Manuscript number JEVS_2019_9_R1
Title Determination of salivary cortisol in donkey stallions Article type Short communication
Abstract
Salivary cortisol provides information about free plasma cortisol concentration and salivary sampling is a non-invasive well-tolerated procedure. The aim of this study was to validate a commercial enzyme immunoassay for the
determination of salivary cortisol in donkeys. Saliva samples were collected in 4 donkey stallions on thirteen non-consecutive days at 8:30 AM to avoid circadian variation. Animals were already accustomed to be handled. Saliva was collected by using a swab inserted at the angle of the lips, placed onto the tongue for 1 min and returned into a polypropylene tube. Tubes were centrifuged and at least 1 ml of saliva was aspirated from each sample and frozen at −20° C until analysis. A commercial enzyme immunoassay kit without extraction was used for determination of cortisol in saliva. Median cortisol concentrations with minimum and maximum value were calculated. Recovery of cortisol standard in donkey saliva was between 97.3% and 99.7% and serial dilution of donkey saliva samples with assay buffer resulted in changes in optical density parallel to the standard curve. Cross-reactivity of the antiserum was 10.4% with 11-deoxycortisol, 5.2% with corticosterone, 0.4% with 11-deoxycorticosterone, 0.2% with cortisone and <0.1% with testosterone, progesterone and estradiol. The intra-assay coefficient of variation was 10.7%, the inter-assay variation was 8.0% and the minimal detectable concentration was 0.01 ng/ml. The results of the present study demonstrate the validity of a commercial kit to determine the concentration of cortisol in donkey saliva, as already reported in other species.
Keywords donkey; salivary cortisol; validation; stress; enzyme immunoassay. Corresponding Author Micaela Sgorbini
Corresponding Author's Institution
Department of Veterinary Sciences, Veterinary teaching Hospital
Order of Authors FRANCESCA BONELLI, Alessandra Rota, Christine Aurich, Ille Natascha, Francesco Camillo, Duccio Panzani, Micaela Sgorbini
Suggested reviewers Fabrizio Rueca, Fulvio Laus
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Cover letter Bonelli et al. cortisol.docx [Cover Letter]
Cortisol in donkeys - Answer to reviewers.docx [Response to Reviewers] Highlights SALIVARY CORTISOL pos revision.docx [Highlights]
DETERMINATION OF SALIVARY CORTISOL IN DONKEY_post revision.docx [Manuscript File]
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Research Data Related to this Submission
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here is our revised paper titled “Determination of salivary cortisol in donkey stallions”, authored by Bonelli et al. The Ethical Committee, University of Pisa, approved this study. This study was supported totally by funds from the University of Pisa (100%) (Progetti di Ricerca di Ateneo: PRA_2016_53). No conflict of interest exits.
Yours sincerely,
1 Reviewer 1 (answers and comments in yellow).
The paper describe the validation of use for a commercial enzyme immunoassay for the determination of salivary cortisol in donkeys. The paper is well written and in my opinion provide useful information to the scientific community. The authors validate a method for evaluation of salivary cortisol in donkey, paving the way for further studies that will investigate the diagnostic use in clinic or in the assessment of well-being.
Minor revision:
Line 6: highlights should convey the main findings and not aims of the paper.
Answer: the highlights have been corrected and the main findings have been better reported. Line 13: Change "The Kit" with "A kit" or revise the sentence.
Answer: The sentence has been revised.
Line 83: Citation number 30 should be changed and "Mat and Met" section consequently revised. (For example see: Pearson and Ouassat, 2000 also cited in Quaresma M. et al, The veterinary Journal, 2013).
Answer: the citation has been changed. Line 93: it is 2017?
Answer: sorry for this mistake, the years were 2015 and 2016, as written in line 92. Line 95: delete "were".
Answer: done.
Line 97: what the authors mean with "special restraint"? Answer: the sentence has been better formulated.
Line 181: change: "...despite in the majority of the samples, the quantity of saliva was good enough for..." with "despite in the majority of the samples the quantity of saliva was good enough for..." Answer: correctio done.
