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Breeding season and transport interactions on the pituitary-adrenocortical and biochemical responses of horses
Accepted Manuscript Breeding season and transport interactions on the pituitary-adrenocortical and biochemical responses of horses Pietro Medica, Cristina Cravana, Giuseppe Bruschetta, Adriana Ferlazzo, Esterina Fazio PII:
S1558-7878(17)30113-2
DOI:
10.1016/j.jveb.2017.11.003
Reference:
JVEB 1100
To appear in:
Journal of Veterinary Behavior
Received Date: 13 June 2017 Revised Date:
12 October 2017
Accepted Date: 9 November 2017
Please cite this article as: Medica, P., Cravana, C., Bruschetta, G., Ferlazzo, A., Fazio, E., Breeding season and transport interactions on the pituitary-adrenocortical and biochemical responses of horses, Journal of Veterinary Behavior (2017), doi: 10.1016/j.jveb.2017.11.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Breeding season and transport interactions on the pituitary-adrenocortical and
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biochemical responses of horses
3 Pietro Medica1, Cristina Cravana1, Giuseppe Bruschetta2, Adriana Ferlazzo1, Esterina Fazio1*
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Department of Veterinary Sciences, 1Unit of Veterinary Physiology, 2Unit of Veterinary
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Biochemistry, Messina University, 98168, Italy.
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*Corresponding author at: Esterina Fazio
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Abstract The aim of this study was to investigate the circulating ACTH and cortisol changes in breeding stallions in response to road transport before and after the breeding season. Creatinine, creatine
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kinase (CK), aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea and lactate
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dehydrogenase (LDH) changes were also considered. Twenty-seven healthy stallions were studied
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before and after transport over a distance of 200 km, before and after the breeding season. On the
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basis of the number of mares covered per stallion during the breeding season, each stallion was
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assigned to two groups: 17 stallions with a poor score:mean: 7.71 ± 4.67 mares per horse (group A);
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10 stallions with a good score, mean: 35.50 ± 10.66 mares per horse (group B). Increases in
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circulating ACTH concentrations were found after the first trip for group A stallions (P < 0.001),
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and after the first trip (P < 0.05) and return trip for group B stallions (P < 0.01). Increases in
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circulating cortisol concentrations were found after transport in both groups A and B (P < 0.001).
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Significant increases, as effect of the transport after the breeding season, were described for all
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biochemical parameters. Two-way ANOVA showed significant differences between groups A and B
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only for increases in cortisol and CK as an effect of post-transport plus the post-breeding season.
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This study showed that the pituitary-adrenocortical axis is efficiently stimulated by transport and that
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the breeding season augments the increase of cortisol and CK values of stallions as an effect of
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mental and physical arousal.
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Keywords: ACTH; biochemical parameters; cortisol; stallion; transport.
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Introduction
56 The transport of live animals during the breeding season has increased the need to assess the
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horse’s welfare and health (Friend, 2001). As reflected in changes in the hypothalamic-pituitary-
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adrenocortical (HPA) axis response, road transport is stressful for horses, and has a detrimental
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effect on welfare and performance (Schmidt et al., 2010a; 2010b). Because of this effect, equine
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stress studies may provide a good stress model (Deichsel et al., 2015). Moreover, the effects of
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transport stress have been widely investigated, evaluating the neuroendocrine (Fazio et al., 2009a;
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2009b; 2013a; 2013b; 2016a; 2016b), functional (Waran and Cuddeford, 1995; Schmidt et al.,
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2010a; 2010b), metabolic (Lindner and Hatzipanagiotou, 1998, Medica et al., 2010; Wessely-
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Szponder et al., 2015), behavioral (Waran, 1993; Tischner and Niezgoda, 2000; Broom, 2005;
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Padalino, 2015) and immunological responses (Stull et al., 2008; Padalino et al., 2017). Transiently
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increased cortisol release after transport over long distances, without negative effects on fertility,
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was recently observed in pony breeding stallions (Deichsel et al., 2015). Moreover, in stallions,
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mean salivary cortisol and plasma testosterone concentrations have been weakly correlated with an
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increase of cortisol concentrations in active sires during the breeding season (Aurich et al., 2015).
