Stage 2 Registered Report: Refinement of tickling protocols to improve positive animal welfare in laboratory rats

May combine two claims.

By the 5th session, all PH rats showed an increase in USV production in contrast to the standard protocol where some rats show little or no increase in USVs.

“By the 5th session, all PH rats showed an increase in USV production in contrast to Figure 2(ii), which used the standard protocol and where some rats show little or no increase in USVs.”

• cites Bombail et al., 2019 sourcePartly supported

  The Results (page not available) report higher average 50 kHz USV production in the fifth than the first tickling session and note individual differences, but do not establish that some rats had little or no increase. The General discussion (page not available) explicitly says the authors did not standardise their tickling method, so this paper does not support describing its procedure as the standard protocol. The statement about all PH rats appears to describe the citing study’s own results, not a finding attributed to this source. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

May combine two claims.

When rats are tickled by the human hand, they often produce 50 kHz USVs, which are understood to indicate positive affect in rats.

“Research (see Bombail et al., 2021; LaFollette et al., 2017 for reviews) has shown that when rats are tickled by the human hand, they often produce 50 kHz USVs, which are understood to indicate positive affect in rats ( e.g., Barker, 2018, Figure 2i).”

• cites Bombail et al., 2021 sourceNot checked — no full text of the source
• cites LaFollette et al., 2017 sourceSupported
• cites Barker, 2018 sourceNot checked — no full text of the source

May combine two claims.

Rats find tickling rewarding, as tickled rats show reduced latency for approaching the human hand and can be trained to perform operant responses for tickling.

“Other evidence suggests that rats find tickling rewarding as in addition to production of USVs, tickled rats show a reduced latency for approaching the human hand and can be trained to perform operant responses for tickling ( Burgdorf & Panksepp, 2001; LaFollette et al., 2017).”

• cites Burgdorf & Panksepp, 2001 sourceNot checked — no full text of the source
• cites LaFollette et al., 2017 sourcePartly supported

  The Results section (‘Approach behavior’; page unavailable) reports that nine of ten experiments assessing approach found positive results, including decreased approach latency. The review also discusses tickling’s reward value, but the passages in our copy do not report rats being trained to perform operant responses for tickling. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

May combine two claims.

USVs in response to tickling are correlated with an independent test of affective state (an affective bias test), indicating that rats vary significantly in their affective response to tickling.

“We have shown that USVs in response to tickling are correlated with an independent test of affective state (an affective bias test) indicating that rats vary significantly in their affective response to tickling ( Hinchcliffe et al., 2020).”

• cites Hinchcliffe et al., 2020 sourceSupported

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

Rat play is boisterous.

“In both PH and standard tickling, movements should reflect that rat play is boisterous ( Cloutier et al., 2018) with finger pressure being firm and movements fast.”

• cites Cloutier et al., 2018 sourceSupported

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

Female rats produce more USVs than males in response to tickling.

“We have also recently found evidence of sex differences with female rats producing more USVs than males in response to tickling ( Tivey et al., 2022).”

• cites Tivey et al., 2022 sourcePartly supported

  The Discussion and Conclusions (page not specified) report that tickled female rats produced more flat 50 kHz USVs associated with play-related behaviours than males. The claim omits those subtype and behavioural qualifiers, implying that females produced more USVs overall; the paper does not establish that broader comparison. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

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

The position of the home cages in the rack was randomised across treatments.

“Due to the varying lux levels and other potentially confounding variables, position of the home cages in the rack was randomised across treatments (see Hammond et al., 2019).”

• cites Hammond et al., 2019 sourceCould not verify

  The passages retrieved from our copy do not address where cages were placed in the rack or whether their positions were randomised across treatments. Searches for cage-rack position, randomisation, and lux levels found no Methods passage that could verify the claim. (LLM, conf=0.70)

  Experimental — accuracy not yet measured

Classified as probably not a substantial claim.

The PH protocol is intended to better mimic the dynamic nature of play, giving the rat more choice over how it is tickled.

“Due to our experiences using the standard tickling protocol we have developed a refined tickling protocol we refer to as playful handling (PH), intended to better mimic the dynamic nature of play, giving the rat more choice over how it is tickled ( Bombail et al., 2019), Figure 1(iii).”

• cites Bombail et al., 2019 sourceSupported

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

Burke et al. (2022) suggested that for male rats being pinned during tickling may be important for production of USVs in comparison to social play where de-vocalisation studies show that it is typically the rat doing the pinning that emits these calls.

“Burke et al. (2022) suggested that for male rats being pinned during tickling may be important for production of USVs in comparison to social play where de-vocalisation studies show that it is typically the rat doing the pinning that emits these calls ( Burke et al., 2018).”

• cites Burke et al., 2022 sourcePartly supported

  Split into 2 atoms — 1 confirmed, 0 partially supported, 0 disputed, 0 misattributed, 0 unverifiable, 1 not checked. Expand the claim to see per-atom verdicts.

