Changes in eye temperature and stress asessment in horses during show dumping competitions

Short Communication Changes in Eye Temperature and Stress Assessment in Horses During Show Jumping Competitions Q11 Mercedes Valera a ,Ester Bartolomé a ,Maria José Sánchez a ,Antonio Molina b ,Nigel Cook c , Al Schaefer d Q1 a Department of Agroforestry Sciences, ETSIA, University of Seville, Seville, Spain b Department of Genetics, University of Cordoba, Mendel Building, Cordoba, Spain c Livestock Welfare Unit, Alberta Agriculture and Rural Development, Lacombe Research Centre, Lacombe, Alberta, Canada Q 2 d Agri-Food Canada, Lacombe Research Centre, Lacombe, Alberta, Canada article info Article history: Received 15 December 2011 Received in revised form 26 February 2012 Accepted 7 March 2012 Available online xxx Keywords: Infrared thermography Performance Salivary cortisol Spanish sport horse Stress abstract High stress levels in horses Q3 during sporting events can influence the animal’s performance in competitions as well as their welfare. This study investigated the use of infrared thermography (IRT) measurement as a rapid, noninvasive, and accurate method for stress assessment in horses during sport competitions and compared IRT with a commonly used physiological method used for stress assessment and salivary cortisol. IRT and salivary cortisol responses were measured 3 hours before the competition, immediately following the competition, and 3 hours after the competition, each day during the 3 days of a show jumping event. Salivary cortisol values ranged from 0.11 4.51 ng/mL in 5-year-old animals to 1.10 2.20 ng/mL in 4-year-old animals, whereas eye temperature values ranged from 35.84 1.16  C in 6-year-old animals to 36.08 1.11  C in 5-year-old animals. The results obtained suggested that IRT measurements constitute an effective method for detecting stress in horses subjected to the acute events of show jumping. Ó2012 Elsevier Inc. All rights reserved. 1. Introduction During competition, horses are exposed to various sources of stress, which could have a negative influence on welfare, and thus, on performance. The relationship between stress and the metabolic system of the animal has been well documented [1,2], and different methods for assessing stress levels have been developed. However, many of the techniques used to measure stress in animals involve invasive procedures, such as blood sampling, that may cause a stress response themselves [3]. One of these techniques is the measurement of cortisol in saliva, that has been highlighted as a more useful method, rather than plasma or urine cortisol, for stress assessment in horses [4], as it is a direct reflection of the free cortisol concentration in blood and a much more sensitive indicator of adrenocortical activity than plasma “total”concentrations. However, cortisol is released after a persistent action of the stressor, highlighting the presence of chronic stress [5], and is affected by several environmental factors, such as the time of day, the moment of feed intake, or how relaxed the horse is in the sampling environment [6]. Infrared thermography (IRT), and specifically, maximum eye temperature measurement, has been demonstrated as a noninvasive tool that can be used to measure acute and chronic stress in other species such as cattle, showing a great potential as a way to assess animal welfare [3]. This method has also been used in horses for the measurement of body temperature and the detection of different limb pathologies [7,8]. Previous studies have focused on the relationship between IRT and the hypothalamic-pituitary-adrenocortical Q4 (HPA) activity (and thus, cortisol release), highlighting the IRT ability to Corresponding author at: Ester Bartolomé, Department of Agroforestry Sciences, ETSIA, University of Seville, Ctra. Utrera km 1, 41013 Sevilla, Spain. E-mail address: [email protected] (E. Bartolomé). Journal of Equine Veterinary Science journal homepage: www.j-evs.com 0737-0806/$ - see front matter Ó2012 Elsevier Inc. All rights reserved. doi:10.1016/j.jevs.2012.03.005 Journal of Equine Veterinary Science xxx (2012) 1-4 FLA 5.1.0 DTD  YJEVS1333_proof  28 March 2012  5:26 pm 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 detect animal responses to stress in cattle [9] and horses [10]. However, no previous studies have been developed using IRT measurements for stress assessment during competition events in horses. The aim of the present study was to determine the usefulness of maximum eye temperature, measured with IRT, as a noninvasive tool to assess acute stress in horses during sport competitions, comparing the results with salivary cortisol levels. 