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Indeed, a blood cortisol increase has been observed in stallions in response to sexual arousal and
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semen collection (Rabb et al., 1989; Veronesi et al., 2010). Furthermore, the shift of energy
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metabolism in a catabolic or anabolic direction after exposure to stressful stimuli is characterized by
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a wide range of biochemical parameter changesthat generally mirror the metabolic state of an
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animal’s tissues, with respect to physical challenges and metabolic imbalances (Abeni et al., 2013;
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Noleto et al., 2016). The hypothesis was that the interaction between transport and the breeding
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season could affect the hormonal and biochemical homeostasis of breeding sires, with the
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expectation of a significant increase of stress markers, including muscle enzymes.
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On these bases, the aim of this study was to investigate the ACTH, cortisol, creatinine, CK, AST,
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ALT, urea and LDH changes of breeding stallions in response to road transport before and after the
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breeding season.
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Animals
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This study was approved by the Ethical Committee for the Care and Use of Animals of Messina
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University, in compliance with the guidelines of the Italian law (D.L. 4/3/2014 n. 26) and the EU
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Directive (Directive 2010/63) on the care and use of animals.
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Consent from the Equine Breeding Reproduction Centre of Catania was obtained for all horses to
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participate in the present study.
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This study was carried out on a total of 27 healthy pure Arabian and Anglo-Arabian breeding
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stallions, ranging in age 11.67 ± 5.02 years and with a mean weight of 435 ± 20 kg.
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The stallions were stabled at the Equine Breeding Reproduction Centre of Catania, Italy (latitude:
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37°30'4 68" N; longitude: 15°4'27 12" E), 380 m above sea level. All the animals were kept
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separate from other horses and were stabled in individual box stalls (4 x 4 m) on straw, under
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natural photoperiod and environmental temperature conditions. The stallions were individually fed
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with pelleted, complete supplement feed and vetch hay twice daily; fresh water and mineral
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supplements were freely available to maintain a physiological body condition. The stallions had
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been accustomed to transport and mating practice during the previous breeding seasons. All
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stallions were cooperative and showed an adequate sexual arousal and performance during the
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breeding routine. During breeding, all stallions achieved and maintained erection, covered the
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mares on the handler’s indication, and ejaculated within one or two mounts. The younger stallions,
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aged 4-5 years and with limited previous experiences of natural mounts, showed a slower and less
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consistent arousal and response for the first few coverings, but achieved ejaculation with one or two
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mounts. Two thirds of the older stallions (> 6 years) showed some precopulatory interaction,
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one mount. These stallions likely recognized routines, equipment or the breeding location. Training
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of both younger and mature breeding stallions involved operant conditioning to gradually encourage
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the stallions to cover a mare, according to the previous seasonal breeding experience.
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Specifically, the breeding activity in previous breeding season, represented by the number of mares
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covered per stallion ranged from 12 ± 2 mares for younger stallions and 31.50 ± 15.50 mares for
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older stallions. The reproductive history in previous years and the typical number of mares covered
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per stallion was in the range of 20 to 50 for older stallions.
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All subjects in this study had a normal sexual behavior, had no history of medical problems, had not
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received any pharmacological treatment for two weeks prior to the study and were healthy. The
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horses were transported from their previous state stud to various private stud farms between the last
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week of March and the last week of April, and returned in the first week of July.
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After the breeding season and on the basis of the mean number of mares successfully covered, paired
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to the mean number of mounts leading to ejaculation for each stallion per mare, the animals were
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assigned to two groups: 17 stallions with a poor score, mean: 7.71 ± 4.67 mares per stallion and
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breeding season (group A); 10 stallions with a good score, mean: 35.50 ± 10.66 mares per stallion
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and breeding season (group B). Stallions of group Awere 11.47 ± 5.01 years and stallions of group B
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were 12 ± 5.27 years.
