PloS one2021; 16(8); e0255618; doi: 10.1371/journal.pone.0255618

A critical evaluation for validation of composite and unidimensional postoperative pain scales in horses.

Abstract: Proper pain therapy requires adequate pain assessment. This study evaluated the reliability and validity of the Unesp-Botucatu horse acute pain scale (UHAPS), the Orthopedic Composite Pain Scale (CPS) and unidimensional scales in horses admitted for orthopedic and soft tissue surgery. Forty-two horses were assessed and videotaped before surgery, up to 4 hours postoperatively, up to 3 hours after analgesic treatment, and 24 hours postoperatively (168 video clips). After six evaluators viewing each edited video clip twice in random order at a 20-day interval, they chose whether analgesia would be indicated and applied the Simple Descriptive, Numeric and Visual Analog scales, CPS, and UHAPS. For all evaluators, intra-observer reliability of UHAPS and CPS ranged from 0.70 to 0.97. Reproducibility was variable among the evaluators and ranged from poor to very good for all scales. Principal component analysis showed a weak association among 50% and 62% of the UHAPS and CPS items, respectively. Criterion validity based on Spearman correlation among all scales was above 0.67. Internal consistency was minimally acceptable (0.51-0.64). Item-total correlation was acceptable (0.3-0.7) for 50% and 38% of UHAPS and CPS items, respectively. UHAPS and CPS were specific (90% and 79% respectively), but both were not sensitive (43 and 38%, respectively). Construct validity (responsiveness) was confirmed for all scales because pain scores increased after surgery. The cut-off point for rescue analgesia was ≥ 5 and ≥ 7 for the UHAPS and CPS, respectively. All scales presented adequate repeatability, criterion validity, and partial responsiveness. Both composite scales showed poor association among items, minimally acceptable internal consistency, and weak sensitivity, indicating that they are suboptimal instruments for assessing postoperative pain. Both composite scales require further refinement with the exclusion of redundant or needless items and reduction of their maximum score applied to each item or should be replaced by other tools.
Publication Date: 2021-08-05 PubMed ID: 34352001PubMed Central: PMC8341545DOI: 10.1371/journal.pone.0255618Google Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
  • Journal Article
  • Research Support
  • Non-U.S. Gov't
  • Validation Study

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The research article aims to assess the reliability and validity of pain scales, namely Unesp-Botucatu horse acute pain scale (UHAPS), Orthopedic Composite Pain Scale (CPS), and unidimensional scales, used in horses following orthopedic and soft tissue surgeries. The findings suggest that the composite scales used are suboptimal and require further development to improve their sensitivity and adequacy in pain assessment.

Methodology

  • 42 horses admitted for orthopedic and soft tissue surgery were assessed and their behaviors were videotaped at four different intervals: before surgery, up to 4 hours post-surgery, up to 3 hours after administering analgesic treatment, and 24 hours post-surgery, resulting in 168 video clips.
  • These clips are then viewed twice in random order, after a gap of 20 days, by six evaluators. Each evaluator would decide if the horses needed analgesia and used the Simple Descriptive, Numeric and Visual Analog scales, CPS, and UHAPS to assess the pain.

Results

  • The intra-observer reliability of UHAPS and CPS ranged from 0.70 to 0.97, indicating that evaluators were consistent with their scores. Reproducibility, or the ability to mimic results by different evaluators, varied from poor to excellent across all scales.
  • Principal Component Analysis showed a weak association among 50% of UHAPS and 62% of the CPS items. This indicates a low level of correlation or relationship between different factors tallied in the pain scales.
  • All scales demonstrated satisfactory criterion validity, where correlation was above 0.67. However, internal consistency, which measures the correlation between different items in a test, was barely adequate (0.51-0.64).
  • Item-total correlation, which assesses the correlation of each test item with the total score, was satisfactory for 50% and 38% of the UHAPS and CPS items, respectively.
  • UHAPS and CPS showed high specificity (ability to correctly identify those without pain; 90% and 79%, respectively), but showed low sensitivity (ability to correctly determine those with pain; 43% and 38%, respectively).
  • All scales provided evidence of construct validity, as pain scores increased after surgery. The cut-off point for rescue analgesia was ≥ 5 for UHAPS and ≥ 7 for CPS.

