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Animals : an open access journal from MDPI2025; 15(24); 3551; doi: 10.3390/ani15243551

Monitoring Weaning Stress in Fillies and Colts on a Thoroughbred Breeding Farm by Cortisol and Blood Inflammatory Markers: The Benefits of Gradual Separation and Social Support.

Abstract: Weaning represents a major developmental milestone for foals, especially when 4- to 7-month-old foals are abruptly separated from their dams. Studies have shown that the post-weaning period is associated with physiological and psychological stress, which may affect immune function. The present study aimed to describe and analyze the pattern of blood inflammatory biomarkers related to the innate immune system. A gradual foal-dam separation procedure was chosen, which included post-weaning social buffering by a familiar adult horse. Twelve Thoroughbred foals (six fillies, six colts) aged 4-6 months were enrolled and divided into a colt and a filly group, and after weaning, were kept as such. Blood samples were collected before and up to 7 days after weaning. Serum cortisol and cytokines (IL-2, IL-6, IL-10, TNF-α, IFN-γ) were quantified using equine-specific ELISA kits. Routine descriptive statistics were used to present the data, and inferences were drawn using a two-way repeated measures ANOVA. The level of significance was set at < 0.05. Results showed significant differences in the concentrations of IL-10 ( < 0.001), IFN-γ ( < 0.01), and TNF-α ( < 0.01) on various days post-weaning. An apparent significant sex effect was not shown, but IL-10 peaked on day 3 in fillies. IFN-γ decreased significantly by day 5 in both sexes. Conversely, TNF-α levels increased significantly by day 5. No significant changes were observed for cortisol.
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Publication Date: 2025-12-10 PubMed ID: 41463836PubMed Central: PMC12729675DOI: 10.3390/ani15243551Google Scholar: Lookup
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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.

Overview

  • This study examined stress responses in 4- to 6-month-old Thoroughbred foals (fillies and colts) during the weaning period by measuring blood inflammatory markers and cortisol.
  • The approach used gradual separation from the dam combined with social support from a familiar adult horse to reduce stress, and the study tracked immune system-related cytokines over 7 days post-weaning.

Introduction and Study Motivation

  • Weaning is a critical and stressful developmental event for foals, especially when separation from the dam is abrupt.
  • Stress during weaning can negatively impact physiological and psychological well-being, including effects on immune function.
  • The study aimed to investigate how a gradual, less abrupt weaning method, combined with social support, influences stress and immune response biomarkers.

Study Design and Methods

  • Participants: Twelve Thoroughbred foals (6 fillies and 6 colts), aged 4 to 6 months.
  • Procedure:
    • Foals were gradually separated from their dams rather than abruptly weaned.
    • Post-weaning, foals were grouped by sex (fillies and colts) and housed accordingly.
    • Each foal was provided social buffering by keeping a familiar adult horse nearby to offer social support.
  • Blood Sampling:
    • Blood samples were collected prior to weaning and daily for 7 days post-weaning.
    • Serum was analyzed for cortisol and inflammatory cytokines associated with the innate immune response: IL-2, IL-6, IL-10, TNF-α, and IFN-γ.
    • Measurement was performed using equine-specific ELISA kits.
  • Statistical Analysis:
    • Two-way repeated measures ANOVA was used to evaluate time (day) and sex effects on biomarker levels.
    • Significance threshold was set at p < 0.05.

Key Results

  • Inflammatory Cytokines:
    • IL-10 (an anti-inflammatory cytokine) levels showed significant changes with a peak on day 3 observed specifically in fillies (p < 0.001).
    • IFN-γ (a pro-inflammatory cytokine involved in immune activation) significantly decreased by day 5 post-weaning in both colts and fillies (p < 0.01).
    • TNF-α (a major inflammatory cytokine) significantly increased by day 5 in both sexes (p < 0.01).
    • No significant changes were found for IL-2 and IL-6 across the days.
  • Cortisol:
    • No significant changes in serum cortisol levels were detected throughout the 7-day period post-weaning, suggesting low acute stress hormone response in this weaning model.
  • Sex Differences:
    • There was no consistent, significant overall sex effect on the inflammatory markers, although IL-10 showed a notable peak specifically in fillies on day 3.

Interpretation and Significance

  • Results suggest that gradual separation combined with social buffering may mitigate extreme stress responses typically observed with abrupt weaning.
  • The lack of significant cortisol change indicates the foals experienced minimal acute physiological stress under this protocol.
  • The observed cytokine fluctuations reflect a regulated immune response during the post-weaning period:
    • The IL-10 peak in fillies may indicate an early anti-inflammatory response to counterbalance immune activation.
    • The decrease of IFN-γ by day 5 could represent a reduction in immune activation as foals adjusted to separation.
    • The increase in TNF-α suggests some ongoing inflammatory activity at day 5, which may be part of normal immune adaptation.
  • Sex-specific cytokine dynamics warrant further investigation to understand differential immune stress responses between male and female foals.
  • The study supports welfare-improving weaning techniques that use gradual separation and social support to help foals transition more healthily.