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Experimental design
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Preliminary procedures (handling, loading, confinement and unloading) were carried out by the
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same staff. Six horses per load on four consecutive days (1st – 4th) and three horses on the last day
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(5th) of the same week were transported. The experimental design was the same for both trips to
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minimize effects of stabling time. Each stallion was always placed at the same location for both
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trips. Feed and water were provided before loading but not during transportation. The breeding
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season started on March 27 and ended on July 7. Covering of mares was performed once daily 5
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covered by the respective stallion. All stallions entered the various small state studs that did not
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keep a stallion specifically for this purpose, on March 25 and 26, and returned to the original state
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stud on July 8. The first covering after arrival regularly took place 24-48 h after transport. The time
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between the last covering and the return to the original state stud was 24 h.
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Natural cover in-hand involved the presentation of the stallion under halter to a mare that showed
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behavioral estrus, approximately 24-48 h prior to ovulation. To get maximal conception rates,
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mares were begun to be bred on the second or third day of receptivity to the stallion. Stallions and
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mares were allowed almost no contact other than brief and hand-directed precopulatory interaction
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immediately before the cover which was always performed in the morning between 7.00 and 9.00
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AM. Immediately before, the stallions were bridled and led into the covering barn, adjacent to their
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stable barn, by the same familiar person; immediately after entering, the stallion was exposed to the
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estrus mare. The stallion was allowed to sniff and touch the mare’s perineal and flank regions until
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the complete erection of the penis. Following successful erection, each stallion responded to the
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covered mare with repeated pelvic thrusts, until obtaining ejaculation. Ejaculation was
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demonstrated by: ragged tail of stallion (tail flagging), the display of rhythmic pulsations of the
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urethra on the ventral surface of the base of the penis. These behaviors were probably due to the
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ability of the stallions used in this study to respond to sub-optimal stimuli, either naturally or as a
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result of conditioning, as described by McDonnell (2000).
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Trailer design
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The commercial trailer (IVECO) used was 9.5 m long and 2.5 m wide, with a ceiling height of 2.5
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m. Six single compartments with swinging gates were available. Stocking density was about 2 m2/
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horse. Rubber padding lined the sides of the trailers from the floor up to an approximate height of
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1.2 m. The same trailer and the same driver were used in this study (one trip per day). The distance
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travelled and the time between loading and unloading were 200 km and about 2 h, respectively. 6
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humidity were 19 °C and 62% (March/April), and 25 °C and 58% (July), respectively. A
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thermohygrometer (Model: Hygrothermograph ST-50, Sekonic Corporation, Tokyo, Japan) was
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used to monitor the temperature and relative humidity inside the trailer. On the first trip temperature
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and relative humidity inside the trailer were 22 °C and 80% after 1 h, and 23 °C and 81% after 2 h;
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when returning temperature and relative humidity were 28 °C and 65% after 1 h, and 29.5 °C and
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62% after 2 h.
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Processing of samples and analytical methods
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Blood sampling was always done by the same person. .
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The first blood samples were collected from the jugular vein immediately before loading, while
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horses were in their single box, at 07:00 AM (baseline samples). Transport started at 08:00 AM.
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The second blood samples were collected immediately after transport and unloading, on arrival at
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the different stud farms (post-transport samples), at 10:00 AM. Blood samples were collected
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immediately before loading (T0), after unloading of the first trip (T1) on arrival at the state stud;
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immediately before loading (T2) and after unloading when returning (T3) on arrival at the origin
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stud farm. This procedure took just few seconds for each horse. Horses were restrained with
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halters. The time points of the blood samples were the same for the first trip and when returning to
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minimize the time effects.
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Blood samples were collected using evacuated tubes (Venoject, Terumo®; Belgium) and were
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immediately refrigerated at 4°C after collection, and subsequently (within 1 h) centrifuged at 3,000g
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for 15 minutes at 0°C. Serum was harvested and stored in polystyrene tubes (Polyst test tube Sorvall
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CW1, Asti, Italy) at -20°C until assayed for cortisol and haematochemical parameters.