Conclusion

  • Though the pain scales demonstrated repeatability, adequate criterion validity, and partial responsiveness, their internal consistency and sensitivity were suboptimal.
  • The association among elements in both UHAPS and CPS was poor, indicating further refinement is required. Unnecessary components could be removed, and the maximum score of each item could be decreased.
  • Alternatively, these tools could be replaced with alternative, more efficacious tools for assessing postoperative pain.

Cite This Article

APA
Barreto da Rocha P, Driessen B, McDonnell SM, Hopster K, Zarucco L, Gozalo-Marcilla M, Hopster-Iversen C, Esteves Trindade PH, Gonzaga da Rocha TK, Taffarel MO, Alonso BB, Schauvliege S, Luna SPL. (2021). A critical evaluation for validation of composite and unidimensional postoperative pain scales in horses. PLoS One, 16(8), e0255618. https://doi.org/10.1371/journal.pone.0255618

Publication

ISSN: 1932-6203
NlmUniqueID: 101285081
Country: United States
Language: English
Volume: 16
Issue: 8
Pages: e0255618
PII: e0255618

Researcher Affiliations

Barreto da Rocha, Paula
  • Department of Surgical Specialties and Anesthesiology, Medical School, Su00e3o Paulo State University (Unesp), Botucatu, Su00e3o Paulo, Brazil.
Driessen, Bernd
  • Department of Clinical Studies, New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, Pennsylvania, United States of America.
McDonnell, Sue M
  • Department of Clinical Studies, New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, Pennsylvania, United States of America.
Hopster, Klaus
  • Department of Clinical Studies, New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, Pennsylvania, United States of America.
Zarucco, Laura
  • Dipartimento di Scienze Veterinarie, Universitu00e0 degli Studi di Torino, Grugliasco, Italy.
Gozalo-Marcilla, Miguel
  • The Royal (Dick) School of Veterinary Studies and the Roslin Institute, The University of Edinburgh, Edinburgh, Midlothian, United Kingdom.
Hopster-Iversen, Charlotte
  • Department of Veterinary Clinical Sciences, Section of Medicine and Surgery, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg, Denmark.
Esteves Trindade, Pedro Henrique
  • Department of Veterinary Surgery and Animal Reproduction, School of Veterinary Medicine and Animal Science, Su00e3o Paulo State University (Unesp), Botucatu, Su00e3o Paulo, Brazil.
Gonzaga da Rocha, Thamiris Kristine
  • Department of Veterinary Surgery and Animal Reproduction, School of Veterinary Medicine and Animal Science, Su00e3o Paulo State University (Unesp), Botucatu, Su00e3o Paulo, Brazil.
Taffarel, Marilda Onghero
  • Department of Veterinary Medicine, Maringu00e1 State University, Maringu00e1, Paranu00e1, Brazil.
Alonso, Bruna Bodini
  • Faculty of Animal Science and Food Engineering, Sao Paulo State University, Botucatu, Brazil.
Schauvliege, Stijn
  • Department of Anesthesiology and Domestic Animal Surgery, Faculty of Veterinary Medicine, Ghent University, Ghent, Belgium.
Luna, Stelio Pacca Loureiro
  • Department of Veterinary Surgery and Animal Reproduction, School of Veterinary Medicine and Animal Science, Su00e3o Paulo State University (Unesp), Botucatu, Su00e3o Paulo, Brazil.

MeSH Terms

  • Animals
  • Female
  • Horse Diseases / diagnosis
  • Horse Diseases / surgery
  • Horses
  • Male
  • Pain Management / methods
  • Pain Management / veterinary
  • Pain Measurement / methods
  • Pain Measurement / veterinary
  • Pain, Postoperative / diagnosis
  • Pain, Postoperative / surgery
  • Pain, Postoperative / veterinary

Conflict of Interest Statement

Professor Bernd Driessen and Associate Professors Klaus Hopster and Laura Zarucco are shareholders of Narkovet Consulting®, LLC, which provided parts of the funding for the present study. However, Narkovet Consulting®, LLC did not in any way or form impact these authors’ adherence to all PLOS ONE policies on sharing data and materials as detailed in the online guide for authors (http://journals.plos.org/plosone/s/competing-interests) or had any influence on data presentation and interpretation. All other authors declare that they have no conflicts of interest.