  Experimental — accuracy not yet measured
• cites Burke et al., 2018 sourceNot checked — no full text of the source

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

Another distinctive difference between social play and tickling in the Burke et al. (2022) study were the correlations between behaviours and sub-types of USVs.

“Another distinctive difference between social play and tickling in the Burke et al., (2022) study were the correlations between behaviours and sub-types of USVs.”

• cites Burke et al., (2022) sourceSupported

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

The literature suggests that individual rats vary consistently in their USV production although most studies use adult male rats.

“Generally, the literature suggests that individual rats vary consistently in their USV production although most studies use adult male rats (e.g., Ahrens et al., 2013; Sundarakrishnan & Clarke, 2022).”

• cites Ahrens et al., 2013 sourceSupported
• cites Sundarakrishnan & Clarke, 2022 sourcePartly supported

  The Introduction (page unavailable) reports temporally stable individual differences in 50-kHz calling, and the Results describe such differences in this study. The Subjects and Strengths and limitations sections say this study used adult male rats, but our copy does not establish that most studies used adult males. (LLM, conf=0.83)

  Experimental — accuracy not yet measured

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

Burke et al. (2022) report that in social play, the strongest associations between behaviour and USVs involve frequency modulated (trill) calls, whereas in tickling the strongest associations involve flat calls.

“Burke et al. (2022) report that in social play, the strongest associations between behaviour and USVs involve frequency modulated (trill) calls, whereas in tickling the strongest associations involve flat calls.”

• cites Burke et al. (2022) sourceSupported

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

The sex effects in the OFT are in agreement with recent findings on sex differences in the OFT.

“The sex effects observed in the OFT were indicative of lower anxiety in females, and this is in agreement with recent findings on sex differences in the OFT ( Knight et al., 2021).”

• cites Knight et al., 2021 sourceSupported

May combine two claims.

In male rats the strongest associations between USVs and behaviour in social play are between approaching, following and nape contact, whereas with tickling the strongest association is with pinning.

“In male rats the strongest associations between USVs and behaviour in social play are between approaching, following and nape contact whereas with tickling the strongest association is with pinning ( Burke et al., 2022; they applied the standard tickling protocol).”

• cites Burke et al., 2022 sourceSupported

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

Sex differences in FCM in rodents are already relatively well established.

“Sex differences in FCM in rodents are already relatively well established ( Cavigelli et al., 2005).”

• cites Cavigelli et al., 2005 sourcePartly supported

  The Sex differences section reports significantly higher fecal corticoid measures in male than female Sprague-Dawley rats. This establishes a sex difference in the rats studied, but the paper does not establish that such differences are well established across rodents generally. (LLM, conf=0.83)

  Experimental — accuracy not yet measured

May combine two claims.

Tivey et al. (2022) found that after 10 days of playful handling, females produced more USVs than males with an indication that female controls also increased calling relative to male controls.

“Tivey et al., (2022) also found that after 10 days of playful handling, females produced more USVs than males with an indication that female controls also increased calling relative to male controls.”

• cites Tivey et al., (2022) sourcePartly supported

  The Methods describe ten days of testing, and the Results (USV production associated with play-related behaviours; page unavailable) report more flat 50 kHz USVs in females than males around certain play behaviours. They also report more flat calls from female than male controls in the second before hopping. The claim omits those subtype and behaviour limits: the paper does not establish that females produced more USVs overall or that female controls’ calling increased over time. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

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

Burke et al. (2022) propose that the lack of correlation between trills and specific tickling behaviour indicates that trills are produced relatively indiscriminately during tickling, potentially due to the lack of a conspecific play partner.

“Burke et al. (2022) propose that the lack of correlation between trills and specific tickling behaviour indicates that trills are produced relatively indiscriminately during tickling, potentially due to the lack of a conspecific play partner.”

• cites Burke et al. (2022) sourcePartly supported

  Split into 2 atoms — 1 confirmed, 0 partially supported, 0 disputed, 0 misattributed, 0 unverifiable, 1 not checked. Expand the claim to see per-atom verdicts.

  Experimental — accuracy not yet measured

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

Tivey et al. (2022) found that playfully handled and control females produced more flat calls associated with play behaviours, performed during the playful handling sessions, than did males; in contrast both sexes produced trills in response to playful handling.

“Tivey et al., (2022) found that playfully handled and control females produced more flat calls associated with play behaviours, performed during the playful handling sessions, than did males; in contrast both sexes produced trills in response to playful handling.”

• cites Tivey et al., (2022) sourcePartly supported

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

  Experimental — accuracy not yet measured

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

Of the 4 cytokines with treatment effects, IL5 and IL12 are classed as pro-inflammatory and IL10 and IL13 as anti-inflammatory.

“Of the 4 cytokines with treatment effects, IL5 and IL12 are classed as pro-inflammatory ( Pelaia et al., 2019; Trinchieri, 1995) and IL10 and IL13 as anti-inflammatory ( Iyer & Cheng, 2012; Kolosowska et al., 2019).”