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Serum ACTH concentrations were analyzed in duplicate using a commercially available
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radioimmunoassay kit (ELSA-ACTH, CIS-Bio International, Gif-sur-Yvette, France), suitable for
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equine use (Ferlazzo et al., 1998). The hormone assay used has a range for the amount of ACTH
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interassay CVs were 6.0 % and 15.0 %, respectively.
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Total serum cortisol concentrations were analyzed in duplicate using a competitive enzyme-linked
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immunoassay (EIA, RADIM, Rome, Italy), suitable for equine use (Ferlazzo et al., 1998). During
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the first incubation, the cortisol sample competed with cortisol conjugated to horseradish peroxidase
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(HRPO) for the specific sites of the antiserum coated on the wells. After incubation, all unbound
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material was removed by aspiration and washing. The enzyme activity bound to the solid phase was
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inversely proportional to cortisol concentration in calibrators and samples and is made evident by
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incubating the wells with a chromogen solution (tetramethylbenzidine) in substrate buffer.
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Colorimetric reading was carried out using a spectrophotometer at 405 nm wave length (Sirio S,
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SEAC, Florence, Italy). Assay sensitivity was 5 ng/mL. The intraassay and interassay CVs were
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4.0% and 6.9%, respectively.
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Serum metabolic parameters (creatinine, CK, urea, AST, ALT, LDH) were assessed using a kinetic
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method by a biochemistry autoanalyzer (SLIM, SEAC, Florence, Italy), using commercially
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available tests of SEAC/Radim kits (Pomezia, Rome, Italy).
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198 Statistics
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Data are presented as mean ± standard deviation (SD). A two-way analysis of variance with
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repeated measures (two way RM-ANOVA) was applied to test for the effects of the different types
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of stimuli: transport, different number of covered mares (Group A and Group B) and sampling time
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(T0, T1, T2 and T3), as well as for the interactions between them, on hormonal concentrations and
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biochemical values. When the F value was significant (P < 0.05), the differences between
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individual means over time were assessed using a post-hoc multiple comparison test (Bonferroni).
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All calculations were performed using the PRISM package (GraphPad Software Inc., San Diego,
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CA, USA).
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Results
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Pituitary-adrenocortical concentrations and metabolic values are presented as mean ( ± SD) in
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Figures 1, 2 and Table 1, respectively.
212 ACTH
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Compared to T0, significant increases in circulating ACTH concentrations at T1 in group A
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(P<0.001), and at T1 (P<0.05) and T3 (P<0.01) in group B were observed. No significant differences
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in ACTH concentrations between the two Groups A and B were observed.
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217 Cortisol
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Compared to T0, significant increases of cortisol concentrations in both groups A and B at T2 (P<0.01)
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and at T3 (P<0.001) were observed.
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The two-way RM-ANOVA indicated that there was a significant interaction between the Groups A and
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B and time on cortisol changes (F= 4.45; P=0.020).
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Specifically, Group A showed a significant interaction between the first trip and when returning and
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time for cortisol (F= 63.35; P=0.00), with higher serum cortisol concentrations when returning at T2
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(P=0.05) and T3 (P=0.01) than at T0 and T1 of the first trip, respectively. Similarly, Group B
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showed a significant interaction between both trips and time for cortisol (F= 120.95; P=0.000), with
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higher serum cortisol concentrations when returning at T2 (P=0.05) and T3 (P=0.01) than at T0 and
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T1 of the first trip, respectively.
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Metabolic parameters
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Compared to T0, significant increases of CK in both groups A and B at T1 (P<0.001), T2 (P<0.01) and
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T3 (P<0.001) were observed. The two way RM-ANOVA indicated that there was a significant
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interaction between the Groups A and B and time on CK changes (F= 3.70; P=0.01).