Conclusions

  • This research highlights that thoughtful management of foal weaning can reduce stress responses measurable via blood biomarkers.
  • Gradual weaning with social buffering decreases cortisol response and modulates inflammatory cytokines, indicating better psychological and immunological welfare during this critical period.
  • These findings can guide breeding farms to implement more welfare-conscious weaning procedures for Thoroughbred foals.

Cite This Article

APA
Deniz Ö, Erol HS, van den Hoven R, Onmaz AC, Aragona F, Fazio F. (2025). Monitoring Weaning Stress in Fillies and Colts on a Thoroughbred Breeding Farm by Cortisol and Blood Inflammatory Markers: The Benefits of Gradual Separation and Social Support. Animals (Basel), 15(24), 3551. https://doi.org/10.3390/ani15243551

Publication

Animals : an open access journal from MDPI
ISSN: 2076-2615
NlmUniqueID: 101635614
Country: Switzerland
Language: English
Volume: 15
Issue: 24
PII: 3551

Researcher Affiliations

Deniz, Ömer
  • Department of Internal Medicine, Faculty of Veterinary Medicine, Kastamonu University, Kastamonu 37150, Türkiye.
Erol, Hüseyin Serkan
  • Department of Biochemistry Balıkesir Türkiye, Faculty of Veterinary Medicine, Balıkesir University, Balıkesir 10100, Türkiye.
van den Hoven, René
  • Department of Companion Animals and Horses, Equine University Clinic for Internal Medicine and Infectious Diseases, Veterinary University of Vienna, Veterinärplatz 1, A-1210 Vienna, Austria.
Onmaz, Ali Cesur
  • Department of Internal Medicine, Faculty of Veterinary Medicine, Erciyes University, Kayseri 38280, Türkiye.
Aragona, Francesca
  • Department of Veterinary Science, University of Messina, Via Giovanni Palatucci, 98168 Messina, Italy.
Fazio, Francesco
  • Department of Veterinary Science, University of Messina, Via Giovanni Palatucci, 98168 Messina, Italy.

Grant Funding

  • KUBAP-01/2024-29 / Kastamonu University

Conflict of Interest Statement

The authors declare no conflicts of interest.

