enrichment on brain plasticity, cognition, and ultrasonic communication in rats. J Comp Neurol 524:1586–1607. 10.1002/cne.23842. [DOI] [PubMed] [Google Scholar] source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § Discussion

    As rats age, play fighting becomes less common.

    “As rats age, play fighting becomes less common.35”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [35] Pellis SM, Pellis VC. 1992. Juvenilized play fighting in subordinate male rats. Aggress Behav 18:449–457. 10.1002/1098-2337(1992)18:6<449::AID-AB2480180607>3.0.CO;2-T. [DOI] [Google Scholar] source

  • May combine two claims.

    Interpretationown data
    § Discussion

    The increase in corticosterone, more numerous 22 kHz calls, and reduction in 50 kHz calls in enriched cages suggest that enriched cages may compromise welfare.

    “Although male rats may be able to stand upright in the enriched cages we used, the increase in corticosterone, more numerous 22 kHz calls, and reduction in 50 kHz calls in the enriched cages suggest that they may compromise welfare.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data
    § Discussion

    Fecal corticosterone was potentially persistently elevated throughout the study, as USV content suggested increased negative affect and decreased positive affect in the enriched condition.

    “although the fecal corticosterone was potentially persistently elevated throughout the study, as USV content, which was assessed every 3 d throughout the study, suggested increased negative affect and decreased positive affect in the enriched condition.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationcitation
    § Discussion

    Different housing conditions may be at least partially responsible for the inability to replicate findings across laboratories.

    “Different housing conditions may be at least partially responsible for the inability to replicate findings across laboratories.15”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [15] Burke DA, Magnuson DSK, Nunn CD, Fentress KG, Wilson ML, Shum-Siu AH, Moore MC, Turner LE, King WW, Onifer SM. 2007. Use of environmentally enriched housing for rats with spinal cord injury: the need for standardization. J Am Assoc Lab Anim Sci 46:34–41. [PubMed] [Google Scholar]

  • May combine two claims.

    Premisecitation
    § Discussion

    Neurologic damage can be potentiated by an elevation in glucocorticoids, potentially complicating comparisons across labs.

    “Neurologic damage can be potentiated by an elevation in glucocorticoids,40 potentially complicating comparisons across labs.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [40] Sapolsky RM. 1996. Stress, glucocorticoids, and damage to the nervous system: the current state of confusion. Stress 1:1–19. 10.3109/10253899609001092 [DOI] [PubMed] [Google Scholar] source

  • Classified as probably not a substantial claim.

    Premisecitation
    § Discussion

    Prolonged elevation in corticosterone levels is implicated in damage to the central nervous system, particularly the hippocampus.

    “Prolonged elevation in corticosterone levels is implicated in damage to the central nervous system, particularly the hippocampus.40”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [40] Sapolsky RM. 1996. Stress, glucocorticoids, and damage to the nervous system: the current state of confusion. Stress 1:1–19. 10.3109/10253899609001092 [DOI] [PubMed] [Google Scholar] source
31 of 31

  • The authors state this as an assumption.

    Differences between the groups would most likely occur at the latest observation point in the study.

    “We assumed that differences between the groups would most likely occur at the latest observation point in the study.”

  • The paper relies on this without support.

    Longer upright standing in enriched cages indicates improved ethologically relevant welfare.

    “Rats in enriched cages stood upright for longer durations as compared with rats in standard housing, suggesting improved ethologically relevant welfare.”
2 of 2

Statistics that don’t add up

Each reported p-value recomputed from its test statistic and degrees of freedom.

  • Inconsistent p-value: F (1, 6) = 34.7, p = .00110

    Minor

    The paper reports p = .0011 here; recomputing from the test statistic and its degrees of freedom gives p = .001. The check cannot tell whether the statistic or the p-value is the misprint.

    Reported p = .0011 · recomputed p = .001

1 of 1

Statistics recomputed

Every reported test result, recomputed from its test statistic and degrees of freedom. A result shows as not adding up only once an independent check has confirmed it.

  • Doesn’t add upF(1, 6) = 34.7reported p = .0011 · recomputed p = .001

    “F (1, 6) = 34.7, p = .00110”

  • Not confirmedF(2, 12) = 13.34reported p = .0009 · recomputed p < .001

    “F (2, 12) = 13.34, p = 9.00e-4”

  • Not confirmedF(1, 6) = 28.8reported p = .0017 · recomputed p = .002

    “F (1, 6) = 28.8, p = .00170”

  • ConsistentF(1, 6) = 5.75reported p = .0535 · recomputed p = .053

    “F (1, 6) = 5.75, p = .0535”

  • ConsistentF(6, 36) = 3.31reported p = .0107 · recomputed p = .011

    “F (6, 36) = 3.31, p = .0107”

  • ConsistentF(6, 36) = 3.27reported p = .0115 · recomputed p = .011

    “F (6, 36) = 3.27, p = .0115”

  • Consistentt(6) = 2.66reported p = .0378 · recomputed p = .038

    “t (6) = 2.66, p = .0378”