2. Material and Methods 2.1. Animals Measurements were obtained from 16 horses (10 mares and 6 males) from the Spanish Sport Horse breed, aged 4 (n ¼7), 5 (n ¼5), and 6 (n ¼4) years. Samples were taken during the 3 days of the final of a show jumping competition. During this period, animals were housed in the show stall boxes at the equestrian center where the competition took place, thereby standardizing environmental and housing conditions. The horses arrived to the Q5 competition place 1 day before the beginning of the study, and they had 19 days off following their previous competitions. The dimension of the boxes was 3 3m 2 , and all the animals were fed with hay, concentrate, and water ad libitum. 2.2. Infrared Thermography One portable IRT camera (ThermaCam i70 0, FLIR Systems AB, Danderyd, Sweden) was used to collect eye images. Images were collected three times per day: 3 hours before the competition (BC), just after the competition (JAC, within 5 minutes after the performance), and 3 hours after the competition (AC) when the animal was resting. Images were collected during the 3 days of the competition, recording a total of nine different collection periods per animal. The left eye of all horses was scanned from a 90  angle and at a distance of 1 m. Several images were taken per animal and per collection period, selecting the image that provided the most optimal operating conditions for analysis (90  angle and 1 m of distance). Environmental temperature and relative humidity were recorded with a digital thermo hygrometer (Extech 44550 Q6 ), every time an eye temperature sample was taken, to calibrate the camera results. Image analysis software Therma Cam Researcher Pro 2.8 SR-2 (FLIR Systems AB, Sweden) was used to determine the maximum temperature within an oval area traced around the eye, including the eyeball and approximately 1 cm surrounding the outside of the eyelids (Fig. 1). This maximum eye temperature was used for the analyses. Fig. 1. Infrared thermography images of the eye region. The images were taken 3 hours before (A), just after (B), and 3 hours after (C) the competition. The circle () indicates the area of the eye (the medial posterior palpebral border of the lower eyelid and the lachrymal caruncle) where the maximum temperature point was obtained. The symbol ( ) indicates the mean point of the photo (automatically settled by the infrared thermography camera), where Rh is the relative humidity, and T.atm is the atmospheric temperature. M. Valera et al. / Journal of Equine Veterinary Science xxx (2012) 1-42 FLA 5.1.0 DTD  YJEVS1333_proof  28 March 2012  5:26 pm 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 2.3. Cortisol Assay Salivary cortisol samples were collected just after each IRT eye recording. Saliva was collected from the mouth of the animal by running a cotton swab between the cheek and the teeth until it was saturated. After collection, swabs were placed in 10-mL collection tubes and centrifuged for 40 minutes at 4,000 rpm. Free cortisol (hydrocortisone and hydroxycorticosterone) in saliva was measured using a double-antibody immunoassay kit (Cortisol Saliva ELISA, SA E-6000, Labor Diagnostika Nord GmbH & Co. KG Q7 ) using an absorbance value of 450 nm and determining the concentration of cortisol from a standard curve fitted with a four-parameter logistic function. 2.4. Statistical Analyses Salivary cortisol was logarithmically transformed to satisfy assumptions of normal distribution. Differences within salivary cortisol and eye temperature measurements due to different factors (age of the animal, day of show jumping competition, and phase within competition day) were assessed using analysis of variance. A least square means comparison analysis was carried out, taking into consideration the significant factors obtained in previous analysis. Intervals between phases were also calculated for salivary cortisol and maximum eye temperature measurements. Spearman rank correlations were performed among salivary cortisol intervals and eye temperature intervals for BC-JAC and JAC-AC phase intervals. Statistical analyses were performed using Statistica v. 8.0 [11] package. 3. Results As shown in Table 1, salivary cortisol showed the lowest values for 5-year-old animals and the highest for 4-year-old animals. When the day of the competition was considered, 5-year-old animals showed the lowest salivary cortisol values on the first competition day and the highest on the second competition day, whereas 6-year-old animals showed lowest eye temperature values on the third competition day, and the 5-year-old animals showed the highest values on the third competition day. The analysis of variance (Table 2) showed significant differences (P<.05) for eye temperature measures taken among BC, JAC, and AC competition levels, whereas it showed no significant differences for salivary cortisol. Cortisol concentrations and eye temperature means during the three phases of the show jumping are presented in Figure 2. Both cortisol and IRT data responded similarly; however, eye temperature showed the greatest changes between BC-JAC and JAC-AC (0.98  C[P¼.0031] and 0.55  C [P¼.3523], respectively), whereas salivary cortisol showed the biggest difference between JAC-AC stages (1.39 ng/mL [P¼.4886]) and a smaller difference between BC and JAC stages (0.76 ng/mL [P¼.8402]). Spearman rank correlations between salivary cortisol intervals and eye temperature intervals showed a nonsignificant correlation of 0.02 for BC-JAC interval and a high and significant (P<.05) correlation of 0.71 for JAC-AC interval. 