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changes (F= 72.75; P=0.001), with higher serum CK concentrations when returning at T2 (P<0.05)
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and T3 (P<0.001) compared to T0 and T1 of the first trip, respectively. Similarly, Group B showed a
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significant interaction between the first trip and when returning and time on CK changes (F= 190.95;
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P=0.0001), with higher serum CK concentrations when returning at T2 (P<0.05) and T3 (P<0.001)
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than at T0 and T1 of the first trip, respectively.
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Also, there was a significant effect of different number of mares covered per stallion and time on CK
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changes (F= 9.33; P<0.005), with higher mean concentrations of CK in Group B than Group A at T2
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(P<0.05).
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Compared to T0, increases were observed at T1 and T3 in creatinine concentrations and ALT
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activities in both Groups A (P < 0.05) and B (P < 0.01); in LDH activities at T1 in both Groups A
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and B (P < 0.001); in urea concentrations in both Groups A and B (P < 0.05); in AST activities at
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T1 (P < 0.01) and T2 (P < 0.001) in both Groups A and B.
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Discussion
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The comparisons of the hormonal results obtained in the current study with published data reported
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for horses did not reveal any marked discrepancies for baseline circulating ACTH (Donaldson et
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al., 2005; Niinistő et al., 2010; Ayala et al., 2012) and cortisol (Waran, 1993; Stull and Rodiek,
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2002; Smiet et al., 2014) concentrations.
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The present study is in line with previous studies, demonstrating that previous travelling experience,
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with the regular transportation, does not reduce the early adrenocortical response to transport,
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perceived as a stressful challenge (Fazio et al., 2008a; 2008b; Schmidt et al., 2010a; 2010b). The
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concomitant increase of ACTH and cortisol concentrations after the 200 km journey, compared to
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the baseline values, confirmed that a pituitary-adrenocortical system response to travel with a
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substantial release of both hormones (Ferlazzo et al., 2012). These increases suggest that the
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season, as suggested by the highest cortisol concentrations observed at T2 and T3 in both Groups A
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and B. Although the cortisol concentrations were clearly elevated in response to transport, the higher
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increase after returning could be due to the additional effects of breeding season, confirming that the
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HPA axis is responsive to horses’ and donkeys’ sexual excitement (Villani et al., 2006; Veronesi et
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al., 2011).
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Nevertheless, no significant differences were observed between Groups A and B, independently of
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the number of mares covered in the breeding season per stallion, nor between different ages of
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stallions. With respect to age effects, it is possible to presume that the breeding season was
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comparable to moderate exercise training that attenuated the effect of age on the cortisol response, as
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observed in horses after acute exertion (Malinowski et al., 2006). No correlations between the
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stallions’ age and fertility were observed. The qualitative and quantitative intra- and inter-stallion
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variations in sperm morphology and fertility showed no consistent differences. Indeed, in both
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Groups A and B the stallions showed an excellent fertility and very high conception rate.
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It is possible to conclude that transport stress induced the greatest pituitary-adrenocortical response,
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which suggests that the transported stallions continuously activated their various, aspecific,
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biological responses regardless of stressful physical and mental stimuli. These data confirm the
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recent effective influence of equine resistant mechanisms to short-term stress, and support the
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hypothesis that breeding stallions can be transported without negatively affecting their fertility
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(Deichsel et al., 2015), as previously reported also for fertility of mares (Berghold et al., 2007). In
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any case, the higher release of cortisol after returning (T3) than on arrival (T1) can be used as a
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reliable marker of stress, confirming that horses are a prey species and respond to stressful
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situations as such as physical or mental stimuli with increased cortisol release (Hada et al., 2003;
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Forkman et al., 2007). Finally, our data confirm the observation of AboEl-Maaty (2011), showing
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that horses held in small state studs, had higher cortisol levels compared to those of the Equine
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Breeding Reproduction Centre, where the welfare conditions are probably better monitored.
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circulating creatinine, urea, ALT and LDH values (Lindner and Hatzipanagiotou, 1998; Wessely-
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Szponder et al., 2015; Noleto et al., 2016; Padalino et al., 2017).