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 53 54 55 56 57 58 59
2 Reviewer 2 (answers anc comments in green)
Determination of salivary cortisol in donkey stallions
Immunoassays have to be evaluated before being used in a given species. The manuscript describes a validation of a commercial enzyme immunoassay for measuring the cortisol concentration in donkey. Data of cortisol concentrations in saliva of 4 donkey stallions measured with this method are included. A denser sampling regime (more frequently than once a day) would have given more information (circadian rhythm and episodic spikes).
Answer: thank you for this comment. The salivary samplings schedule used in this paper was mandatory because the samples have been performed at the same time for two different protocols (please see Rota et al., 2018. Effect of housing system on reproductive behaviour and on some endocrinological and seminal parameters of donkey stallions. Reprod Dom Anim, 53(1): 40-47.
The manuscript may benefit if the scopes of applications of the different matrices (blood, saliva, urine and faeces) are discussed, as not in all cases measuring cortisol in saliva is the best way to monitor cortisol production. In the discussion it would be desirable to compare cortisol concentrations in the saliva of donkyes and horses.
Answer: the comparison between saliva cortisol concentrations of donkeys and horses has been added to the discussion section (please see lines 178-184, in grey).
Material and methods: how much saliva did you use per well?
Answer: the amount of saliva collected was at least 1 ml (please see line 100), because this the minimum amount needed for all the laboratory procedures.
Reviewer 3 (answer and comments in blue)
This is a nice manuscript that needs a few improvements in the english language. See comments below. 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118
3 In general, the biggest question is why no comparison to blood cortisol levels was done?
Answer: Thank you for this comment. The salivary cortisol evaluation has been performed to verify the effect of housing or semen collection in donkey stallion (please see Rota et al., 2018. Effect of housing system on reproductive behaviour and on some endocrinological and seminal parameters of donkey stallions. Reprod Dom Anim, 53(1): 40-47). Our study was performed as the validation of the kit used in the previous study and the sampling scheduling (saliva and blood) was mandatory in the way it has been performed.
How do you explain the high values of cortisol in your study compared to horses? Any ideas why basal levels in donkeys could be that much higher? How do you explain the big individual differences for the samples?
Answer: An explanation about the reason why in donkeys we found higher values of saliva cortisol has been added to the discussion section (please see 178-184, grey).
Highlights: Line 15 – donkeys. Answer: correction done.
Abstract: Line 18 - well-tolerated Answer: correction done.
Introduction:
Line 39/40 – belated physiological responses to a stressor Answer: correction done.
Line 56-58 – repitition of lines 50/51 , rephrase or emphasize why putting more than once 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179
4 Answer: the repetition has been delated.
Line 61 – other equids??? Why do I need to mention them? Line 61 - … the donkey achieved a …
Answer: correction done. Line 63 – donkeys have become Answer: correction done.
Line 78 – cleaned daily Answer: correction done.
Line 80 - according to NRC Answer: correction done.
Line 81 to 83 – move to results section. How was body weight determined? Neccessary fort he paper?
Lines 82-85 have been delated.
Line 87 – all 4 jacks were subjected to semen collection Answer: correction done.
Line 97 – special restraint? Please xplain in more detail what type of action was taken. Answer: the sentence has been rewerded.
Line 100 – 700g? 1800g? not clear how centrifuged. 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238
5 Answer: sorry for this mistake. The saliva samples have been centrifuged at 1000g for 10 min, as in the paper by Rota et al 2018, Reprod Dom Anim, 53(1): 40-47.
Table 1 – explain individual differences in discussion. Values are pretty high in genereal compared to basal lines in horses for example.
Line 149 – always stress?
Answer: “always” has been changed with “often” (Peeters et al. 2011, Equine Vet J, 43(4): 487-93.
Line 152 – delete „well“ Answer: correction done.
Line 160 - change none to no effect Answer: correction done.
Line 161-163 – reference 39 is about mice, not horses. Please check and use correct reference. Answer: a new reference has been added.