4. Discussion and Conclusion Previous studies investigating eye temperature responses in cattle showed that during the first few seconds of the stressor, eye temperature dropped rapidly (acute phase) because of a sympathetic response as part of the autonomic nervous system reaction [9,12]. Then, if the stressor persisted for a longer time, the hypothalamicpituitary-adrenal (HPA) axis produced an adrenocorticotropic hormone release and thus a cortisol response that Table 1 Mean SE salivary cortisol concentrations (log transformed) and eye temperature responses of horses of different ages (4 years old: n ¼7; 5 years old: n ¼5; 6 years old: n ¼4) for every competition day (CD) Q 9 CD Log Cortisol (ng/mL) Eye Temperature (  C) Mean SE Range Mean SE Range 4 years old (n ¼7) 10.13 0.69 1.35 36.03 1.43 2.80 2 2.02 1.11 2.56 35.70 0.91 2.40 3 0.81 3.17 9.42 35.97 0.89 2.60 Mean 1.10 2.20 9.42 35.87 0.95 3.00 5 years old (n ¼5) 11.30 4.42 9.22 35.94 0.72 2.10 2 4.06 0.78 1.10 36.55 2.47 3.50 3 0.41 1.49 3.62 36.48 1.72 3.80 Mean 0.11 4.51 9.42 36.08 1.11 3.50 6 years old (n ¼4) 1 0.48 3.57 8.65 35.64 1.06 2.90 2 0.55 2.01 7.19 35.94 1.24 3.70 3 0.22 0.17 0.24 35.25 0.78 1.10 Mean 0.53 2.47 8.66 35.84 1.16 3.70 Table 2 Univariate ANOVA for eye temperature and salivary cortisol (logarithmically transformed), with age, competition day, and phase within competition day as factors Log Cortisol (ng/mL) Eye Temperature (  C) FPFP Intercept 0,00 Q10 n.s. 18414,74 * Age 0,10 n.s. 0,30 n.s. Phase 0,49 n.s. 4,27 *** Competition day 1,50 n.s. 0,02 n.s. ANOVA, analysis of variance. *P<.001, ** P<.01, *** P<.05, n.s. ¼not significant. Fig. 2. Least square means for cortisol level ( ) and eye temperature ( ) values, measured 3 hours before the competition (BC), just after the competition (JAC), and 3 hours after the competition (AC), for horses of different ages (4 years old: n ¼7; 5 years old: n ¼5; and 6 years old: n ¼4). M. Valera et al. / Journal of Equine Veterinary Science xxx (2012) 1-4 3 FLA 5.1.0 DTD  YJEVS1333_proof  28 March 2012  5:26 pm 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 could be maintained from minutes to hours (chronic phase). However, recent studies by the same authors [13,14] showed no relationship between the increase in eye temperature and HPA axis activation. However, the thermal measurements in the current study were taken from the lachrymal caruncle. As such, this anatomical area is very sensitive to both pain and stress events affecting an animal, and these observed changes may be partly because of HPA activation. In contrast, Becker-Birk et al. [15] found that age was a key factor on the stress responses during the competition, as well as on the recovery time. Our results supported the results obtained by BeckerBirk et al. [15] and furthermore displayed different results depending on the method of stress assessment (IRT or salivary cortisol). The higher eye temperature of 4-year-old horses may be because of the stress of the novel situation, and the lower eye temperature of 6-year-old horses may be because of their previous experience at competitions and likely habituation to the event. We only found significant correlations between maximum eye temperature and cortisol increases between two phases, so that the higher the eye temperature difference among the third and second phases (3 hours after and just after the competition, respectively), the higher the cortisol interval. These results suggested that both techniques measure a physiological response to external stressors, despite possible differences in the origin of the stressor. Although Stewart et al. [9] found no evidence of a relationship between IRT and HPA in stressed cattle, Cook et al. [10] and Warren et al. [16] found significant correlations between maximum eye temperature and both salivary and plasma cortisol concentrations after an injection of adrenocorticotropic hormone in horses. Considering that cortisol causes several thermogenic reactions in tissue metabolism, including gluconeogenesis, glycolysis, proteolysis, and lipolysis, it is conceivable that changes in eye temperature as seen in the present study may be driven at least in part by an HPA axis response to stimulation. However, the