• cites Pelaia et al., 2019 sourceSupported
• cites Trinchieri, 1995 sourceNot checked — no full text of the source
• cites Iyer & Cheng, 2012 sourceSupported

The quote only partly states this claim.

There are sex differences in response to tickling.

“We report the novel finding of sex differences in response to tickling.”

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

In the dataset, 22 kHz USVs were too rare to analyse.

“22 kHz analysis: In our dataset, these events were too rare to analyse.”

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

Solitary play behaviour occurred too rarely to analyse.

“Anticipatory behaviour in the tickling arena, in the minute preceding treatment administration, was analysed (solitary play behaviour occurred too rarely to analyse).”

May combine two claims.

50 kHz USVs were used as a measure of positive affect and were shown to be responsive to treatments.

“50 kHz ultrasonic vocalisations (USVs) used as a measure of positive affect and shown to be responsive to treatments.”

May combine two claims.

In males, controls produced fewer 50 kHz USVs than P0 (T=-4.71, p=0.001), P1 (T=-6.59, p<0.001) and P4 males (T=-6.05, p<0.001).

“In males, controls produced fewer 50 kHz USVs than P0 (T = -4.71, p = 0.001), P1 (T = -6.59, p < 0.001) and P4 males (T = -6.05, p <0.001”

May combine two claims.

The % change in FCM over the treatment phase (TP1 to TP2) differed significantly by sex (F1,113=6.54, p=0.012) but not treatment.

“The % change in FCM over the treatment phase (TP1 to TP2) differed significantly by sex (F 1,113= 6.54, p = 0.012) but not treatment”

May combine two claims.

Within-individual variance in USVs was higher in the P0 treatment compared to the Control (mean difference=0.105, 95% CI 0.017-0.195) and P4 (mean difference=0.123, 95% CI 0.039-0.209) treatments.

“Notably, within-individual variance was higher in the P0 treatment compared to the Control (mean difference = 0.105, 95 % CI 0.017-0.195) and P4 (mean difference = 0.123, 95 % CI 0.039-0.209) treatments”

May combine two claims.

22-kHz USVs were observed during early habituation for some individuals, but no 22-kHz USVs were observed during the treatment phase.

“22-kHz USVs were observed during early habituation for some individuals, however no 22-kHz USV were observed during the treatment phase.”

May combine two claims.

There were no differences in repeatability of USVs between treatments, as the credible intervals for repeatability differences included zero.

“However, there were no differences in repeatability between treatments, as the credible intervals for repeatability differences included zero.”

May combine two claims.

In the model including day, additional two-way interactions of sex*day (F5,599.05=16.78, p<0.001) and treatment*day (F15,599.05=2.42, p=0.002) were observed.

“and additional two-way interactions of sex * day (F 5,599.05 = 16.78, p < 0.001) and treatment * day (F 15,599.05 = 2.42, p = 0.002) were observed.”

The quote only partly states this claim.

No effects of treatment were observed on behavioural transitions during the anticipation phase.

“No effects of treatment were observed.”

May combine two claims.

Including day in the model strengthened the fit, with R-sq adj = 38% without day and 83% with day.

“without day R-sq adj = 38%, with day R-sq adj = 83%”

May combine two claims.

Comparisons between treatments did not reveal significant differences in among-individual variance in 50 kHz USVs, as all 95% credible intervals for the differences included zero.

“Comparisons between treatments did not reveal significant differences in among-individual variance, as all 95 % credible intervals for the differences included zero.”

May combine two claims.

Weak negative correlations were observed between USV production on the final day of treatment and % distance travelled in the outer zone (r2=-0.334) and % time in outer zone (r2=-0.362).

“Weak negative correlations were observed between USV production on the final day of treatment and % distance travelled in the outer zone (r 2 = -0.334) and % time in outer zone (r 2 = -0.362).”

May combine two claims.

Delta frequency increased over days, with significant differences observed between Days 1 and 3 (T=6.68, p<0.001) and Days 3 and 6 (T=3.49, p=0.002).

“Delta frequency increased over days, with significant differences observed between Days 1 and 3 (T = 6.68, p < 0.001) and Days 3 and 6 (T = 3.49, p = 0.002).”

May combine two claims.

Sinuosity was higher in P1 and P0 than in P4 and control, and at Day 6 compared to Day 1.

“Differences determined by non-overlapping confidence intervals indicate sinuosity to be higher in P1 and P0 than in P4 and control and at Day 6 compared to Day 1.”

May combine two claims.

Females in the P1 group produced significantly more 50 kHz USVs than those in the P4 (T=2.19, p=0.03) and control groups (T=3.67, p=0.016), but not more than those in the P0 group.

“Females in the P1 group produced significantly more 50 kHz USVs than those in the P4 (T = 2.19, p = 0.03) and control groups (T = 3.67, p = 0.016), but not more than those in the P0 group.”