Conclusions
Line 180-182 – please rephrase
Answer: the sentence has been rephrased.
References:
Line 285 - correct references, spelling mistakes Answer: correction done.
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Highlights
The sampling procedure was easy to perform and stress-free for the animals.
The commercial kit used to determine the concentration of cortisol in donkey saliva was valid.
Cortisol evaluation in saliva could be considered highly suitable in donkeys 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 53 54 55 56 57 58
1 1 Determination of salivary cortisol in donkey stallions
2 Short title: Salivary cortisol in donkeys
3 Francesca Bonellia, Alessandra Rotaa, Christine Aurichb, Natascha Illeb, Francesco Camilloa, Duccio
4 Panzania, Micaela Sgorbinia
5 aDepartment of Veterinary Sciences, via Livornese snc, 56122 San Piero a Grado (PI), Italy
6 bCentre for Artificial Insemination and Embryo Transfer, University of Veterinary Sciences, Vienna,
7 Austria. 8
9 Corresponding Author: 10 Micaela Sgorbini
11 Department of Veterinary Sciences 12 via Livornese snc
13 56122 San Piero a Grado (PI), Italy 14 micaela.sgorbini@unipi.it 15 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 53 54 55
2 16 Abstract
17 Salivary cortisol provides information about free plasma cortisol concentration and 18 salivary sampling is a non-invasive well-tolerated procedure. The aim of this study was to 19 validate a commercial enzyme immunoassay for the determination of salivary cortisol in 20 donkeys.
21 Saliva samples were collected in 4 donkey stallions on thirteen non-consecutive days at 22 8:30 AM to avoid circadian variation. Animals were already accustomed to be handled. Saliva 23 was collected by using a swab inserted at the angle of the lips, placed onto the tongue for 1 min 24 and returned into a polypropylene tube. Tubes were centrifuged and at least 1 ml of saliva was 25 aspirated from each sample and frozen at −20° C until analysis. A commercial enzyme 26 immunoassay kit without extraction was used for determination of cortisol in saliva. Median 27 cortisol concentrations with minimum and maximum value were calculated.
28 Recovery of cortisol standard in donkey saliva was between 97.3% and 99.7% and serial 29 dilution of donkey saliva samples with assay buffer resulted in changes in optical density parallel 30 to the standard curve. Cross-reactivity of the antiserum was 10.4% with 11-deoxycortisol, 5.2% 31 with corticosterone, 0.4% with 11-deoxycorticosterone, 0.2% with cortisone and <0.1% with 32 testosterone, progesterone and estradiol. The intra-assay coefficient of variation was 10.7%, the 33 inter-assay variation was 8.0% and the minimal detectable concentration was 0.01 ng/ml.
34 The results of the present study demonstrate the validity of a commercial kit to determine 35 the concentration of cortisol in donkey saliva, as already reported in other species.
36 Key words: donkey; salivary cortisol; validation; stress; enzyme immunoassay. 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110
3 37 Introduction
38 Domestic animals react to stress through physiological responses, which are the results of 39 individual emotional reactivity [1]. The hypothalamic-pituitary-adrenocortical (HPA) system is 40 activated in response to a stressor. While early response is under catecholaminergic control, late 41 physiological response to stressor is commonly assessed by determination of glucocorticoid 42 concentrations, such as cortisol.
43 An increase in hypothalamic-pituitary-adrenocortical activity indicates a physiological response 44 to different stressors, and measurement of plasma corticosteroids is frequently used to study 45 middle and longtime stress response [2].
46 Cortisol was primarily obtained from blood, plasma or urine. Blood sampling itself (by syringe or 47 by catheterization) can produce stress in the animal causing a rise in cortisol levels [3,4]. Urine 48 and fecal samples can be easily and non-invasively collected without submitting animals to stress 49 [5,6], but the volume/unit weight varies between animals and species [5] and time periods 50 between application of the stressor and collection of the urine or feces cannot be easily 51 controlled. On the other hand the animals are hardly affected by saliva sample collection [7,8], 52 thus saliva can be considered a non-invasive monitoring of HPA functioning in animals.