timing of such responses as well as the relationship between cortisol and IRT response is complex and merits further examination. Previous studies investigating cortisol responses in horses have shown some evidence of recovery at 2 hours after competition and a salivary cortisol peak concentration of 2.2 0.4 ng/mL [15] that, when log transformation was applied, resulted in a value similar to that obtained in this study (0.34 ng/mL). Furthermore, the present results could be due to the possibility that 3 hours after competition was enough time for recovery in these sport horses. In contrast, the small differences found between the two first phases (BC and JAC) for salivary cortisol measurement could be because cortisol concentration was influenced by circadian rhythm [6], showing a peak early in the morning that could bias the results of these analysis, as all the measurements from the first phase were taken between 8:00 AM and 10:00 AM . These results suggest that IRT can detect differences in stress between phases within a competition. Furthermore, given the noninvasive and rapid nature of collecting infrared measures, IRT is an appropriate tool to assess the stress produced in horses during sport competitions. References [1] Minton JE. System in models of acute stress in domestic farm animals. Function of the hypothalamic-pituitary-adrenal axis and the sympathetic nervous. J Anim Sci 1994;72:1891-8. [2] Salak-Johnson JL, McGlone JJ. Making sense of apparently conflicting data: stress and immunity in swine and cattle. J Anim Sci 2007;85:81-8. [3] Stewart M, Webster JR, Schaefer AL, Cook NJ, Scott SL. Infrared thermography as a non-invasive tool to study animal welfare. Anim Welfare 2005;14:319-25. [4] Schmidt A, Möstl E, Aurich J, Neuhauser S, Aurich C. Comparison of cortisol and cortisone levels in blood plasma and saliva and cortisol metabolite concentrations in faeces for stress analysis in horses. In: Proceedings 5th International Equitation Science Conference (ISES); 2009; Sydney, Australia. [5] Schaefer AL, Matthews LR, Cook NJ, Webster J, Scott SL. Novel noninvasive measures of animal welfare. Animal welfare and behaviour: from science to solution. ISAE Conference. Hamilton, New Zealand; 2002. Q 8 [6] Irvine CHG, Alexander SL. Factors affecting the circadian rhythm in plasma cortisol concentrations in the horse. Domest Anim Endocrinol 1994;11:227-38. [7] Johnson SR, Rao S, Hussey SB, Morley PS, Traub-Dargatz JL. Thermographic eye temperature as an index to body temperature in ponies. J Equine Vet Sci 2011;31:63-6. [8] Jones WE. Thermography. J Equine Vet Sci 1998;18:422-3. [9] Stewart M, Webster JR, Verkerk GA, Schaefer AL, Colyn JJ, Stafford KJ. Non-invasive measurement of stress in dairy cows using infrared thermography. Physiol Behav 2007;92:520-5. [10] Cook NJ, Schaefer AL, Warren L, Burwash L, Anderson M, Baron V. Adrenocortical and metabolic responses to ACTH injection in horses: an assessment by salivary cortisol and infrared thermography of the eye. Can J Anim Sci 2001;81:621. [11] Statistica software, v. 8.0. Statsoft, Inc. www.statsoft.com.2007. [12] Stewart M, Stafford KJ, Dowling SK, Schaefer AL, Webster JR. Eye temperature and heart rate variability of calves disbudded with or without local anaesthetic. Physiol Behav 2008;93:789-97. [13] Stewart M, Verkerk GA, Stafford KJ, Schaefer AL, Webster JR. Noninvasive assessment of autonomic activity for evaluation of pain in calves, using surgical castration as a model. J Dairy Sci 2010;93: 3602-9. [14] Stewart M, Webster JR, Stafford KJ, Schaefer AL, Verkerk GA. Effects of an epinephrine infusion on eye temperature and heart rate variability in bull calves. J Dairy Sci 2010;93:5252-7. [15] Becker-Birck M, Schmidt A, Biau S, Möstl E, Morillon B, Aurich J, et al. Cortisol release in sport horses participating in equestrian competitions and events. In: Proceedings 6th International Equitation Science Conference (ISES); 2010; Uppsala, Sweden. [16] Warren LK, Cook NJ, Schaefer AL, Burwash L, Anderson M, Baron V, et al. The use of salivary cortisol as an index of stress in horses. In: Proceedings 17th Symposium Equine Nutrition and Physiology Society; 2001; Lexington, Kentucky. M. Valera et al. / Journal of Equine Veterinary Science xxx (2012) 1-44 FLA 5.1.0 DTD  YJEVS1333_proof  28 March 2012  5:26 pm 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 Our reference: YJEVS 1333 P-authorquery-v9 AUTHOR QUERY FORM Journal: YJEVS Article Number: 1333 Please e-mail or fax your responses and any corrections to: E-mail: [email protected] Fax: 717-738-9479 or 717-738-9478 Dear Author, Please check your proof carefully and mark all corrections at the appropriate place in the proof (e.g., by using on-screen annotation in the PDF file) or compile them in a separate list. 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