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The metabolic demand increases after workload and exercise, according to the intensity, duration
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and/or distance, as well as the training state, and may induce changes in bloodstream biochemical
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and endocrinological parameters that can affect the horse’s performance (Wanderly et al., 2015)
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The fact that the highest serum CK, AST, ALT and LDH, associated with creatinine and urea
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increases after transport, can be attributed to the skeletal muscle stress, generated during transport
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by the physical activity of posture, and the consequences of the effort made during breeding
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activity plus postural support. The highest increase of creatinine concentrations and CK activities
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showed that transport is comparable to physical exercise, since it involves muscle activity. These
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increases can be evaluated on the basis of the training state of horses (Overgaard et al., 2004;
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Wessely-Szponder et al., 2015). Moreover, it is well known that under routine physiological
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conditions skeletal muscle fibres release a significant CK even in the absence of muscle damage
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(Muñoz et al., 2002). The CK pattern confirmed that there was a rapid increase in its activity
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within 4-12 h from the damaging stimulus (Valberg et al., 1993), and may reflect the muscular
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effort of standing up during transport. Moreover, the highest CK, AST and LDH activities
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observed after transport could be attributed to the challenges of membrane permeability, or to the
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increases of their synthesis or decreases of their clearance, according to physical workload (Boyd,
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1985; Wessely-Szponder et al., 2015).
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It is therefore feasible that the adrenergic system, involved during the first stages of transport,
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induced a consequent increase of enzyme activities, representing a stronger stress-causing factor
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than others (Tischner and Niezgoda, 2000; Thomassian et al., 2007).
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The CK pathway related to skeletal muscle metabolism proved to be a sensitive marker in relation to
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muscle workload, as shown by the higher values after the return. On the other hand, the increases of
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AST, ALT and LDH activities are not in line with changes observed in horses during transport,
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usinga different orientation (Padalino et al., 2012).
313 Conclusion
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The hypothesis that there is a temporal interaction between transport and the breeding season of
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stallions was confirmed by the significant increase of stress markers, including muscle enzymes.
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This study showed that the pituitary-adrenocortical axis is efficiently stimulated by transport and that
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the breeding season strengthens the increase of cortisol and CK values of stallions as an effect of
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mental and physical arousal.
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From a practical point of view, the evaluation of the reference values and related changes of
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endocrine and biochemical variables of stallions at the start of breeding season represents an
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additional requirement for future implementation of reliable markers for the seasonal breeders’
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status. The potential positive effectof normal behavior on reproductive success remains an important
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matter for discussion.
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325 Acknowledgments
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The authors thank the team of Equine Breeding Reproduction Centre of Catania, Italy, veterinary
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practitioners and breeding farm managers who collaborated with this study. This research did not
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receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
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Ethical considerations
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This study was approved by the Ethical Committee for the Care and Use of Animals of
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University of Messina, in compliance with the guidelines of the Italian law on the care and
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use of animals (D.L. 4/3/2014 n. 26) and the EU Directive (Directive 2010/63).
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and AST activities in plasma of Standardbred horses during exercise. Pferdeilkunde 14, 456-460.
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2010a. Changes in cortisol release and heart rate variability in sport horses during long-distance
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road transport. Domest. Anim. Endocrinol. 38, 179-189.
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heart rate variability in horses during road transport. Horm. Behav. 57, 209-215. 18
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W., Wijnberg, L.D., van der Kolk, J.H. 2014. Effect of different head and neck positions on
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Nicoletti, J.L.M. 2007. Atividades séricas da aspartato aminotransferase, creatina quinase e lactato
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Valberg, S., Johnson, L., Lindholm, A., Holmgren, N. 1993. Muscle histopathology and plasma
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aspartate aminotransferase, creatine kinase and myoglobin changes with exercise in horses with
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recurrent exertional rhabdomyolysis. Equine Vet. J. 25, 11-16.