References

This article includes 34 references
  1. Smith BH. Mammalian Life History: Weaning and Tooth Emergence in a Seasonal World.. Biology 2024;13:612.
    doi: 10.3390/biology13080612pmc: PMC11351645pubmed: 39194550google scholar: lookup
  2. Huijsmans TERG, Courtiol A, Van Soom A, Smits K, Rousset F, Wauters J, Hildebrandt TB. Quantifying Maternal Investment in Mammals Using Allometry.. Commun. Biol. 2024;7:475.
    doi: 10.1038/s42003-024-06165-xpmc: PMC11026411pubmed: 38637653google scholar: lookup
  3. Skibiel AL, Downing LM, Orr TJ, Hood WR. The Evolution of the Nutrient Composition of Mammalian Milks.. J. Anim. Ecol. 2013;82:1254–1264.
    doi: 10.1111/1365-2656.12095pubmed: 23895187google scholar: lookup
  4. Henry S, Sigurjónsdóttir H, Klapper A, Joubert J, Montier G, Hausberger M. Domestic Foal Weaning: Need for Re-Thinking Breeding Practices?. Animals 2020;10:361.
    doi: 10.3390/ani10020361pmc: PMC7070483pubmed: 32102206google scholar: lookup
  5. Delank K, Reese S, Erhard M, Wöhr A-C. Behavioral and Hormonal Assessment of Stress in Foals (Equus caballus) throughout the Weaning Process.. PLoS ONE 2023;18:e0280078.
    doi: 10.1371/journal.pone.0280078pmc: PMC9821722pubmed: 36607910google scholar: lookup
  6. Hoffman RM, Kronfeld DS, Holland JL, Greiwe-Crandell KM. Preweaning Diet and Stall Weaning Method Influences on Stress Response in Foals.. J. Anim. Sci. 1995;73:2922–2930.
    doi: 10.2527/1995.73102922xpubmed: 8617662google scholar: lookup
  7. Malinowski K, Hallquist NA, Helyar L, Sherman AR, Scanes CG. Effect of Different Separation Protocols between Mares and Foals on Plasma Cortisol and Cell-Mediated Immune Response.. J. Equine Vet. Sci. 1990;10:363–368.
    doi: 10.1016/S0737-0806(06)80098-5google scholar: lookup
  8. Aragona F, Rizzo M, Arfuso F, Arrigo F, Fazio F, Giudice E, Faggio C, Piccione G, Giannetto C. Preliminary Insights on the Daily Rhythm of CRP and IL-6 in Athletic Horses.. J. Equine Vet. Sci. 2025;154:105702.
    doi: 10.1016/j.jevs.2025.105702pubmed: 40939749google scholar: lookup
  9. Aragona F, Rizzo M, Giudice E, Fazio F, Costa A, Di Bella B, De Caro S, Arfuso F, Briglia M, Piccione G. Circadian Oscillation of Leukocyte Subpopulations and Inflammatory Cytokines over a 24-H Period in Horses.. Vet. Sci. 2025;12:386.
    doi: 10.3390/vetsci12040386pmc: PMC12031226pubmed: 40284888google scholar: lookup
  10. . Clinical Biochemistry of Domestic Animals.. .
  11. Aragona F, Fazio F, Piccione G, Giannetto C. Chronophysiology of Domestic Animals.. Chronobiol. Int. 2024;41:888–903.
    doi: 10.1080/07420528.2024.2360723pubmed: 38832548google scholar: lookup
  12. Henry S, Zanella AJ, Sankey C, Richard-Yris M-A, Marko A, Hausberger M. Adults May Be Used to Alleviate Weaning Stress in Domestic Foals (Equus caballus). Physiol. Behav. 2012;106:428–438.
    doi: 10.1016/j.physbeh.2012.02.025pubmed: 22406387google scholar: lookup
  13. Möstl E, Messmann S, Bagu E, Robia C, Palme R. Measurement of Glucocorticoid Metabolite Concentrations in Faeces of Domestic Livestock.. Zentralbl Vet. A. 1999;46:621–631.
    doi: 10.1046/j.1439-0442.1999.00256.xpubmed: 10638300google scholar: lookup
  14. Otovic P, Hutchinson E. Limits to Using HPA Axis Activity as an Indication of Animal Welfare.. ALTEX-Altern. Anim. Exp. 2014;32:41–50.
    doi: 10.14573/altex.1406161pubmed: 25418851google scholar: lookup
  15. Jimeno B, Verhulst S. Meta-Analysis Reveals Glucocorticoid Levels Reflect Variation in Metabolic Rate, Not “Stress”.. Elife 2023;12:RP88205.
    doi: 10.7554/eLife.88205pmc: PMC10611431pubmed: 37889839google scholar: lookup
  16. Sapolsky RM, Romero LM, Munck AU. How Do Glucocorticoids Influence Stress Responses? Integrating Permissive, Suppressive, Stimulatory, and Preparative Actions.. Endocr. Rev. 2000;21:55–89.
    doi: 10.1210/edrv.21.1.0389pubmed: 10696570google scholar: lookup
  17. Adams AA, Horohov DW. The Effect of an Immunomodulator (Parapoxvirus ovis) on Cell-Mediated Immunity (CMI) in Abruptly Weaned Foals.. Vet. Immunol. Immunopathol. 2013;153:118–122.
    doi: 10.1016/j.vetimm.2012.11.020pubmed: 23312290google scholar: lookup
  18. Schein M, Wittmann DH, Holzheimer R, Condon RE. Hypothesis: Compartmentalization of Cytokines in Intraabdominal Infection.. Surgery. 1996;119:694–700.
    doi: 10.1016/S0039-6060(96)80195-4pubmed: 8650611google scholar: lookup