References

This article includes 50 references
  1. Molony V, Kent JE. Assessment of acute pain in farm animals using behavioral and physiological measurements.. J Anim Sci 1997 Jan;75(1):266-72.
    doi: 10.2527/1997.751266xpubmed: 9027575google scholar: lookup
  2. de Grauw JC, van Loon JP. Systematic pain assessment in horses.. Vet J 2016 Mar;209:14-22.
    doi: 10.1016/j.tvjl.2015.07.030pubmed: 26831169google scholar: lookup
  3. Torcivia C, McDonnell S. In-Person Caretaker Visits Disrupt Ongoing Discomfort Behavior in Hospitalized Equine Orthopedic Surgical Patients.. Animals (Basel) 2020 Jan 27;10(2).
    doi: 10.3390/ani10020210pmc: PMC7070845pubmed: 32012670google scholar: lookup
  4. Lindegaard C, Thomsen MH, Larsen S, Andersen PH. Analgesic efficacy of intra-articular morphine in experimentally induced radiocarpal synovitis in horses.. Vet Anaesth Analg 2010 Mar;37(2):171-85.
  5. Ashley FH, Waterman-Pearson AE, Whay HR. Behavioural assessment of pain in horses and donkeys: application to clinical practice and future studies.. Equine Vet J 2005 Nov;37(6):565-75.
    doi: 10.2746/042516405775314826pubmed: 16295937google scholar: lookup
  6. Sutton GA, Paltiel O, Soffer M, Turner D. Validation of two behaviour-based pain scales for horses with acute colic.. Vet J 2013 Sep;197(3):646-50.
    doi: 10.1016/j.tvjl.2013.04.007pubmed: 23993390google scholar: lookup
  7. Sutton GA, Dahan R, Turner D, Paltiel O. A behaviour-based pain scale for horses with acute colic: scale construction.. Vet J 2013 Jun;196(3):394-401.
    doi: 10.1016/j.tvjl.2012.10.008pubmed: 23141961google scholar: lookup
  8. Bussiu00e8res G, Jacques C, Lainay O, Beauchamp G, Leblond A, Cadoru00e9 JL, Desmaiziu00e8res LM, Cuvelliez SG, Troncy E. Development of a composite orthopaedic pain scale in horses.. Res Vet Sci 2008 Oct;85(2):294-306.
    doi: 10.1016/j.rvsc.2007.10.011pubmed: 18061637google scholar: lookup
  9. van Loon JPAM, Van Dierendonck MC. Pain assessment in horses after orthopaedic surgery and with orthopaedic trauma.. Vet J 2019 Apr;246:85-91.
    doi: 10.1016/j.tvjl.2019.02.001pubmed: 30902195google scholar: lookup
  10. Dalla Costa E, Minero M, Lebelt D, Stucke D, Canali E, Leach MC. Development of the Horse Grimace Scale (HGS) as a pain assessment tool in horses undergoing routine castration.. PLoS One 2014;9(3):e92281.
  11. van Loon JP, Van Dierendonck MC. Monitoring acute equine visceral pain with the Equine Utrecht University Scale for Composite Pain Assessment (EQUUS-COMPASS) and the Equine Utrecht University Scale for Facial Assessment of Pain (EQUUS-FAP): A scale-construction study.. Vet J 2015 Dec;206(3):356-64.
    doi: 10.1016/j.tvjl.2015.08.023pubmed: 26526526google scholar: lookup
  12. Taffarel MO, Luna SP, de Oliveira FA, Cardoso GS, Alonso Jde M, Pantoja JC, Brondani JT, Love E, Taylor P, White K, Murrell JC. Refinement and partial validation of the UNESP-Botucatu multidimensional composite pain scale for assessing postoperative pain in horses.. BMC Vet Res 2015 Apr 1;11:83.
    doi: 10.1186/s12917-015-0395-8pmc: PMC4393617pubmed: 25888751google scholar: lookup
  13. Dyson S, Berger J, Ellis AD, Mullard J. Development of an ethogram for a pain scoring system in ridden horses and its application to determine the presence of musculoskeletal pain. J Vet Behav Clin Appl Res 2018;23:47u201357. doi: 10.1016/j.jveb.2017.10.008
  14. Gleerup KB, Forkman B, Lindegaard C, Andersen PH. An equine pain face.. Vet Anaesth Analg 2015 Jan;42(1):103-14.
    doi: 10.1111/vaa.12212pmc: PMC4312484pubmed: 25082060google scholar: lookup
  15. van Loon JPAM, Back W, Hellebrekers LJ, van Weeren PR. Application of a composite pain scale to objectively monitor horses with somatic and visceral pain under hospital conditions. J Equine Vet Sci 2010;30:641u20139. doi: 10.1016/j.jevs.2010.09.011