May combine two claims.

Rats in the P1 treatment exhibited more fluctuations in USVs over time compared to the P4 treatment (mean difference=0.073, 95% CI 0.002-0.138), although the 2.5% credible interval is close to zero, indicating a marginal difference.

“Similarly, rats in the P1 treatment also exhibited more fluctuations in USVs over time compared to the P4 (mean difference = 0.073, 95 % CI 0.002-0.138) treatment, although the 2.5 % credible interval is close to zero, indicating a marginal difference.”

May combine two claims.

Female controls, P0 and P1 all produced more 50 kHz USVs during treatment than their male counterparts within the same treatment (Control, t=9.14, p<0.001; P0, t=5.57, p<0.001; P1, t=3.27, p=0.024).

“Female controls, P0 and P1 all produced more 50 kHz USVs during treatment than their male counterparts within the same treatment (Control, t = 9.14, p < 0.001; P0, t = 5.57, p < 0.001; P1, t = 3.27, p = 0.024).”

May combine two claims.

Males and females did not differ in their 50 kHz USV production on Days 1 (T=0.44, p=1.00) and 2 (T=2.35, p=0.45).

“Males and females did not differ in their 50 kHz USV production on Days 1 (T = 0.44, p = 1.00) and 2 (T = 2.35, p = 0.45; Figure 5).”

May combine two claims.

Comparisons between treatments did not reveal significant differences in among-individual variance in FCM, as all 95% credible intervals for the differences included zero.

“Comparisons between treatments did not reveal significant differences in among-individual variance, as all 95 % credible intervals for the differences included zero.”

May combine two claims.

A primary analysis not including day indicated significant main effects of sex (F1,113=20.26, p<0.001) and treatment (F3,113=19.49, p<0.001) on mean 50-kHz USV production.

“A primary analysis not including day as a factor indicated significant main effects of sex (F 1,113 = 20.26, p < 0.001) and treatment (F 3,113 = 19.49, p < 0.001) on mean 50-kHz USV production”

May combine two claims.

On Days 5 and 6, female rats did not differ and produced more 50 kHz USVs than all days and groups bar female rats on Day 4.

“On Days 5 and 6, female rats did not differ and produced more 50 kHz USVs than all days and groups bar female rats on Day 4.”

May combine two claims.

Across all four treatments, rats did not exhibit among-individual variance in FCM, indicating that the average amount of FCM excreted did not vary across individuals in any treatment groups.

“Across all four treatments, rats did not exhibit among-individual variance in FCM, indicating that the average amount of FCM excreted did not vary across individuals in any treatment groups ( Table 3).”

Probably not a substantial claim: may not pass S1 (asserted), S3 (load-bearing), S4 (based).

No effects of treatment were observed on FCM concentration.

“No effects of treatment were observed.”

May combine two claims.

No statistically significant effect of cohort or cage was observed on 50 kHz USV production.

“No statistically significant effect of cohort or cage was observed on 50 kHz USV production.”

May combine two claims.

Female anticipatory USVs increased over time, while male anticipatory USVs did not.

“Female anticipatory USVs increased over time, while male anticipatory USVs did not ( Figure 6 panel A).”

May combine two claims.

50 kHz USV production increased over the treatment phase in a dose dependent manner that differed by sex.

“50 kHz USV production increased over the treatment phase in a dose dependent manner that differed by sex ( Figure 5).”

May combine two claims.

Tickled individuals produced more 50 kHz USVs than controls (p<0.001), with the highest number produced by the P1 group.

“tickled individuals produced more 50 kHz USVs than controls (p < 0.001), with the highest number of USVs being produced by the P1 group.”

May combine two claims.

50 kHz USV production continued to rise in females to Day 5, while males reached a plateau at Day 3.

“50 kHz USV production continued to rise in females to Day 5, while males reached a plateau at Day 3.”

May combine two claims.

Transitions were lower on day 1 than on days 3 (T=-6.02, p<0.001) and 6 (T=-4.75, p<0.001), primarily driven by the increase in transitions observed in females on days 3 and 6 through the interaction of sex*day.

“Transitions were lower on day 1 than on days 3 (T = -6.02, p < 0.001) and 6 (T = -4.75, p < 0.001), primarily driven by the increase in transitions observed in females on days 3 and 6 through the interaction of sex * day ( Figure 7).”

May combine two claims.

At TP1, no differences were found between sexes or allocated treatment groups in FCM.

“At TP1, no differences were found between sexes or allocated treatment groups ( Figure 8, Panel A).”

May combine two claims.

For IL10, the order of mean concentrations was Control < P1< P4 < P0, with differences derived from post-hoc comparisons occurring between Control and P4 (T=2.82, p=0.028), and Control and P0 (T=3.23, p=0.009).

“For IL 10, the order of mean concentrations was Control < P1< P4 < P0, with the differences derived from post-hoc comparisons occurring between Control and P4 (T = 2.82, p = 0.028), and Control and P0 (T = 3.23, p = 0.009).”