  • ConsistentF(1, 6) = 9.84reported p = .0202 · recomputed p = .020

    “F (1, 6) = 9.84, p = .0202”

  • ConsistentF(6, 36) = 0.767reported p = .601 · recomputed p = .601

    “F (6, 36) = 0.767, p = .601”

  • ConsistentF(6, 36) = 0.69reported p = .659 · recomputed p = .659

    “F (6, 36) = 0.690, p = .659”

  • ConsistentF(1, 6) = 5reported p = .0667 · recomputed p = .067

    “F (1, 6) = 5.00, p = .0667”

  • ConsistentF(2, 12) = 1.5reported p = .262 · recomputed p = .262

    “F (2, 12) = 1.50, p = .262”

  • ConsistentF(2, 12) = 3.5reported p = .0635 · recomputed p = .063

    “F (2, 12) = 3.50, p = .0635”

  • ConsistentF(1, 6) = 4.99reported p = .0669 · recomputed p = .067

    “F (1, 6) = 4.99, p = .0669”

  • ConsistentF(2, 12) = 2reported p = .178 · recomputed p = .178

    “F (2, 12) = 2.00, p = .178”

  • ConsistentF(2, 12) = 3.42reported p = .0668 · recomputed p = .067

    “F (2, 12) = 3.42, p = .0668”

  • ConsistentF(2, 12) = 2.42reported p = .131 · recomputed p = .131

    “F (2, 12) = 2.42, p = .131”

  • ConsistentF(2, 12) = 4.74reported p = .0304 · recomputed p = .030

    “F (2, 12) = 4.74, p = .0304”

  • ConsistentF(1, 6) = 0.599reported p = .468 · recomputed p = .468

    “F (1, 6) = 0.599, p = .468”

  • ConsistentF(2, 12) = 0.429reported p = .661 · recomputed p = .661

    “F (2, 12) = 0.429, p = .661”

  • ConsistentF(2, 12) = 2.22reported p = .151 · recomputed p = .151

    “F (2, 12) = 2.22, p = .151”

  • ConsistentF(1, 6) = 17.4reported p = .0059 · recomputed p = .006

    “F (1, 6) = 17.4, p = .00590”

  • ConsistentF(2, 12) = 10.8reported p = .002 · recomputed p = .002

    “F (2, 12) = 10.8, p = .0020”

  • ConsistentF(1, 6) = 6.5reported p = .0435 · recomputed p = .044

    “F (1, 6) = 6.50, p = .0435”

  • ConsistentF(2, 12) = 3.3reported p = .0723 · recomputed p = .072

    “F (2, 12) = 3.30, p = .0723”

  • ConsistentF(2, 12) = 3.49reported p = .0638 · recomputed p = .064

    “F (2, 12) = 3.49, p = .0638”

  • ConsistentF(1, 6) = 1.43reported p = .276 · recomputed p = .277

    “F (1, 6) = 1.43, p = .276”

  • ConsistentF(2, 12) = 2.24reported p = .149 · recomputed p = .149

    “F (2, 12) = 2.24, p = .149”

  • ConsistentF(2, 12) = 1.43reported p = .277 · recomputed p = .277

    “F (2, 12) = 1.43, p = .277”

  • ConsistentF(1, 6) = 6.12reported p = .0482 · recomputed p = .048

    “F (1, 6) = 6.12, p = .0482”

  • ConsistentF(2, 12) = 2.04reported p = .173 · recomputed p = .173

    “F (2, 12) = 2.04, p = .173”

  • ConsistentF(2, 12) = 2.5reported p = .124 · recomputed p = .124

    “F (2, 12) = 2.50, p = .124”

  • ConsistentF(1, 6) = 5.03reported p = .066 · recomputed p = .066

    “F (1, 6) = 5.03, p = .0660”

  • ConsistentF(2, 12) = 0.767reported p = .486 · recomputed p = .486

    “F (2, 12) = 0.767, p = .486”

  • ConsistentF(2, 12) = 0.775reported p = .483 · recomputed p = .482

    “F (2, 12) = 0.775, p = .483”

  • ConsistentF(1, 6) = 5.79reported p = .0528 · recomputed p = .053

    “F (1, 6) = 5.79, p = .0528”

  • ConsistentF(2, 12) = 0.234reported p = .795 · recomputed p = .795

    “F (2, 12) = 0.234, p = .795”

  • ConsistentF(1, 6) = 1.76reported p = .233 · recomputed p = .233

    “F (1, 6) = 1.76, p = .233”

  • ConsistentF(2, 12) = 0.988reported p = .401 · recomputed p = .401

    “F (2, 12) = 0.988, p = .401”

  • ConsistentF(2, 12) = 1.02reported p = .391 · recomputed p = .390

    “F (2, 12) = 1.02, p = .391”

  • ConsistentF(1, 6) = 3.6reported p = .107 · recomputed p = .107

    “F (1, 6) = 3.60, p = .107”

  • ConsistentF(2, 12) = 0.583reported p = .573 · recomputed p = .573

    “F (2, 12) = 0.583, p = .573”