53 Cortisol is passively diffused into the salivary glands, not bound to proteins and its concentration 54 is not affected by salivary flow rate. Therefore, the salivary cortisol provides direct information 55 about free plasma cortisol concentration, which is the only biologically active fraction in the 56 organism, owing to it being able to bind to cell receptors [9,10].
57 Furthermore, the collection of saliva is a non-invasive procedure, which animals usually tolerate 58 better than blood sampling. For all of these reasons, the interest in measurement of salivary 59 cortisol concentration in animals has increased during the past few years.
60 Salivary cortisol concentration has been evaluated in human [8,11,12], dogs [13-16], cattle [3,17-114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167
4 61 19], pigs [20,21] and horses [9,10,22-24].
62 In the last decades other equids, the donkey, achieved a relevant position in the human society 63 thanks to their employment in animal-assisted therapy [25] and milk production [26-28]. 64 Therefore, the welfare and stress responses of donkeys have become an interesting field of 65 research related to its behavior. To the authors’ knowledge there are no validated commercial kits 66 for the determination of salivary cortisol levels in donkeys. We followed the hypothesis that 67 determination of cortisol concentration in donkey saliva is possible. It was the aim of this study 68 to validate a commercial enzyme immunoassay for the determination of salivary cortisol 69 concentrations in donkeys.
70
71 Materials and methods
72 This study was conducted between September 2015 and February 2016 and was approved by the 73 Ethical Committee (OPBA) of the University of Pisa, according to D.lvo 26/2014. Four Amiata 74 donkey stallions (jacks), aged 3 years (born between May and July 2012) were included in this 75 study.
76 The jacks were kept together in a paddock (10 x 20 mt) for three months (September-November 77 2015). Then donkeys were housed for three months (December 2015-January 2016) in single 78 boxes (3.5 x 3.5 mt) with a small outside paddock (3.5 x 6 mt), where they could see each other, 79 but had no physical contact. Boxes were cleaned daily and bedded with straw. All the donkeys 80 had free access to water from a water bowl and were fed meadow hay ad libitum and commercial 81 concentrate (Equifioc®, Molitoria Val di Serchio, Lucca, Italy), according to NRC
82 recommendations for adult donkeys [29]. Weight was 280.3±30.4 kg when donkeys were moved 83 to boxes and 283.5±27.0 kg at the end of the study, with a difference of −3, +1, +10 and +5 kg in 84 the single animals, while Body Condition Score (BCS) was always evaluated between 5 and 6/9 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222
5 85 [30].
86 Saliva samples were collected on thirteen non-consecutive days at 8:30 AM to avoid circadian 87 variation [10] using a cotton-based swab (Salivette®, Sarstedt, Numbrecht-Rommelsdorf,
88 Germany) as described in horses [22,23]. Sampling time for cortisol evaluation was scheduled in 89 line with another project running simultaneously [31]. Briefly, all the 4 jacks were subjected to 90 semen collections thrice weekly (Tuesday, Thursday and Saturday) during 3 weeks in the month 91 of November 2015 and February 2016. One saliva sampling took place the week before the 92 beginning of semen collection (November 2015), while the other 12 samplings were carried out 93 every Tuesday and Saturday when semen collection was scheduled (6 samplings in November 94 2015 and 6 samplings in February 2016). Thus, 7 saliva sampling were done in November 2015 95 and 6 in February 2016.
96 Samplings were always made before feeding and cleaning procedures of the boxes and before 97 semen collection, when scheduled, with animals were at rest. Special care was used to minimize 98 stress during the sampling procedure, in particular, the same operator handled the animals 99 throughout the study and only manual restrain during saliva samplings was applied. The swab 100 was grasped with a surgical clamp, inserted at the angle of the lips into the mouth of the donkey 101 and placed gently onto the tongue for 1 min and afterwards returned into a polypropylene tube. 102 Tubes were then centrifuged for 10 min at 1000 g, at least 1 ml of saliva was aspirated from each 103 sample and the obtained saliva was frozen at −20° C until analysis.