May combine two claims.

For IL5, the order of median concentrations was Control < P1 < P4 < P0 with differences derived from non-overlapping confidence intervals occurring between control and P4 individuals.

“For IL5, the order of median concentrations was Control < P1 < P4 < P0 with the differences derived from non-overlapping confidence intervals occurring between control and P4 individuals.”

May combine two claims.

For IL12, the order of mean concentrations was Control < P1 < P4 < P0, with differences derived from post-hoc comparisons occurring between control and P0 (T=2.76, p=0.034).

“For IL12, the order of mean concentrations was Control < P1 < P4 < P0, with the differences derived from post-hoc comparisons occurring between control and P0 (T = 2.76, p = 0.034).”

May combine two claims.

For IL13, the order of median concentrations was Control < P1 < P0 < P4 with differences derived from non-overlapping confidence intervals occurring between control and P0 individuals.

“For IL13, the order of median concentrations was Control < P1 < P0 < P4 with the differences derived from non-overlapping confidence intervals occurring between control and P0 individuals.”

May combine two claims.

There was a significant sex by treatment interaction on total 50 kHz USVs, indicating that females were responsible for the effect.

“However, a significant sex by treatment interaction indicates that it was females who were responsible for this effect.”

May combine two claims.

No correlations were observed between any cytokines tested and faecal corticosteroid metabolites, or total USV production.

“No correlations were observed between any cytokines tested and faecal corticosteroid metabolites, or total USV production.”

May combine two claims.

Although in males the tickling treatments did not differ significantly, the one pin per session treatment was the numerically highest treatment and significantly different from controls in terms of USVs.

“Although in males the tickling treatments did not differ significantly, the one pin per session treatment was the numerically highest treatment and significantly different from controls in terms of USVs.”

May combine two claims.

Control animals had consistently the highest concentrations of cytokines, followed by P1, with P0 and P4 alternating with having the lowest concentrations.

“We found treatment effects on 4 out of 12 cytokines, with control animals having consistently the highest concentrations followed by P1 with P0 and P4 alternating with having the lowest concentrations.”

Classified as probably not a substantial claim.

The experimental design was a pseudo randomised control trial where every week each of the eight cages of two male or two female rats was assigned to one of four treatments (control, P0, P1, P4).

“Our experimental design was a pseudo randomised control trial where every week, each of the eight cages of two male or two female rats would be assigned to one of four treatments (control, P0, P1, P4).”

May combine two claims.

Females preferred tickling where the amount of pinning is minimised relative to males that showed no preference across tickling treatments.

“Females preferred tickling where the amount of pinning is minimised relative to males that showed no preference across tickling treatments.”

• Also stated in: § Conclusions · boosted

  “Demonstration of the benefits of considering sex differences where the objective is to improve welfare with female juvenile Wistar rats preferring tickling with less pinning relative to males.”

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

All animal work was carried out in accordance with the U.K. Animals (Scientific Procedures) Act 1986 and reported in compliance with the ARRIVE guidelines.

“All animal work was carried out at the Roslin Institute, Edinburgh, U.K., in accordance with the U.K. Animals (Scientific Procedures) Act 1986 and reported in compliance with the ARRIVE guidelines ( Bombail, 2022).”

• cites Bombail, 2022 sourceNot checked — no full text of the source

May combine two claims.

This work did not involve aversive procedures and was not legally required to have UK Home Office approval.

“This work did not involve aversive procedures (and is deemed less harmful and stressful than skilled insertion of a hypodermic needle according to good veterinary practice) and therefore not legally required to have UK Home Office approval.”

May combine two claims.

The work was confirmed subthreshold to regulation under the U.K. Animals (Scientific Procedures) Act 1986 and approved by the University of Edinburgh's Animal Welfare and Ethical Review Body.

“Following consideration by the University of Edinburgh’s Animal Welfare and Ethical Review Body (reference NC/W001209/1, 26 July 2022, Establishment number X212DDDBD), it was confirmed the work was subthreshold to regulation under the U.K. Animals (Scientific Procedures) Act 1986, and the committee approved it as detailed in this paper.”

May combine two claims.

Rat tickling is proposed as an effective and practical approach to improve laboratory rat welfare.

“For laboratory rats, a positive handling approach referred to as rat tickling is proposed as an effective and practical approach to improve their welfare ( https://www.nc3rs.org.uk/rat-tickling ; https://na3rsc.org/rodent-handling/).”

• cites https://www.nc3rs.org.uk/rat-ticklingNot matched

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

• cites https://na3rsc.org/rodent-handling/Not matched

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

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

The study was approved by the Veterinary Ethical Review Committee of the Royal (Dick) School of Veterinary Studies, University of Edinburgh.

“This study was also approved by the Veterinary Ethical Review Committee of the Royal (Dick) School of Veterinary Studies, University of Edinburgh (reference 92.22, 2 September 2022).”

