Analyze Diet
Frontiers in veterinary science2025; 12; 1574351; doi: 10.3389/fvets.2025.1574351

Physiological and behavioral parameters of pain and stress in mares during and after transvaginal ultrasound-guided follicular aspiration.

Abstract: The use of transvaginal ultrasound-guided follicle aspiration (TVA) for oocyte collection has become a widely used procedure in horses for embryo production (IVEP). The TVA procedure is characterized by various manipulations, which are physical restraint, perineal preparation, transrectal palpation, insertion of an intravaginal device, and needle punctures. These repeated transvaginal and transovarian punctures have raised concerns about their potential effects on mare welfare. Our study aimed to investigate the effects of TVA manipulations, and especially puncturing, on pain and stress in mares in a commercial set-up. Unassigned: Therefore, eight mares were subjected to three TVA procedures: two with puncturing (P) and one without (P, control). Before, during and after all TVA procedures, blood was collected for serum glucose, lactate and cortisol levels, and facial pain scores were assessed. An electrocardiogram (ECG) was recorded for each mare before and during the procedure to measure heart rate (HR) and heart rate variability (HRV). Parameters in response to manipulations and puncturing were analyzed using linear mixed effect models for each outcome variable at different timepoints during the procedure. Unassigned: Results revealed that puncturing during TVA did not significantly influence serum metabolite levels, facial pain scores, HR, or HRV over the complete procedure ( > 0.05). Notably, HR did not increase at the moment of puncturing ( > 0.05), and no significant changes in HRV parameters between P and P were detected ( > 0.05). Both P and P procedures triggered significant increases in cortisol and lactate levels, facial pain scores, and HR during restraint in stocks and perineal preparation compared to the day before and the day after TVA ( < 0.05). Interestingly, even without puncturing, manipulations in the P procedure were sufficient to induce significant elevations in metabolite levels and facial pain scores compared to the day before and after ( < 0.05). Unassigned: These results indicate that the TVA procedure induces stress responses in mares, predominantly associated with all manipulations specific to TVA, while the effect of puncturing itself was minimal. All effects were acute, with parameters returning to baseline when measured 24 h later.
Publication Date: 2025-04-09 PubMed ID: 40271484PubMed Central: PMC12014748DOI: 10.3389/fvets.2025.1574351Google 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

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.

This study investigates the effects of a common horse fertility procedure, transvaginal ultrasound-guided follicle aspiration (TVA), on mare welfare. It finds that while the procedure does cause some short-term stress, it doesn’t induce significant pain or long-term stress, regardless of whether the follicle aspiration is performed or not.

Objectives and Methodology

  • The primary goal of the research was to analyze the impact of TVA, specifically the needle punctures involved in the procedure, on the pain and stress levels in horses. The secondary purpose was to understand whether other aspects of the procedure cause stress or pain.
  • The study was carried out on eight mares. Each mare was subjected to three TVA procedures, two with puncturing and one without (which served as a control). All stages in the procedure were scrutinized, including physical restraint, perineal preparation, transrectal palpation, and the insertion of the intravaginal device.
  • Several parameters were measured before, during, and after the procedure, such as serum glucose, lactate and cortisol levels, facial pain scores, and heart-related parameters (HR and HRV) via an electrocardiogram. These varying parameters were analyzed using linear mixed effect models at different intervals during the procedure.

Results and Findings

  • The results showed that puncturing during TVA did not significantly affect serum metabolites, facial pain scores, heart rate, or heart rate variability.
  • Both TVA procedures with and without puncturing led to a momentary increase in cortisol and lactate levels, facial pain scores, and heart rate during the restraint and preparation stages compared to the levels observed the day before and after the procedure.
  • Surprisingly, even without puncturing, the TVA procedure’s manipulations led to a significant rise in metabolite levels and facial pain scores compared to the levels seen before and the day after the procedure.
  • It was underscored that the effects were acute and short-term, with all parameters returning to their initial state 24 hours post-procedure.