  16. Sutton GA, Atamna R, Steinman A, Mair TS. Comparison of three acute colic pain scales: Reliability, validity and usability.. Vet J 2019 Apr;246:71-77.
    doi: 10.1016/j.tvjl.2019.01.004pubmed: 30902193google scholar: lookup
  17. Dalla Costa E, Stucke D, Dai F, Minero M, Leach MC, Lebelt D. Using the Horse Grimace Scale (HGS) to Assess Pain Associated with Acute Laminitis in Horses (Equus caballus).. Animals (Basel) 2016 Aug 3;6(8).
    doi: 10.3390/ani6080047pmc: PMC4997272pubmed: 27527224google scholar: lookup
  18. Maskato Y, Dugdale AHA, Singer ER, Kelmer G, Sutton GA. Prospective Feasibility and Revalidation of the Equine Acute Abdominal Pain Scale (EAAPS) in Clinical Cases of Colic in Horses.. Animals (Basel) 2020 Nov 29;10(12).
    doi: 10.3390/ani10122242pmc: PMC7760242pubmed: 33260428google scholar: lookup
  19. Streiner DL, Norman GR, Cairney J. Health measurement scales: a practical guide to their development and use. 5th ed. Oxford: Oxford University Press; 2015.
  20. Trindade PHE, Taffarel MO, Luna SPL. Spontaneous Behaviors of Post-Orchiectomy Pain in Horses Regardless of the Effects of Time of Day, Anesthesia, and Analgesia.. Animals (Basel) 2021 May 31;11(6).
    doi: 10.3390/ani11061629pmc: PMC8230028pubmed: 34072875google scholar: lookup
  21. de Oliveira FA, Luna SP, do Amaral JB, Rodrigues KA, Sant'Anna AC, Daolio M, Brondani JT. Validation of the UNESP-Botucatu unidimensional composite pain scale for assessing postoperative pain in cattle.. BMC Vet Res 2014 Sep 6;10:200.
    doi: 10.1186/s12917-014-0200-0pmc: PMC4172785pubmed: 25192598google scholar: lookup
  22. Brondani JT, Mama KR, Luna SP, Wright BD, Niyom S, Ambrosio J, Vogel PR, Padovani CR. Validation of the English version of the UNESP-Botucatu multidimensional composite pain scale for assessing postoperative pain in cats.. BMC Vet Res 2013 Jul 17;9:143.
    doi: 10.1186/1746-6148-9-143pmc: PMC3722032pubmed: 23867090google scholar: lookup
  23. Luna SPL, de Arau00fajo AL, da Nu00f3brega Neto PI, Brondani JT, de Oliveira FA, Azeru00eado LMDS, Telles FG, Trindade PHE. Validation of the UNESP-Botucatu pig composite acute pain scale (UPAPS).. PLoS One 2020;15(6):e0233552.
  24. Silva NEOF, Trindade PHE, Oliveira AR, Taffarel MO, Moreira MAP, Denadai R, Rocha PB, Luna SPL. Validation of the Unesp-Botucatu composite scale to assess acute postoperative abdominal pain in sheep (USAPS).. PLoS One 2020;15(10):e0239622.
  25. Brondani JT, Luna SP, Padovani CR. Refinement and initial validation of a multidimensional composite scale for use in assessing acute postoperative pain in cats.. Am J Vet Res 2011 Feb;72(2):174-83.
    doi: 10.2460/ajvr.72.2.174pubmed: 21281191google scholar: lookup
  26. Kaiser HF. The varimax criterion for analytic rotation in factor analysis. Psychometrika 1958;23:187u2013200. doi: 10.1007/BF02289233
    doi: 10.1007/BF02289233google scholar: lookup
  27. Altman D. Practical Statistics for Medical Research. London, UK: Chapman and Hall/CRC; 1991.
  28. Schuster C. A Note on the interpretation of weighted kappa and its relations to other rater agreement statistics for metric scales. Educ Psychol Meas 2004;64:243u201353. doi: 10.1177/0013164403260197
    doi: 10.1177/0013164403260197google scholar: lookup
  29. Landis JR, Koch GG. The measurement of observer agreement for categorical data.. Biometrics 1977 Mar;33(1):159-74.
    pubmed: 843571
  30. Cohen J. Weighted kappa: nominal scale agreement with provision for scaled disagreement or partial credit.. Psychol Bull 1968 Oct;70(4):213-20.
    doi: 10.1037/h0026256pubmed: 19673146google scholar: lookup
  31. Altman D. Some common problems in medical research. In: Practical Statistics for Medical Research. London, UK: Chapman and Hall/CRC; 1991. p. 404u20138.
  32. DeVellis RF. Scale development: theory and applications. 4th ed. Thousand Oaks: SAGE Publications Inc; 2017.