Classified as probably not a substantial claim.

The cage was fitted as a random effect for all treatments and both sexes.

“Therefore we fitted the cage as a random effect for all treatments and both sexes.”

• AssumptionRests on an assumption: Within a cage, individuals cannot be considered independent because they might influence each other in response to the treatment through emotional contagion.

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

Due to time constraints, manual categorisation of USVs was not performed.

“Due to time constraints, we were not able to manually categorise USVs identified in this study.”

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

No animals were required to be excluded from the study for health reasons.

“No animals were required to be excluded from the study for health reasons (e.g. injury or infection).”

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

Quantitative behavioural analysis, collection of USVs, analysis of physiological samples, and collation and analysis of behavioural data were blinded.

“As individual rat’s behaviour was observed in the anticipation phase but not during the tickling sessions, we were able to blind the quantitative behavioural analysis as well as all other aspects including collection of USVs, analysis of physiological samples, and collation and analysis of behavioural data.”

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

Home cage pair behaviour was not analysed.

“Home cage pair behaviour (incorrectly refered to as anticipation in the Stage One paper) was not analysed.”

Classified as probably not a substantial claim.

Rat treatment allocation was spread evenly across all litters to prevent bias due to genetic background and early life experience.

“Rat treatment allocation was spread evenly across all litters to prevent bias due to genetic background and early life experience ( i.e., each of the four individuals from each sex was assigned to each of the four treatments).”

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

The play arena litter contained odorants from several rats, standardising, diluting and minimising the impact of individual olfactory cues.

“We used this method to guarantee the litter contained odorants from several rats, thereby standardising, diluting and minimising the impact of individual olfactory cues.”

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

Rat tickling with the human hand was developed to study positive emotions and USVs in rats by mimicking playful interactions between rats.

“Rat tickling with the human hand was developed to study positive emotions and ultrasonic vocalisations (USVs) in rats by mimicking playful interactions between rats ( Panksepp, 1998, Figure 1i).”

• cites Panksepp, 1998Not matched

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

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

Adjustments in treatment distribution were made to balance body weights evenly across treatments.

“For each sex, rat weight was averaged for each treatment, and to avoid potential bias caused by uneven distribution of body weight between treatment groups, adjustments in treatment distribution were made to balance evenly weights across treatments”

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

Rats were not excluded based on their USV production.

“We are aware of the high variability in USV response among rats, and are experienced in recording it, and therefore did not exclude rats based on their USV production.”

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

A total of 128 rats (64 males and 64 females) were used in this study, in accordance with the power analysis.

“A total of 128 rats (64 males and 64 females) were used in this study, in accordance with our power analysis detailed below.”

Classified as probably not a substantial claim.

Due to paucity of data, power calculations could not be generated for the exploratory hypothesis.

“Due to the paucity of data on the impact PH might have on the variables we will measure, we could not generate power calculations and pre-register this hypothesis, this is therefore exploratory work.”

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

Prior to analysis, the data were visualised and tested for the assumptions of the cognate statistical approaches (data distribution normality or heteroskedasticity), and when necessary, non-parametric alternatives or square root transformation were used.

“Prior to analysis, we visualised and tested the data for the assumptions of the cognate statistical approaches (data distribution normality or heteroskedasticity), and when necessary, we used non-parametric alternatives or data transformation (square root transformation).”

Classified as probably not a substantial claim.

Pairs were assigned to a treatment using a random list generator.

“Pairs were assigned to a treatment using a random list generator ( https://www.random.org/lists/).”

• cites https://www.random.org/lists/Not matched

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

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

Rat ID 13-1 (female control) failed to provide a faecal sample for timepoint 2.

“Rat ID 13-1 (Female – Control) failed to provide a sample for timepoint 2.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims. The quote only partly states this claim.

One individual (female P1) displayed sickness behaviour on the day scheduled to be killed and was excluded from analysis of measures collected on the final day; prior to that final day, the animal fell within the parameters of its own group.

“This individual was excluded from analysis of measures collected on the final day (prior to this final day, the animal fell within the parameters of its own group, suggesting there was no remarkable impact on earlier measures).”

May combine two claims.

DeepSqueak USV identification showed strong concordance with manual identification without systematic bias (mean difference -3.8 ± 15.8 USV, within 1.96 SD).

“We found strong concordance between methods without evidence of systematic bias (average of USV count difference between methods was -3.8 ± 15.8 USV, data points were within 1.96 standard deviations and exhibited no bias).”

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

Only animals from experienced parents were used, reducing variability in the early life environment for the pups.

“Only animals from experienced parents (sire/dam will have been sired/nursed at least one previous litter) were used. This reduced variability in the early life environment for the pups in case of effects of differences in parental experience.”

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

The faecal corticosteroid assay utilises an antibody for metabolite fractions containing 5a-pregnane-3b,11b,21-triol-20-one.