  • ConsistentF(2, 12) = 0.25reported p = .783 · recomputed p = .783

    “F (2, 12) = 0.250, p = .783”

  • ConsistentF(1, 6) = 7.89reported p = .0308 · recomputed p = .031

    “F (1, 6) = 7.89, p = .0308”

  • ConsistentF(2, 12) = 3.31reported p = .0719 · recomputed p = .072

    “F (2, 12) = 3.31, p = .0719”

  • ConsistentF(1, 6) = 0.6reported p = .468 · recomputed p = .468

    “F (1, 6) = 0.600, p = .468”

  • ConsistentF(2, 12) = 0.857reported p = .449 · recomputed p = .449

    “F (2, 12) = 0.857, p = .449”

  • ConsistentF(1, 6) = 5.13reported p = .0642 · recomputed p = .064

    “F (1, 6) = 5.13, p = .0642”

  • ConsistentF(2, 12) = 0.386reported p = .688 · recomputed p = .688

    “F (2, 12) = 0.386, p = .688”

  • ConsistentF(2, 12) = 0.144reported p = .867 · recomputed p = .867

    “F (2, 12) = 0.144, p = .867”

  • ConsistentF(1, 6) = 1.28reported p = .301 · recomputed p = .301

    “F (1, 6) = 1.28, p = .301”

  • ConsistentF(2, 12) = 3.19reported p = .0777 · recomputed p = .077

    “F (2, 12) = 3.19, p = .0777”

  • ConsistentF(2, 12) = 5.41reported p = .0212 · recomputed p = .021

    “F (2, 12) = 5.41, p = .0212”

  • Consistentt(6) = 2.13reported p = .0775 · recomputed p = .077

    “t (6) = 2.13, p = .0775”

  • ConsistentF(1, 6) = 23.3reported p = .0029 · recomputed p = .003

    “F (1,6) = 23.3, p = .00290”

  • Consistentt(11) = 3.7reported p = .0035 · recomputed p = .004

    “t (11) = 3.70, p = .00350”

  • ConsistentF(5, 30) = 0.592reported p = .706 · recomputed p = .706

    “F (5, 30) = 0.592, p = .706”

  • ConsistentF(5, 30) = 0.496reported p = .777 · recomputed p = .777

    “F (5, 30) = 0.496, p = .777”

  • ConsistentF(5, 30) = 1.15reported p = .358 · recomputed p = .356

    “F (5, 30) = 1.15, p = .358”

  • ConsistentF(5, 30) = 0.911reported p = .487 · recomputed p = .487

    “F (5, 30) = 0.911, p = .487”

60 of 60

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Fighting described as elevated despite a nonsignificant comparison

    Minor

    The paper reports no detected difference in fighting duration (P = 0.0775), but later presents higher fighting in enriched cages as an observed effect. The direction of the measured difference may be higher, but the reported test does not support a detected increase.

  • Fecal corticosterone sample counts do not match t-test degrees of freedom

    Minor

    The stated 7 enriched and 8 standard samples give 13 degrees of freedom for the specified unpaired two-sample Student t test, not the reported 11. The reported degrees of freedom imply that only 13 samples were included.

2 of 2

Cites

Flagged first, then by citations

Research works printed but not linked: 4

  1. J Am Assoc Lab Anim Sci, 47, 30
  2. Lab Anim, 36, 28
  3. Open J Soc Sci, 04, 93
  4. Lab Anim, 39, 45

Cited by

Sorted by citations

Authors
Bigelow, L. J., Cohen, A. J., Pimm, R., Knight, J. B., & Bernard, P. B.
Affiliations
University of Prince Edward Island (CA)
Venue
Journal of the American Association for Laboratory Animal Science
Publisher
American Association for Laboratory Animal Science
Published
2022-03-01
Volume
61
Issue
2
Pages
140-148
ISSN
1559-6109 (ISSN-L)
DOI
10.30802/aalas-jaalas-21-000024
PMC
PMC8956216
OpenAlex
W4210310168
Semantic Scholar
6f8683956ee0f4d9af523c8fa9160ae255d63f01
Cited by
9
References
7
Citations by year
2026: 2 · 2025: 5 · 2024: 1 · 2022: 1
FWCI
1.70
Type
journal article
Language
en
Open access
Locations
  • Journal of the American Association for Laboratory Animal Science (publishedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (submittedVersion)
Topics
  • Health Sciences › Veterinary › Small Animals › Animal Behavior and Welfare Studies
  • Health Sciences › Veterinary › Small Animals › Animal testing and alternatives
  • Health Sciences › Veterinary › Small Animals › Veterinary Pharmacology and Anesthesia
Keywords
ultrasonic vocalizations, environmental enrichment, animal welfare, fecal corticosterone, 50-kHz calls, standing, fighting behavior, animal stress, home cage behavior, housing standardization
MeSH terms
Animals, Animal Welfare, Animals, Laboratory, Behavior, Animal, Housing, Animal, Humans, Ultrasonics, Vocalization, Animal, Rats

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-12.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-12.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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