104 A commercial enzyme immunoassay kit without extraction (Demeditec Diagnostics, Kiel-105 Wellsee, Germany) was used for determination of cortisol in saliva. The assay was validated for 106 asinine saliva in the laboratory of the unit for Obstetrics, Gynecology and Andrology, 107 Department for Small Animals and Horses, Vetmeduni (Vienna, Austria). Cross-reactivity of the 108 antiserum was determined at 50% binding with deoxycortisol, corticosterone, 11-226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279
6 109 deoxycorticosterone, cortisone, testosterone, progesterone and estradiol-17β. Recovery of cortisol 110 standards added to donkey saliva was tested. Parallelism between serial dilutions of saliva 111 samples with assay buffer and respective changes in optical density with respect to the standard 112 curve were determined. The intra-assay and inter-assay coefficients of variation were determined 113 from duplicates of control saliva with low and high cortisol concentrations run in each assay 114 (n=5). The minimal detectable concentration was defined as 2 standard deviations from zero 115 binding.
116 The Graph Pad Prism 6 (USA) software was used for statistical analysis. Data were analyzed for 117 normal distribution by Kolmogorov-Smirnov test. Because not all data were normally distributed, 118 results were expressed as median values of cortisol concentration and range calculated for each 119 day of sampling. Values are given as ng/ml.
120
121 Results
122 Saliva samples could be easily collected in all the donkeys enrolled in this study. The sampling 123 procedure was well accepted by all the animals. In one donkey (n. 4 table 1), the amount of saliva 124 collected was occasionally too small for analysis (samples 2nd, 6th and 9th).
125 Recovery of cortisol standard in donkey saliva was between 97.3% and 99.7% and serial dilution 126 of donkey saliva samples with assay buffer resulted in changes in optical density parallel to the 127 standard curve. Cross-reactivity of the antiserum at 50% binding was 10.4% with 11-128 deoxycortisol, 5.2% with corticosterone, 0.4% with 11-deoxycorticosterone, 0.2% with cortisone 129 and <0.1% with testosterone, progesterone and estradiol. The intra-assay coefficient of variation 130 was 10.7%, the inter-assay variation was 8.0% and the minimal detectable concentration was 0.01 131 ng/ml.
132 Results of cortisol concentrations determined in saliva collected from donkeys on different days 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334
7 133 are presented in Table 1.
134
Salivary cortisol concentration (ng/ml)
Donkeys I II III IV V VI VII VIII IX X XI XII XIII 1 3.9 3.6 10.8 3.6 6.2 1.5 4.6 3.4 5.8 5.4 4.6 5.6 4.8 2 0.5 7.25 8.7 8.11 7.5 1.73 1.7 4.7 5.9 2.9 8.1 1.9 1.9 3 9.4 5.36 20.6 7.72 1.0 6.6 2.8 3.7 9.2 3.0 5.0 6.9 2.1 4 3.1 * 4.7 10.38 9.5 * 9.7 5.6 * 3.7 11.8 4.2 2.7 Median 3 5 9 7.5 6.6 1 3 3.5 5 3 6.5 4.5 2 Range 0-9 3-7 4-20 3-10 1-9 1-6 1-9 3-5 5-9 2-5 4-11 1-6 1-4 135
136 Tab 1. Results of salivary cortisol concentration obtained in 4 individual donkey stallions on 137 13 non-consecutive days. Legend: *Amount of saliva not sufficient for cortisol analysis.
138
139 Discussion
140 The production of cortisol is regulated by the corticotrophic axis and can be altered in different 141 circumstances. Cortisol concentrations can rise in stressful situations, thus it is considered to be 142 an indicator of animal welfare [4]. Although traditionally cortisol concentrations have been 143 determined in plasma or serum, their determination in samples easily collectable under field 144 conditions (saliva, milk, urine, feces and hair) can be of interest for animal welfare studies in 145 different species [8,32-35]. Thus, in our study, a commercial kit assay system, originally used in 146 humans for measuring concentration of cortisol, was adapted and validated for donkey saliva 147 samples.