Conclusions

  • The study concluded that TVA does induce some short-term stress in mares, but these effects result mainly from the manipulations associated with the procedure, not the puncturing. Therefore, the welfare impact of TVA, as performed in this commercial setting, seems minimal.
  • It was highlighted that all the observed effects were temporary, suggesting that mares recover from the procedure within 24 hours.
  • This research can prove valuable for improving existing protocol for TVA and potentially mitigating the stress caused by the procedure.

Cite This Article

APA
Van den Branden E, Salamone M, Broothaers K, Peere S, Polfliet E, Dewulf M, Van Steenkiste G, van Loon G, Smits K, Govaere J. (2025). Physiological and behavioral parameters of pain and stress in mares during and after transvaginal ultrasound-guided follicular aspiration. Front Vet Sci, 12, 1574351. https://doi.org/10.3389/fvets.2025.1574351

Publication

ISSN: 2297-1769
NlmUniqueID: 101666658
Country: Switzerland
Language: English
Volume: 12
Pages: 1574351
PII: 1574351

Researcher Affiliations

Van den Branden, Emma
  • Department of Internal Medicine, Reproduction, and Population Medicine, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium.
Salamone, Matthieu
  • Department of Internal Medicine, Reproduction, and Population Medicine, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium.
  • Division of Animal and Human Health Engineering, Department of Biosystems, KU Leuven, Leuven, Belgium.
Broothaers, Klaartje
  • Department of Internal Medicine, Reproduction, and Population Medicine, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium.
Peere, Sofie
  • Department of Internal Medicine, Reproduction, and Population Medicine, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium.
Polfliet, Ellen
  • Department of Internal Medicine, Reproduction, and Population Medicine, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium.
Dewulf, Manon
  • Department of Internal Medicine, Reproduction, and Population Medicine, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium.
Van Steenkiste, Glenn
  • Equine Cardioteam Ghent, Department of Internal Medicine, Reproduction, and Population Medicine, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium.
van Loon, Gunther
  • Equine Cardioteam Ghent, Department of Internal Medicine, Reproduction, and Population Medicine, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium.
Smits, Katrien
  • Department of Internal Medicine, Reproduction, and Population Medicine, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium.
Govaere, Jan
  • Department of Internal Medicine, Reproduction, and Population Medicine, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

