Is Your Horse in Pain? A New Saliva Test Could Offer Answers
"Researchers explore how salivary alpha-amylase and cortisol levels can help detect acute abdominal pain in horses, providing a non-invasive way to assess their well-being."
Recognizing pain in horses can be challenging, especially when dealing with acute abdominal issues like colic. Traditional methods often rely on subjective assessments and invasive procedures, creating a need for more objective and stress-free evaluation techniques.
A recent study has explored the potential of salivary alpha-amylase (sAA) and salivary cortisol as biomarkers for pain and stress in horses experiencing acute abdominal disease. These substances, easily collected through saliva, could provide valuable insights into a horse's condition without the need for blood samples.
This article delves into the findings of this research, explaining how sAA and cortisol levels in saliva can indicate pain-induced stress in horses. We'll explore the study's methodology, key results, and the implications for improved equine welfare.
Measuring Stress in Horses Through Saliva Analysis
A study published May 17, 2024 (Scientific Reports, s41598-024-62172-2) investigated stress levels in domestic horses that were reared in the wild and then moved to human-controlled housing, using saliva analysis to compare the same horses in the wild and in a paddock. The work was premised on the idea that a horse kept in the wild has a lower level of stress than under other housing systems, and reported the concentrations of salivary alpha-amylase (sAA), butyrylcholinesterase (BChol), and cortisol collected from those horses. Giving researchers across the equine-specific and equine assisted services industries a minimally invasive way to collect equine saliva is expected to let them account for a horse's well-being with greater accuracy and intentionality.
Why Saliva Is Gaining Ground Over Blood Tests
Recognition and assessment of pain in horses remain fundamental yet challenging aspects of equine clinical practice and welfare, because unlike human patients, horses cannot self-report pain, so clinicians must rely on behavioural observation, physiological indicators, and structured assessment tools. Saliva has generated growing interest in horses partly because it can be collected easily by non-trained staff without causing pain, discomfort, or stress. A groundbreaking line of work uses saliva samples to measure stress and pain in horses with acute abdominal issues, offering a less invasive alternative to traditional blood tests, and a modified bit device has been shown to provide a feasible and valid method for equine salivary sample collection.
From Umbrella Tests to Facial Expression Scales
Early foundational work in this field includes a 2019 MDPI study (Animals, 9, 993) in which saliva samples were collected from nine riding horses during a test that involved opening an umbrella, with sampling at a basal time point in the stall (T1), in the test indoor arena (T2), at a time of stress (T3), and 30 minutes (T4) and 60 minutes (T5) later. The researchers found that saliva analytes correlate with horses' behavioural responses, opening the possibility of wider use of selected biomarkers such as butyrylcholinesterase and alpha-amylase for evaluating acute stress in horses. In parallel, researchers developed a protocol for assessing and measuring horse pain as interpreted through facial expressions, setting a foundation of work in equine pain assessment.
Decoding Saliva: How the Study Measured Pain in Horses
The study, published in BMC Veterinary Research, involved analyzing saliva samples from 19 horses diagnosed with acute abdominal disease and comparing them to a control group of 11 healthy horses. The goal was to determine if there were significant differences in sAA and cortisol levels between the two groups.
- Salivary alpha-amylase (sAA) activity: Measured using a colorimetric commercial kit, following established scientific methods.
- Salivary cortisol levels: Analyzed with an immunoassay system, ensuring accurate and reliable measurements.
- Statistical analysis: Employed to compare the data between the two groups and identify any significant correlations between biomarker levels and pain scores.
New Tools and Collaborations Push the Field Forward
In February 2025, researchers presented the development and validation of a new method for detecting oxytocin in equine saliva based on AlphaLISA technology, with a limit of detection of 31.7 pg/mL and a limit of quantification of 50.5 pg/mL, sensitivity that allows detection of low concentrations of oxytocin in saliva. Around the same time, a research collaboration between Aberdeen-based biotechnology company Formartine Bio and the University of the West of Scotland (UWS) set out to improve early detection of health issues in horses using a non-invasive saliva testing approach, on the premise that saliva can reveal early signs of inflammation, metabolic stress, immune response, or injury. Related August 2026 research reported that an AI system can read pain on a horse's face almost perfectly, though it does not always focus where expert veterinarians do.