  19. Deniz Ö, Aragona F, Murphy BA, Tümer KÇ, Bozacı S, Fazio F. Climate Change Impact on Blood Haemogram in the Horse: A Three-Year Preliminary Study.. Front. Vet. Sci. 2024;11:1482268.
    doi: 10.3389/fvets.2024.1482268pmc: PMC11683153pubmed: 39736933google scholar: lookup
  20. Deniz Ö, Aragona F, Pezzino G, Cancellieri E, Bozaci S, Tümer KÇ, Fazio F. Modeling Climate Change Effects on Some Biochemical Parameters in Horse.. Res. Vet. Sci. 2025;189:105630.
    doi: 10.1016/j.rvsc.2025.105630pubmed: 40168832google scholar: lookup
  21. Duaso J, Perez-Ecija A, Martínez E, Navarro A, De Las Heras A, Mendoza FJ. Assessment of Common Hematologic Parameters and Novel Hematologic Ratios for Predicting Piroplasmosis Infection in Horses.. Animals. 2025;15:1485.
    doi: 10.3390/ani15101485pmc: PMC12108503pubmed: 40427362google scholar: lookup
  22. Massányi M, Halo M, Massányi P, Mlyneková E, Greń A, Formicki G, Halo M. Changes in Haematological and Biochemical Parameters in Blood Serum of Horses during Exposition to Workload Stress.. Heliyon. 2022;8:e12241.
    doi: 10.1016/j.heliyon.2022.e12241pmc: PMC9758420pubmed: 36536901google scholar: lookup
  23. Anhold H, Candon R, Chan D-S, Amos W. A Comparison of Elevated Blood Parameter Values in a Population of Thoroughbred Racehorses.. J. Equine Vet. Sci. 2014;34:651–655.
    doi: 10.1016/j.jevs.2013.12.005google scholar: lookup
  24. Piccione G, Arfuso F, Giudice E, Aragona F, Pugliatti P, Panzera MF, Zumbo A, Monteverde V, Bartolo V, Barbera A. Dynamic Adaptation of Hematological Parameters, Albumin, and Non-Esterified Fatty Acids in Saddlebred and Standardbred Horses During Exercise.. Animals. 2025;15:300.
    doi: 10.3390/ani15030300pmc: PMC11816135pubmed: 39943070google scholar: lookup
  25. Cywinska A, Turlo A, Witkowski L, Szarska E, Winnicka A. Changes in Blood Cytokine Concentrations in Horses after Long-Distance Endurance Rides.. Med. Weter. 2014;70:568–571.
  26. Aragona F, Giannetto C, Piccione G, Arfuso F, Arrigo F, Costa A, De Caro S, Cannuli A, Fazio F. Effect of Time of Day and Physical Exercise on Inflammatory Biomarkers in Athletic Horses.. Front. Vet. Sci. 2025;12:1608770.
    doi: 10.3389/fvets.2025.1608770pmc: PMC12175248pubmed: 40534779google scholar: lookup
  27. Deniz Ö, Ekinci G, Onmaz AC, Derelli FM, Fazio F, Aragona F, Hoven R van den. Monitoring of Inflammatory Blood Biomarkers in Foals with Rhodococcus Equi Pneumonia during Antimicrobial Treatment.. J. Equine Vet. Sci. 2024;138:105103.
    doi: 10.1016/j.jevs.2024.105103pubmed: 38797250google scholar: lookup
  28. Moons C, Laughlin K, Zanella A. Effects of short-term maternal separations on weaning stress in foals.. Appl. Anim. Behav. Sci. 2005;91:321–323.
    doi: 10.1016/j.applanim.2004.10.007google scholar: lookup
  29. Wulf M, Beythien E, Ille N, Aurich J, Aurich C. The stress response of 6-month-old horses to abrupt weaning is influenced by their sex.. J. Vet. Behav. 2018;23:19–24.
    doi: 10.1016/j.jveb.2017.10.010google scholar: lookup
  30. Mak TW, Saunders ME. The Immune Response: Basic and Clinical Principals.. Elsevier Academic Press; San Diego, CA, USA: 2006. Cytokines and Cytokine Receptors; pp. 464–516.
  31. Varga N-I, Bagiu IC, Vulcanescu DD, Lazureanu V, Turaiche M, Rosca O, Bota AV, Horhat FG. IL-6 Baseline Values and Dynamic Changes in Predicting Sepsis Mortality: A Systematic Review and Meta-Analysis.. Biomolecules. 2025;15:407.
    doi: 10.3390/biom15030407pmc: PMC11940105pubmed: 40149943google scholar: lookup
  32. Tisoncik JR, Korth MJ, Simmons CP, Farrar J, Martin TR, Katze MG. Into the Eye of the Cytokine Storm.. Microbiol. Mol. Biol. Rev. 2012;76:16–32.
    doi: 10.1128/MMBR.05015-11pmc: PMC3294426pubmed: 22390970google scholar: lookup
  33. Chua RL, Lukassen S, Trump S, Hennig BP, Wendisch D, Pott F, Debnath O, Thürmann L, Kurth F, Völker MT. COVID-19 Severity Correlates with Airway Epithelium–Immune Cell Interactions Identified by Single-Cell Analysis.. Nat. Biotechnol. 2020;38:970–979.
    doi: 10.1038/s41587-020-0602-4pubmed: 32591762google scholar: lookup
  34. Haeusler GM, Garnham AL, Li-Wai-Suen CS, Clark JE, Babl FE, Allaway Z, Slavin MA, Mechinaud F, Smyth GK, Phillips B. Blood Transcriptomics Identifies Immune Signatures Indicative of Infectious Complications in Childhood Cancer Patients with Febrile Neutropenia.. Clin. Transl. Immunol. 2022;11:e1383.
    doi: 10.1002/cti2.1383pmc: PMC9113042pubmed: 35602885google scholar: lookup

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