References

This article includes 69 references
  1. Lazzari G, Colleoni S, Crotti G, Turini P, Fiorini G, Barandalla M. Laboratory production of equine embryos. J Equine Vet Sci (2020) 89:103097.
    doi: 10.1016/j.jevs.2020.103097pubmed: 32563445google scholar: lookup
  2. Claes A, Stout TAE. Success rate in a clinical equine in vitro embryo production program. Theriogenology (2022) 187:215–8.
  3. Galli C, Colleoni S, Duchi R, Lagutina I, Lazzari G. Equine assisted reproduction and embryo technologies. Anim Reprod (2013) 10:334–43.
  4. Galli C, Colleoni S, Duchi R, Lagutina I, Lazzari G. Developmental competence of equine oocytes and embryos obtained by in vitro procedures ranging from in vitro maturation and ICSI to embryo culture, cryopreservation and somatic cell nuclear transfer. Anim Reprod Sci (2007) 98:39–55.
  5. Martin-Pelaez S, Stout TAE, Leemans B, Dini P, Claes AN. Ovum pick up in a clinical program can induce mild transient discomfort in mares. AAEP Proceedings (2022) 68:280–1.
  6. Morris LHA. The development of in vitro embryo production in the horse. Equine Vet J (2018) 50:712–20.
    doi: 10.1111/evj.12839pubmed: 29654624google scholar: lookup
  7. Viana JHM. Statistics of embryo production and transfer in domestic farm animals. Embryo Technology Newsletter (2022) 40:22–40.
  8. Vanderwall DK, Hyde KJ, Woods GL. Effect of repeated transvaginal ultrasound-guided follicle aspiration on fertility in mares. J Am Vet Med Assoc (2006) 228:248–50.
    doi: 10.2460/javma.228.2.248pubmed: 16426200google scholar: lookup
  9. Stout TAE. Clinical application of in vitro embryo production in the horse. J Equine Vet Sci (2020) 89:103011.
    doi: 10.1016/j.jevs.2020.103011pubmed: 32563449google scholar: lookup
  10. Seyhan A, Ata B, Son WY, Dahan MH, Tan SL. Comparison of complication rates and pain scores after transvaginal ultrasound-guided oocyte pickup procedures for in vitro maturation and in vitro fertilization cycles. Fertil Steril (2014) 101:705–9.
  11. Gilboa D, Seidman L, Kimiagarov P, Noni A, Doron R, Seidman DS. Why do women choose to undergo oocyte aspiration without sedation or analgesia?. Reprodu Fertil (2021) 2:89–94.
    doi: 10.1530/RAF-20-0064pmc: PMC8812437pubmed: 35128445google scholar: lookup
  12. D’Angelo A, Panayotidis C, Amso N, Marci R, Matorras R, Onofriescu M. Recommendations for good practice in ultrasound: oocyte pick up†. Hum Reprod Open (2019) 2019:hoz025.
    doi: 10.1093/hropen/hoz025pmc: PMC6903452pubmed: 31844683google scholar: lookup
  13. Awonuga A, Waterstone J, Oyesanya O, Curson R, Nargund G, Parsons J. A prospective randomized study comparing needles of different diameters for transvaginal ultrasound-directed follicle aspiration. Fertil Steril (1996) 65:109–13.
    doi: 10.1016/S0015-0282(16)58036-4pubmed: 8557123google scholar: lookup
  14. Velez IC, Arnold C, Jacobson CC, Norris JD, Choi YH, Edwards JF. Effects of repeated transvaginal aspiration of immature follicles on mare health and ovarian status. Equine Vet J (2012) 44:78–83.
  15. Bøgh IB, Brink P, Jensen HE, Lehn-Jensen H, Greve T. Ovarian function and morphology in the mare after multiple follicular punctures. Equine Vet J (2003) 35:575–9.
    doi: 10.2746/042516403775467243pubmed: 14515957google scholar: lookup
  16. Fernández-Hernández P, Valero-González M, Fuentes-Romero B, Iglesias-García M, Ezquerra-Calvo LJ, Martín-Cuervo M. Resolution of two cases of ovarian abscesses in mares subjected to ovum pick up. Equine Vet J (2024) 56:751–8.
    doi: 10.1111/evj.14031pubmed: 38083902google scholar: lookup