Caveats and Open Questions Remain
Even as equine saliva testing advances, the approach is not without limitations. Saliva biomarker concentrations can be influenced by collection method, time of day, and normal individual variation, and research in this area is still largely at an early exploratory stage. Findings from small, preliminary studies should therefore be interpreted cautiously rather than treated as settled. That caution is why investigators tend to position saliva-based measures as a complement to, rather than a substitute for, careful clinical observation and structured pain assessment.
Saliva Versus Blood, and Beyond
A 2010 study comparing salivary and serum cortisol assays in horses across a wide range of concentrations used an adrenocorticotropic hormone (ACTH) stimulation test, drawing blood through an i.v. catheter and collecting saliva with swabs, in order to validate salivary cortisol for stress assessment in horses. Saliva-based tools have also moved into commercial use, as illustrated by EquiSal Tapeworm, an ELISA-based saliva test developed and marketed by Austin Davis Biologics that measures tapeworm molecule-specific antibodies shown to correlate with tapeworm burdens. Beyond diagnostic testing, pain recognition in horses matters for equine welfare, quality of life, accurate diagnosis and treatment, condition monitoring, horse and human safety, and performance, while recent editorial guidance emphasizes cautious opioid selection at the lowest effective dose in postoperative pain management.
Saliva: A Promising Tool for Assessing Equine Pain
The study's findings revealed that horses with acute abdominal disease had significantly higher sAA and cortisol levels in their saliva compared to healthy horses. Importantly, sAA activity showed a strong correlation with the EAAPS-1 pain scale, indicating its potential as a reliable indicator of pain.
A Converging Picture of Equine Pain
Taken together, the emerging evidence points toward a future in which equine pain and stress can be assessed using multiple complementary tools, from behavioural observation and facial expression scales to structured clinical assessments and increasingly sensitive saliva-based biomarkers. The appeal of saliva is that it offers a less invasive route to measuring physiological states, which suits an animal that cannot describe its own discomfort. That said, experts caution that no single marker is likely to provide a complete answer, and interpretations should always be weighed alongside clinical judgment. The field appears to be converging on a model where technology supports, rather than replaces, the humans responsible for a horse's care.
Where Saliva Diagnostics Could Be Headed
Looking ahead, saliva-based equine diagnostics could evolve toward routine, point-of-care screening for pain, stress, and early disease in everyday equine practice. Broader panels combining multiple analytes may eventually offer more reliable signals than single biomarkers. Validation in larger and more diverse horse populations will be crucial before such tests are widely adopted. If those hurdles are cleared, minimally invasive saliva collection could become a standard part of equine welfare monitoring and preventive care.
Welfare, Standardisation, and the Road to Adoption
Wider adoption of saliva-based testing faces systemic challenges, including standardisation of collection methods and laboratory analysis, variability between breeds and management settings, and the need for validation across clinical contexts. Equine welfare is also shaped by broader housing and management decisions, not only by diagnostic tools. Economic constraints, training requirements, and uneven access to veterinary technology could slow uptake in different regions and sectors of the industry. Addressing these factors will be integral to translating promising research into meaningful improvements in horse welfare.
What New Testing Means for Owners and Vets
For horse owners and veterinarians, the promise of less invasive testing is a practical one: easier, lower-stress sampling that could encourage earlier detection and more proactive care. Recognizing pain early is directly tied to quality of life, accurate diagnosis and treatment, and the safety of both horses and the people handling them. If saliva-based approaches mature as expected, they could give owners and professionals a more accessible window into an animal's wellbeing. In the nearer term, even the research itself matters, giving those who care for horses a stronger evidence base for everyday decisions.
While salivary cortisol levels were elevated in horses with colic, they did not correlate as strongly with the pain scale. This suggests that sAA may be a more sensitive marker of pain-induced stress in these cases.
These preliminary results pave the way for further research into the use of saliva as a non-invasive diagnostic tool for assessing pain and stress in horses. By understanding the relationship between salivary biomarkers and equine health, veterinarians can improve animal welfare and provide more effective treatment strategies.