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Veronesi, M.C., Tosi, U., Villani, M., Govoni, N., Faustini, M., Kindahl, H. 2010. Oxytocin,
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vasopressin, prostaglandin F2alpha, hormone, testosterone, estrone sulfate, and cortisol plasma
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concentrations after sexual stimulation in stallions. Theriogenology 73, 460-467.
490 Veronesi, M.C., De Amicis, I., Panzani, S., Kindahl, H., Govoni, N., Probo, M., Carluccio, A. 2011.
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PGF2α, LH, testosterone, oestrone sulphate, and cortisol plasma concentrations around sexual
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stimulation in jackass. Theriogenology 75, 1489-1498.
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Villani, M., Cairoli, F., Kindahl, H., Galeati, G., Faustini, M., Carluccio, A., Veronesi, M.C. 2006.
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Effects of mating on plasma concentrations of testosterone, cortisol, oestrone sulphate and 15-
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ketodihydro-PGF2alfa in stallions. Reprod. Domest. Anim. 1, 544-548.
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Wanderly, E.K., Bem, B.S.C., Melo, S.K.M., González, J.C., Manso, H.E.C.C.C., Manso Fiho,
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H.C. 2015. Haematological and biochemical changes in Mangalaga Marchador horses after a four-
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beat gait challenge in three different distances. J. Equine Vet. Sci. 35, 269-273.
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of horses. Appl. Anim. Behav. Sci. 43, 71-81.
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Table 1 - Mean values (mean ± SD) of the bloodstream biochemical parameters of stallions before and after transports
Groups
Returning Before T2
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Parameters
The first trip Before T0 After T1
After T3
325.66 ± 40.23c 328.96 ± 24.85c
243.10 ± 33.15 bA 303.10 ± 32.17 bAα
creatinine (mg/dL) A B
1.34 ± 0.40 1.39 ± 0.46
2.07 ± 0.68a 2.13 ± 0.56b
1.38 ± 0.62 2.13 ± 0.56b
2.10 ± 0.17 a 2.44 ± 0.33b
urea (mg/dL)
A B
21.27 ± 5.22 20.42 ± 6.59
27.54 ± 8.29a 27.21 ± 5.39a
20.93 ± 5.18 20.81 ± 3.11
28.08 ± 4.15 a 28.19 ± 4.08 a
AST (U/L)
A B
107.64 ± 25.03 103.56 ± 35.51
287.62 ± 55.51b 272.33 ± 55.50b
111.05 ± 38.05 114.09 ± 33.20
383.02 ± 22.13 c 339.03 ± 23.69 c
ALT (U/L)
A B
20.70 ± 6.12 20.73 ± 8.38
30.10 ± 6.39a 29.23 ± 6.23b
22.33 ± 3.34 24.22 ± 4.14
27.10 ± 3.24a 30.39 ± 3.27b
LDH (U/L)
A B
164.79 ± 45.47 170.91 ± 38.05
289.95 ± 42.49c 192.82 ± 50.02c
155.23 ± 20.04 181.33 ± 23.13
275.45 ± 30.15c 301.25 ± 30.44c
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433.10 ± 43.18cB 452.10 ± 43.12cB
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121.33 ± 20.51 131.11 ± 31.59
CK (U/L)
Superscripts indicate significant (aP < 0.05; bP < 0.01; cP < 0.001) differences vs T0. Superscripts indicate significant (AP < 0.05; BP < 0.01) differences vs the first trip. Superscript indicates significant (αP < 0.05) difference vs Group A.
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Figure 1 - Circulating ACTH concentrations (mean ± SD) of stallions before and after transports
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Asterisks indicate significant (*P < 0.05; **P < 0.01; ***P < 0.001)) differences vs T0.