  17. Campbell MLH, Sandøe P. Welfare in horse breeding. Vet Rec (2015) 176:436–40.
    doi: 10.1136/vr.102814pmc: PMC4431322pubmed: 25908746google scholar: lookup
  18. Petyim S, Båge R, Madej A, Larsson B. Ovum pick-up in dairy heifers: does it affect animal well-being?. Reprod Domest Anim (2007) 42:623–32.
  19. Chastant-Maillard S, Quinton H, Lauffenburger J, Cordonnier-Lefort N, Richard C, Marchal J. Consequences of transvaginal follicular puncture on well-being in cows. Reproduction (2003) 125:555–63.
    doi: 10.1530/rep.0.1250555pubmed: 12683926google scholar: lookup
  20. Alam M, Dobson H. Effect of various veterinary procedures on plasma concentrations of cortisol, luteinising hormone and prostaglandin F2 alpha metabolite in the cow. Vet Rec (1986) 118:7–10.
    doi: 10.1136/vr.118.1.7pubmed: 3946066google scholar: lookup
  21. Oltedal A, Gaustad AH, Peltoniemi O, Björkman S, Skaare A, Oropeza-Moe M. Experiences with transvaginal ovum pick-up (OPU) in sows. Theriogenology (2024) 214:157–65.
  22. Schönbom H, Kassens A, Hopster-Iversen C, Klewitz J, Piechotta M, Martinsson G. Influence of transrectal and transabdominal ultrasound examination on salivary cortisol, heart rate, and heart rate variability in mares. Theriogenology (2015) 83:749–56.
  23. Diego R, Douet C, Reigner F, Blard T, Cognié J, Deleuze S. Influence of transvaginal ultrasound-guided follicular punctures in the mare on heart rate, respiratory rate, facial expression changes, and salivary cortisol as pain scoring. Theriogenology (2016) 86:1757–63.
  24. Hernández-Avalos I, Mota-Rojas D, Mendoza-Flores JE, Casas-Alvarado A, Flores-Padilla K, Miranda-Cortes AE. Nociceptive pain and anxiety in equines: physiological and behavioral alterations. Vet World (2021) 14:2984–95.
  25. Rietmann TR, Stuart AEA, Bernasconi P, Staᆲher M, Auer JA, Weishaupt MA. Assessment of mental stress in warmblood horses: heart rate variability in comparison to heart rate and selected behavioural parameters. Appl Anim Behav Sci (2004) 88:121–36.
  26. Ishizaka S, Aurich JE, Ille N, Aurich C, Nagel C. Acute physiological stress response of horses to different potential short-term stressors. J Equine Vet Sci (2017) 54:81–6.
  27. König V, Borstel U, Visser EK, Hall C. Indicators of stress in equitation. Appl Anim Behav Sci (2017) 190:43–56.
  28. Squires EJ. Applied animal endocrinology. Wallingford: CABI; (2003).
  29. Malancus RN, Rusu RO, Ailincăi LI. Stress levels in Hanoverian horses used in competition and other recreational activities. Rev Rom Med Vet (2022) 32:56–8.
  30. van Loon JPAM, 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) 206:356–64.
    doi: 10.1016/j.tvjl.2015.08.023pubmed: 26526526google scholar: lookup
  31. Covalesky ME, Russoniello CR, Malinowski K. Effects of show-jumping performance stress on plasma cortisol and lactate concentrations and heart rate and behavior in horses. J Equine Vet Sci (1992) 12:244–51.
  32. Massányi M, Halo M, Mlyneková E, Kováčiková E, Tokárová K, Greń A. The effect of training load stress on salivary cortisol concentrations, health parameters and hematological parameters in horses. Heliyon (2023) 9:e19037.
  33. Bruschetta G, Zanghì G, Giunta RP, Ferlazzo AM, Satué K, D’Ascola A. Short road transport and slaughter stress affects the expression profile of serotonin receptors, adrenocortical, and Hematochemical responses in horses. Vet Sci (2024) 11:113.
    doi: 10.3390/vetsci11030113pmc: PMC10974429pubmed: 38535847google scholar: lookup
  34. Aurich C, Aurich J. Effects of stress on reproductive functions in the horse. Pferdeheilkunde (2008) 24:99–102.
