34Startle is a fast response elicited by sudden acoustic, tactile or visual stimuli in a variety of animal species 35 and in humans. The magnitude of startle response can be modulated by external and internal variables and 36 can be a useful tool to study the sensory-motor integration in animals. Different stimuli have been used to 37 induce startle in horses, which makes it difficult to compare the responses to these different approaches. 38The present study uses ultra-short-term heart rate variability (HRV) analysis to characterize the cardiac 39 autonomic modulation, reactivity assessment and blood cortisol measurements to describe the behavioural 40 and endocrine responses to a simple, easy to replicate, effective and safe method of startle (an umbrella is 41 abruptly opened near the horse). The ultra-short-term (64 s) heart rate (HR) series were interpolated (4 Hz) 42 and divided into 256 points segments then the spectra calculated (Fast Fourier Transform). The spectra 43 were then integrated into low (LF; 0.01-0.07 Hz; Index of Cardiac Sympathetic Modulation) and high (HF; 44 0.07-0.50 Hz; Index of Cardiac Parasympathetic Modulation) frequency bands. Following the startle test, 45 the HR (p=0.0101), the power of the LF band of the cardiac interval spectrum (p=0.0002) and the LF/HF 46 ratio (p=0.0066) were found to be higher, whereas the power of the HF band of the cardiac interval 47 spectrum was found to be lower (p=0.0002). Also, the horses showed a noticeable escape response, with 48 latency of reaction varying from 0.28 to 1.28 s, duration of reaction ranging from 1.52 to 7.92 s and escape 49 distance covered varying from 3.43 to 9.97 m. However, the endocrine measurements failed to reveal 50 significant changes in the cortisol levels after the startle test. We conclude that the startle test used in the 51 current study was effective to produce changes in behavioural parameters and cardiac autonomic 52 modulation of the horses and can therefore be an appropriate tool for neurobiological studies. 53Furthermore, the use of ultra-short segments (64 s) for HRV analysis appears to be effective and promising 54 for the detection of mental stress in horses.

Startle is a fast response elicited by sudden acoustic, tactile, or visual stimuli in a variety of animals and in humans. As the magnitude of startle response can be modulated by external and internal variables, it can be a useful tool to study reaction to stress. Our study evaluated whether acupuncture can change cardiac autonomic modulation (heart rate variability); and behavioural (reactivity) and endocrine (cortisol levels) parameters in response to startle. Brazilian Sport horses (n = 6) were subjected to a model of startle in which an umbrella was abruptly opened near the horse. Before startle, the horses were subjected to a 20-minute session of acupuncture in acupoints GV1, HT7, GV20, and BL52 (ACUP) and in nonpoints (NP) or left undisturbed (CTL). For analysis of the heart rate variability, ultrashort-term (64 s) heart rate series were interpolated (4 Hz) and divided into 256-point segments and the spectra integrated into low (LF; 0.01–0.07 Hz; index of sympathetic modulation) and high (HF; 0.07–0.50 Hz; index of parasympathetic modulation) frequency bands. Acupuncture (ACUP) changed the sympathovagal balance with a shift towards parasympathetic modulation, reducing the prompt startle-induced increase in LF/HF and reducing cortisol levels 30 min after startle. However, acupuncture elicited no changes in behavioural parameters.

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