“The faecal corticosteroid assay utilises an antibody for metabolite fractions containing 5a-pregnane-3b,11b,21-triol-20-one, first described by Touma et al. (2003).”

• cites Touma et al. (2003) sourceNot checked — no full text of the source

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

No outlier removal based on the Grubb's test was performed.

“No outlier removal (based on the Grubb’s test ( Grubbs, 1969)) was performed.”

• cites Grubbs, 1969 sourceNot checked — no full text of the source

Classified as probably not a substantial claim.

Activation of the cortical area in response to touch influences USV production.

“The similar amount of physical contact for all individuals is important, since activation of the cortical area in response to touch has been shown to influence USV production ( Ishiyama & Brecht, 2016).”

• cites Ishiyama & Brecht, 2016 sourceNot checked — no full text of the source

Classified as probably not a substantial claim.

22 kHz USVs are indicators of negative affective state.

“We also pre-registered analysing 22 kHz USVs as indicators of negative affective state ( Brudzynski, 2013).”

• cites Brudzynski, 2013 sourceNot checked — no full text of the source

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

Bonferroni correction was used where appropriate to account for potential Type I errors that may arise from multiple comparisons.

“To account for potential Type I errors that may arise from multiple comparisons, we used Bonferroni correction where appropriate.”

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

The order of EPM and OF testing was pseudo-randomised.

“In Phase 2, the next day after the final playful or control interaction, rats underwent behaviour testing with Elevated Plus Maze (EPM) and Open Field (OF), their order of passage was pseudo-randomised.”

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

For physiological markers, data points within the dynamic range of the assay were used and replicate quality was checked.

“For physiological markers, we used data points within the dynamic range of the assay and checked replicate quality.”

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

The handler was not blinded during the preparatory and experimental phases because they needed to know which handling method they would be using with each rat.

“We were not able to blind the handler during the preparatory and experimental phases, as they needed to know which handling method they would be using with each rat.”

Classified as probably not a substantial claim.

Negative affective states inhibit play behaviour.

“The rats were habituated to handling and the experimental recording arena, to lessen any anxiety or fear due to novelty, as negative affective states inhibit play behaviour (reviewed in Ahloy-Dallaire et al., 2018).”

• cites Ahloy-Dallaire et al., 2018 sourceNot checked — no full text of the source

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

The standard curve for each plate was checked for outliers; no outlier removal was necessary.

“The standard curve created by the software for each plate was checked for outliers as recommended; however, no outlier removal was necessary.”

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

The order of testing of cages, and rats within cage, was pseudo-randomised using a random list generator.

“The order of testing of cages, and rats within cage were pseudo-randomised (a sequence for cage and individuals generated using a random list generator).”

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

Retrospective power analysis using the variability within this data set to calculate effect of Treatment x Sex X Day (Between x Between X Within), with an intraclass correlation coefficient by cage of 0.42, returned a power level value of 0.8697, exceeding the predicted value of 0.80.

“Retrospective power analysis using the variability within this data set to calculate effect of Treatment x Sex X Day (Between x Between X Within), with an intraclass correlation coefficient by cage of 0.42, returned a power level value of 0.8697, exceeding our predicted value of 0.80.”

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

The expression of social play in the five minutes prior to the anticipatory phase was too infrequent for analysis.

“The expression of social play in the five minutes prior to the anticipatory phase was too infrequent for analysis.”

Classified as probably not a substantial claim.

To avoid inflating estimates of variance components, the model incorporated fixed effects for treatment, sex, and day, as well as random effects for cohort and cage.

“To avoid inflating our estimates of variance components, the model also incorporated fixed effects for treatment, sex, and day, as well as random effects for cohort and cage.”

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

Uninformative or weak priors were used on all parameters, including wide normal priors for fixed effects and Half-Student priors for variance parameters.

“We used uninformative or weak priors on all parameters ( Gelman et al., 2013) which included wide normal priors for fixed effects, and Half-Student priors for variance parameters.”

• cites Gelman et al., 2013Not matched

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

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

Models for estimating variance components were run using four MCMC chains, each with 8,500 iterations, including a burn-in of 1,000 iterations and a thinning interval of 100.

“The models for estimating variance components were run using four Markov Chain Monte Carlo (MCMC) chains, each with 8,500 iterations, including a burn-in of 1,000 iterations and a thinning interval of 100.”

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

A full exploration of among- and within-individual variance by treatment was not possible as the data were too constrained for the model complexity; a population level analysis to assess total variance in the model was performed.

“A full exploration of among- and within-individual variance by treatment was not possible as the data were too constrained for the model complexity, however a population level analysis to assess total variance in the model was performed.”

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

One explanation for the sex by treatment interaction is that it reflects sex differences in social play behaviour.

“One explanation for this sex by treatment interaction is that it reflects sex differences in social play behaviour.”

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

One explanation for females' greater lateral rotation is females' greater capacities to sense the approach of an attacker and hence being more able to perform longer distance defence moves.