148 The evaluation of salivary cortisol has some advantages in large animals. Cortisol concentration 149 in saliva corresponds to the free fraction of cortisol in plasma, thus salivary cortisol represents a 150 better indicator of the possible effects of the corticotrophic axis on the animal organism than 151 plasma cortisol [17]. Moreover, blood collection often produces stress in the animal that can 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391
8 152 cause cortisol levels to rise [3,8]. On the other hand, saliva sampling, especially in already 153 handled animals, can be an easy and stress-free procedure as confirmed by our results.
154 All donkeys included in the study well tolerated the procedures and the amount of saliva 155 collected was good enough in the majority of the samples. The results of the present study 156 demonstrate the validity of a commercial kit to determine the concentration of cortisol in donkey 157 saliva, as already reported in dogs [13-15] and horses [9,10,22,23,37].
158 In the specificity tests, all the cross reactions observed were in line with the manufactory 159 performance characteristic declared. A very low cross-reaction has been found with 11-160 deoxycortisol (10.4%) and corticosterone (5.2%). The 11-deoxycortisol is a direct precursor of 161 cortisol and is found in plasma at lower concentrations than cortisol. Literature showed that the 162 11-deoxycortisol have no glucocorticoid effect and almost no mineral corticoid effect [38]. 163 Corticosterone is the predominant glucocorticoid produced in some species (i.e. mice, rats and 164 birds) [39]. Even if corticosterone is also synthesized in cortisol-dominant species, like equids, its 165 glucocorticoid activity in these species is still under assessment [40]. Cross-reactivity with 11-166 deoxycorticosterone, cortisone and with testosterone, progesterone and estradiol were negligible. 167 Intra- and inter-assay coefficients of variation are used to control the potential analytic error in 168 biological assay systems [41]. Our results showed a slightly increased intra- and inter-assay CV 169 % compared with the manufactory performance characteristic declaration (intra-assay CV of 3.8-170 5.8% and inter-assay CV of 6.4-6.2%). However, the validation results indicated that the method 171 was adequate for salivary cortisol measurements given that it is generally accepted that the CV 172 must be lower than 20% for analytical determinations [41]. The minimal detectable concentration 173 found in our study was slightly below those indicated by the manufactory guidelines (0.01 ng/ml 174 vs 0.024 ng/ml, respectively) and published in cows [17,42], but similar to results in pigs [20] and
175 horses [10]. 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446
9 176 The cortisol concentration in the saliva obtained in donkey stallions was higher respect to equine 177 mares or geldings [9,22,23], but similar to equine foals during the immediate postnatal period 178 [37].
179 In a recent study [10], saliva cortisol concentration has been assessed in equine stallion during the 180 winter-time (November-February). Saliva cortisol concentration obtained in jacks was higher 181 than in stallion [10]. Donkeys enrolled in this study were similar to horses included in the 182 previous study concerning sex and age. Moreover the saliva cortisol concentration was assessed 183 during the same season and using the same method. Thus, the higher saliva cortisol concentration 184 obtained in donkeys might be related to species.
185
186 Conclusions
187 In conclusion, given the advantages of the stress-free collection and processing of saliva and the 188 good performance of the assay, the use of cortisol detection in saliva could be considered highly 189 suitable in donkey stress research. This study has some limitation. Salivary and blood cortisol 190 concentrations have not been compared. However, when this was done in horses, the relationship 191 was very good [9,10]. Also, despite in the majority of the samples, the quantity of saliva was 192 good enough for running the assay, an improvement in saliva collection procedure need to be 193 addressed.
194
195 Acknowledgements
196 The study was funded by University of Pisa (Progetti di Ricerca di Ateneo: PRA_2016_53). We 197 are grateful to Ente Terre Regionali Toscane for allowing us to employ the animals for this study. 198 199 Fundings 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503
10 200 This research did not receive any specific grant from funding agencies in the public, commercial, 201 or not-for-profit sectors. 202 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558
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