  33. Scherer M, Blozik E, Himmel W, Laptinskaya D, Kochen MM, Herrmann-Lingen C. Psychometric properties of a German version of the neck pain and disability scale.. Eur Spine J 2008 Jul;17(7):922-9.
    doi: 10.1007/s00586-008-0677-ypmc: PMC2443271pubmed: 18437433google scholar: lookup
  34. Deyo RA, Diehr P, Patrick DL. Reproducibility and responsiveness of health status measures. Statistics and strategies for evaluation.. Control Clin Trials 1991 Aug;12(4 Suppl):142S-158S.
    doi: 10.1016/s0197-2456(05)80019-4pubmed: 1663851google scholar: lookup
  35. Streiner DL, Cairney J. What's under the ROC? An introduction to receiver operating characteristics curves.. Can J Psychiatry 2007 Feb;52(2):121-8.
    doi: 10.1177/070674370705200210pubmed: 17375868google scholar: lookup
  36. u0160imundiu0107 AM. Measures of Diagnostic Accuracy: Basic Definitions.. EJIFCC 2009 Jan;19(4):203-11.
    pmc: PMC4975285pubmed: 27683318
  37. Streiner DL. Starting at the beginning: an introduction to coefficient alpha and internal consistency.. J Pers Assess 2003 Feb;80(1):99-103.
    doi: 10.1207/S15327752JPA8001_18pubmed: 12584072google scholar: lookup
  38. Roughan JV., Flecknell PA. Training in behaviour-based post-operative pain scoring in ratsu2014An evaluation based on improved recognition of analgesic requirements. Appl Anim Behav Sci 2006;96:327u201342. doi: 10.1016/j.applanim.2005.06.012
  39. Holton LL, Scott EM, Nolan AM, Reid J, Welsh E, Flaherty D. Comparison of three methods used for assessment of pain in dogs.. J Am Vet Med Assoc 1998 Jan 1;212(1):61-6.
    pubmed: 9426779
  40. Kaufman AB, Rosenthal R. Can you believe my eyes? The importance of interobserver reliability statistics in observations of animal behaviour. Anim. Behav 2009; 78(6):1487u20131491. doi: 10.1016/j.anbehav.2009.09.014
  41. Ferrell BA, Stein WM, Beck JC. The Geriatric Pain Measure: validity, reliability and factor analysis.. J Am Geriatr Soc 2000 Dec;48(12):1669-73.
  42. Hesselgard K, Larsson S, Romner B, Stru00f6mblad LG, Reinstrup P. Validity and reliability of the Behavioural Observational Pain Scale for postoperative pain measurement in children 1-7 years of age.. Pediatr Crit Care Med 2007 Mar;8(2):102-8.
  43. Gauvain-Piquard A, Rodary C, Rezvani A, Serbouti S. The development of the DEGR(R): A scale to assess pain in young children with cancer.. Eur J Pain 1999 Jun;3(2):165-176.
    doi: 10.1053/eujp.1999.0118pubmed: 10700346google scholar: lookup
  44. Liang MH. Longitudinal construct validity: establishment of clinical meaning in patient evaluative instruments.. Med Care 2000 Sep;38(9 Suppl):II84-90.
    pubmed: 10982093
  45. Maitho TE, Lees P, Taylor JB. Absorption and pharmacokinetics of phenylbutazone in Welsh Mountain ponies.. J Vet Pharmacol Ther 1986 Mar;9(1):26-39.
  46. Gerring EL, Lees P, Taylor JB. Pharmacokinetics of phenylbutazone and its metabolites in the horse.. Equine Vet J 1981 Jul;13(3):152-7.
  47. Manteca X, Deag JM. Use of physiological measures to assess individual differences in reactivity. Appl Anim Behav Sci 1993;37:265u201370. doi: 10.1016/0168-1591(93)90116-7
  48. McCann JS, Heird JC, Bell RW, Lutherer LO. Normal and more highly reactive horses. I. Heart rate, respiration rate and behavioral observations. Appl Anim Behav Sci 1988;19:201u201314. doi: 10.1016/0168-1591(88)90001-9
  49. van Loon JP, Jonckheer-Sheehy VS, Back W, van Weeren PR, Hellebrekers LJ. Monitoring equine visceral pain with a composite pain scale score and correlation with survival after emergency gastrointestinal surgery.. Vet J 2014 Apr;200(1):109-15.
    doi: 10.1016/j.tvjl.2014.01.003pubmed: 24491373google scholar: lookup
  50. American Educational Research Association, American Psychological Association N, Council on Measurement in Education JC on S for E and, (U.S.) PT. Standards for educational and psychological testing. Washington, DC: American Educational Research Association; 2014.