“one explanation of this being females’ greater capacities to sense the approach of an attacker ( Pellis et al., 1994) and hence being more able to perform longer distance defence moves ( Pellis et al., 1989, 1997).”

• cites Pellis et al., 1994 sourceNot checked — no full text of the source
• cites Pellis et al., 1989, 1997Not matched

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

May combine two claims.

Studies of social play in juvenile rats have revealed sex differences in playful defence behaviours, with males more likely to adopt a supine posture and females more likely to evade through lateral rotation.

“Studies of social play in juvenile rats have revealed sex differences in playful defence behaviours, with males more likely to adopt a supine posture and females more likely to evade through lateral rotation ( Pellis & McKenna, 1992; Pellis & Pellis, 1990).”

• cites Pellis & McKenna, 1992 sourceNot checked — no full text of the source
• cites Pellis & Pellis, 1990 sourceNot checked — no full text of the source

May combine two claims.

The results suggest that females prefer tickling where pinning is minimised, whereas males do not discriminate across treatments varying in amount of pinning, on the basis of USV production.

“These results suggest that females prefer tickling where pinning is minimised, whereas males do not discriminate across treatments varying in amount of pinning, on the basis of USV production, a well-validated indicator of positive affective states in rats ( Burgdorf & Panksepp, 2001).”

• cites Burgdorf & Panksepp, 2001 sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing).

Direct comparisons between social play and tickling to interpret USV production need to be done with caution, as these are potentially not equivalent experiences for the rat.

“However, direct comparisons between social play and tickling to interpret USV production need to be done with caution, as these are potentially not equivalent experiences for the rat.”

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

Variations in call sub-types could be crucial when interpreting rats' affective response to different forms of play.

“Variations in call sub-types could be crucial when interpreting rats’ affective response to different forms of play.”

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

Trill calls are seen to be most clearly associated with positive affect whereas flat calls are thought to be more involved in social communication.

“Trill calls are seen to be most clearly associated with positive affect ( Burgdorf et al., 2008) whereas flat calls are thought to be more involved in social communication ( Wöhr et al., 2008).”

• cites Burgdorf et al., 2008 sourceNot checked — no full text of the source
• cites Wöhr et al., 2008Not matched

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

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

There are known sex differences in call sub-types during playful handling.

“There are also known to be sex differences in call sub-types during playful handling.”

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

The data on sinuosity could also be consistent with higher levels of trill calls in the treatments with reduced levels of pinning.

“The data on sinuosity could also be consistent with higher levels of trill calls in the treatments with reduced levels of pinning.”

• AssumptionRests on an assumption: Lower delta frequencies and lower sinuosity scores reflect flat calls, and higher delta frequency and higher sinuosity scores reflect trill calls.

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

The higher delta frequency in females could reflect their overall higher trill call rate in response to control and treatment conditions.

“The higher delta frequency in females could reflect their overall higher trill call rate in response to control and treatment conditions.”

• AssumptionRests on an assumption: Lower delta frequencies and lower sinuosity scores reflect flat calls, and higher delta frequency and higher sinuosity scores reflect trill calls.

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

The lower delta frequency in the control groups and the increasing delta frequency over days in the treatment groups could suggest increases in trill calls with exposure to heterospecific play.

“In line with this, the lower delta frequency in the control groups and the increasing delta frequency over days in the treatment groups, could suggest increases in trill calls with exposure to heterospecific play.”

• AssumptionRests on an assumption: Lower delta frequencies and lower sinuosity scores reflect flat calls, and higher delta frequency and higher sinuosity scores reflect trill calls.

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

The pseudo-randomisation procedure was more deterministic than the computer-run randomisation algorithm.

“Our pseudo-randomisation procedure is therefore more deterministic than the computer-run randomisation algorithm.”

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

Use of a single male handler constitutes a limitation, especially in light of recent reports about experimenter sex affecting rodents.

“Finally, we accept that use of a single male handler constitutes a limitation, especially in the light of recent reports about experimenter sex affecting rodents.”

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

Animals were assigned to treatments according to preset criteria to homogenise factors assumed to affect treatment response (sex, litter of origin, social cage environment, body weight).

“We assigned animals to treatments according to preset criteria to homogenise factors we assume might affect treatment response (sex, litter of origin and social cage environment, body weight).”

• AssumptionThe authors state this as an assumption: Sex, litter of origin, social cage environment, and body weight might affect treatment response.

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

The experimenter was aware of the treatments administered as the rats were handled and was therefore not blinded to the treatments.

“Additionally, the experimenter was aware of the treatments administered as the rats were handled. He was therefore not blinded to the treatments.”

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

A different researcher, who had not handled the rats and was blinded to their treatment allocation, performed the faecal corticosteroid metabolite assays and also performed the statistical analysis, thus reducing potential biases in analysis.

“A different researcher, who had not handled the rats and was blinded to their treatment allocation, performed the faecal corticosteroid metabolite assays and also performed the statistical analysis thus reducing potential biases in analysis.”