    doi: 10.21836/PEM20080121google scholar: lookup
  35. Sikorska U, Maśko M, Ciesielska A, Zdrojkowski Ł, Domino M. Role of cortisol in Horse’s welfare and health. Agriculture (2023) 13:2219.
    doi: 10.3390/agriculture13122219pubmed: 0google scholar: lookup
  36. Broux B, De Clercq D, Decloedt A, Ven S, Vera L, van Steenkiste G. Heart rate variability parameters in horses distinguish atrial fibrillation from sinus rhythm before and after successful electrical cardioversion. Equine Vet J (2017) 49:723–8.
    doi: 10.1111/evj.12684pubmed: 28323361google scholar: lookup
  37. Van Steenkiste G, van Loon G, Crevecoeur G. Transfer learning in ECG classification from human to horse using a novel parallel neural network architecture. Sci Rep (2020) 10:186.
    doi: 10.1038/s41598-019-57025-2pmc: PMC6957496pubmed: 31932667google scholar: lookup
  38. R Core Team. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. (2020).
  39. Wickham H, Averick M, Bryan J, Chang W, McGowan LD, François R. Welcome to the tidyverse. J Open Source Softw (2019) 4:1686.
    doi: 10.21105/joss.01686google scholar: lookup
  40. Waring E, Quinn M, McNamara A, Arino de la Rubia E, Zhu H, Ellis S. skimr: compact and flexible summaries of data. (2022).
  41. Bates D, Mächler M, Bolker B, Walker S. Fitting linear mixed-effects models using lme4. J Stat Softw (2015) 67:1–48.
    doi: 10.18637/jss.v067.i01pubmed: 0google scholar: lookup
  42. Kuznetsova A, Brockhoff PB, Christensen RHB. lmerTest package: tests in linear mixed effects models. J Stat Softw (2017) 82:1–26.
    doi: 10.18637/jss.v082.i13google scholar: lookup
  43. Lüdecke D, Ben-Shachar MS, Patil I, Waggoner P, Makowski D. Performance: an R package for assessment, comparison and testing of statistical models. J Open Source Softw (2021) 6:3139.
    doi: 10.21105/joss.03139google scholar: lookup
  44. Lüdecke D. sjPlot: data visualization for statistics in social science. (2024).
  45. Pedersen TL. Ggforce: accelerating “ggplot2.”. (2024).
  46. Lenth R V. Emmeans: estimated marginal means, aka least-squares means. (2024).
  47. Kassambara A. Ggpubr: “ggplot2” based publication ready plots. (2023).
  48. Green P, MacLeod CJ. Simr: an R package for power analysis of generalised linear mixed models by simulation. Methods Ecol Evol (2016) 7:493–8.
    doi: 10.1111/2041-210X.12504pubmed: 0google scholar: lookup
  49. Haffner JC, Fecteau KA, Eiler H, Tserendorj T, Hoffman RM, Oliver JW. Blood steroid concentrations in domestic Mongolian horses. J Vet Diagn Invest (2010) 22:537–43.
    doi: 10.1177/104063871002200407pubmed: 20622223google scholar: lookup
  50. Kirchmeier A, van Herwaarden AE, van der Kolk JH, Sauer FJ, Gerber V. Plasma steroid profiles before and after ACTH stimulation test in healthy horses. Domest Anim Endocrinol (2020) 72:106419–5.
  51. Marc M, Parvizi N, Ellendorff F, Kallweit E, Elsaesser F. Plasma cortisol and ACTH concentrations in the warmblood horse in response to a standardized treadmill exercise test as physiological markers for evaluation of training status. J Anim Sci (2000) 78:1936–46.
    doi: 10.2527/2000.7871936xpubmed: 10907837google scholar: lookup
  52. Tennent-Brown B. Blood lactate measurement and interpretation in critically ill equine adults and neonates. Vet Clin North Am Equi Pract (2014) 30:399–413.
    doi: 10.1016/j.cveq.2014.04.006pubmed: 25016498google scholar: lookup
  53. Kaneko JJ, Harvey JW, Bruss ML. Clinical biochemistry of domestic animals. 6th ed. Amsterdam: Elsevier; (2008).