Citations

This article has been cited 6 times.
  1. Fonseca MW, Trindade PHE, Pinho RH, Justo AA, Tomacheuski RM, Silva NEOFD, Gonu00e7alves HC, Luna SPL. Development and Validation of the Unesp-Botucatu Goat Acute Pain Scale.. Animals (Basel) 2023 Jun 28;13(13).
    doi: 10.3390/ani13132136pubmed: 37443934google scholar: lookup
  2. Tomacheuski RM, Oliveira AR, Trindade PHE, Oliveira FA, Candido CP, Teixeira Neto FJ, Steagall PV, Luna SPL. Reliability and Validity of UNESP-Botucatu Cattle Pain Scale and Cow Pain Scale in Bos taurus and Bos indicus Bulls to Assess Postoperative Pain of Surgical Orchiectomy.. Animals (Basel) 2023 Jan 20;13(3).
    doi: 10.3390/ani13030364pubmed: 36766253google scholar: lookup
  3. Trindade PHE, Mello JFSR, Silva NEOF, Luna SPL. Improving Ovine Behavioral Pain Diagnosis by Implementing Statistical Weightings Based on Logistic Regression and Random Forest Algorithms.. Animals (Basel) 2022 Oct 26;12(21).
    doi: 10.3390/ani12212940pubmed: 36359065google scholar: lookup
  4. Ask K, Andersen PH, Tamminen LM, Rhodin M, Hernlund E. Performance of four equine pain scales and their association to movement asymmetry in horses with induced orthopedic pain.. Front Vet Sci 2022;9:938022.
    doi: 10.3389/fvets.2022.938022pubmed: 36032285google scholar: lookup
  5. Luna SPL, Trindade PHE, Monteiro BP, Crosignani N, Della Rocca G, Ruel HLM, Yamashita K, Kronen P, Tseng CT, Teixeira L, Steagall PV. Multilingual validation of the short form of the Unesp-Botucatu Feline Pain Scale (UFEPS-SF).. PeerJ 2022;10:e13134.
    doi: 10.7717/peerj.13134pubmed: 35345592google scholar: lookup
  6. Lanci A, Benedetti B, Freccero F, Castagnetti C, Mariella J, van Loon JPAM, Padalino B. Development of a Composite Pain Scale in Foals: A Pilot Study.. Animals (Basel) 2022 Feb 11;12(4).
    doi: 10.3390/ani12040439pubmed: 35203146google scholar: lookup