  54. Papas M, Govaere J, Peere S, Gerits I, Van de Velde M, Angel-Velez D. Anti-müllerian hormone and opu-icsi outcome in the mare. Animals (2021) 11:2004.
    doi: 10.3390/ani11072004pmc: PMC8300260pubmed: 34359132google scholar: lookup
  55. Le Breton A, Lewis N. Equine ART and antral follicle count: can we deepen our understanding to improve outcomes?. Reprod Domest Anim (2024) 59:e14625.
    doi: 10.1111/rda.14625pubmed: 39233592google scholar: lookup
  56. Jacobson CC, Choi YH, Hayden SS, Hinrichs K. Recovery of mare oocytes on a fixed biweekly schedule, and resulting blastocyst formation after intracytoplasmic sperm injection. Theriogenology (2010) 73:1116–26.
  57. Galli C, Duchi R, Colleoni S, Lagutina I, Lazzari G. Ovum pick up, intracytoplasmic sperm injection and somatic cell nuclear transfer in cattle, buffalo and horses: from the research laboratory to clinical practice. Theriogenology (2014) 81:138–51.
  58. Schmidt A, Biau S, Möstl E, Becker-Birck M, Morillon B, Aurich J. Changes in cortisol release and heart rate variability in sport horses during long-distance road transport. Domest Anim Endocrinol (2010) 38:179–89.
  59. Becker-Birck M, Schmidt A, Lasarzik J, Aurich J, Möstl E, Aurich C. Cortisol release and heart rate variability in sport horses participating in equestrian competitions. J Vet Behav (2013) 8:87–94.
  60. Morton AJ, Varney CR, Ekiri AB, Grosche A. Cardiovascular effects of N-butylscopolammonium bromide and xylazine in horses. Equine Vet J (2011) 43:117–22.
  61. Hallman I, Tapio H, Raekallio M, Karikoski N. Effect of constant rate infusion of detomidine with and without vatinoxan on blood glucose and insulin concentrations in horses. Vet Anaesth Analg (2024) 51:144–51.
    doi: 10.1016/j.vaa.2023.11.005pubmed: 38103967google scholar: lookup
  62. Buhl R, Ersbøll AK, Larsen NH, Eriksen L, Koch J. The effects of detomidine, romifidine or acepromazine on echocardiographic measurements and cardiac function in normal horses. Vet Anaesth Analg (2007) 34:1–8.
  63. Sellon DC, Roberts MC, Blikslager AT, Ulibarri C, Papich MG. Effects of continuous rate intravenous infusion of butorphanol on physiologic and outcome variables in horses after celiotomy. J Vet Intern Med (2004) 18:555–63.
  64. Röder M, Heuwieser W, Borchardt S, Plenio JL, Palme R, Sutter F. The effect of transdermal flunixin meglumine on blood cortisol levels in dairy calves after cautery disbudding with local anesthesia. J Dairy Sci (2022) 105:3468–76.
    doi: 10.3168/jds.2021-21257pubmed: 35123779google scholar: lookup
  65. Wilson D, Bohart G, Evans A, Robertson S, Rondenay Y. Retrospective analysis of detomidine infusion for standing chemical restraint in 51 horses. Vet Anaesth Analg (2002) 29:54–7.
  66. de Oliveira AR, Gozalo-Marcilla M, Ringer SK, Schauvliege S, Fonseca MW, Trindade PHE. Development and validation of the facial scale (FaceSed) to evaluate sedation in horses. PLoS One (2021) 16:e0251909.
  67. Robinson EP, Natalini CC. Epidural anesthesia and analgesia in horses. Vet Clin N Am Equine Pract (2002) 18:61–82.
    doi: 10.1016/S0749-0739(02)00010-Xpubmed: 12064183google scholar: lookup
  68. Michielsen AJHC, Schauvliege S. Epidurale anesthesie en analgesie bij paarden. Vlaams Diergeneeskd Tijdschr (2019) 88:233–40.
    doi: 10.21825/vdt.v88i4.16013google scholar: lookup
  69. McNicholas CP, Madden T, Zhao Q, Secura G, Allsworth JE, Peipert JF. Cervical lidocaine for IUD insertional pain: a randomized controlled trial. Am J Obstet Gynecol (2012) 207:384.e1–6.
    doi: 10.1016/j.ajog.2012.09.018pmc: PMC3492878pubmed: 23107081google scholar: lookup

Citations

This article has been cited 0 times.