Figure 2 - Circulating cortisol concentrations (mean ± SD) of stallions before and after transports Asterisks indicate significant (*P < 0.01; **P < 0.001) differences vs T0
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Symbols indicate significant (●P < 0.05; ●●P < 0.01) differences vs the first trip
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ACCEPTED MANUSCRIPT HIGHLIGHTS ● The horses’ pituitary-adrenocortical responses to transport were evaluated ● The exploration of biochemical parameters was also considered. ● The concomitant increases of ACTH and cortisol after transport were observed.
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● Significant increases of all biochemical parameters after transport were described
S1558-7878(17)30113-2
DOI:
10.1016/j.jveb.2017.11.003
Reference:
JVEB 1100
To appear in:
Journal of Veterinary Behavior
Received Date: 13 June 2017 Revised Date:
12 October 2017
Accepted Date: 9 November 2017
Please cite this article as: Medica, P., Cravana, C., Bruschetta, G., Ferlazzo, A., Fazio, E., Breeding season and transport interactions on the pituitary-adrenocortical and biochemical responses of horses, Journal of Veterinary Behavior (2017), doi: 10.1016/j.jveb.2017.11.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
Breeding season and transport interactions on the pituitary-adrenocortical and
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biochemical responses of horses
3 Pietro Medica1, Cristina Cravana1, Giuseppe Bruschetta2, Adriana Ferlazzo1, Esterina Fazio1*
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Department of Veterinary Sciences, 1Unit of Veterinary Physiology, 2Unit of Veterinary
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Biochemistry, Messina University, 98168, Italy.
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*Corresponding author at: Esterina Fazio
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Abstract The aim of this study was to investigate the circulating ACTH and cortisol changes in breeding stallions in response to road transport before and after the breeding season. Creatinine, creatine
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kinase (CK), aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea and lactate
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dehydrogenase (LDH) changes were also considered. Twenty-seven healthy stallions were studied
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before and after transport over a distance of 200 km, before and after the breeding season. On the
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basis of the number of mares covered per stallion during the breeding season, each stallion was
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assigned to two groups: 17 stallions with a poor score:mean: 7.71 ± 4.67 mares per horse (group A);
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10 stallions with a good score, mean: 35.50 ± 10.66 mares per horse (group B). Increases in
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circulating ACTH concentrations were found after the first trip for group A stallions (P < 0.001),
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and after the first trip (P < 0.05) and return trip for group B stallions (P < 0.01). Increases in
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circulating cortisol concentrations were found after transport in both groups A and B (P < 0.001).
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Significant increases, as effect of the transport after the breeding season, were described for all
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biochemical parameters. Two-way ANOVA showed significant differences between groups A and B
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only for increases in cortisol and CK as an effect of post-transport plus the post-breeding season.
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This study showed that the pituitary-adrenocortical axis is efficiently stimulated by transport and that
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the breeding season augments the increase of cortisol and CK values of stallions as an effect of
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mental and physical arousal.
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Keywords: ACTH; biochemical parameters; cortisol; stallion; transport.
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Introduction
56 The transport of live animals during the breeding season has increased the need to assess the
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horse’s welfare and health (Friend, 2001). As reflected in changes in the hypothalamic-pituitary-
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adrenocortical (HPA) axis response, road transport is stressful for horses, and has a detrimental
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effect on welfare and performance (Schmidt et al., 2010a; 2010b). Because of this effect, equine
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stress studies may provide a good stress model (Deichsel et al., 2015). Moreover, the effects of
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transport stress have been widely investigated, evaluating the neuroendocrine (Fazio et al., 2009a;
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2009b; 2013a; 2013b; 2016a; 2016b), functional (Waran and Cuddeford, 1995; Schmidt et al.,
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2010a; 2010b), metabolic (Lindner and Hatzipanagiotou, 1998, Medica et al., 2010; Wessely-
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Szponder et al., 2015), behavioral (Waran, 1993; Tischner and Niezgoda, 2000; Broom, 2005;
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Padalino, 2015) and immunological responses (Stull et al., 2008; Padalino et al., 2017). Transiently
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increased cortisol release after transport over long distances, without negative effects on fertility,
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was recently observed in pony breeding stallions (Deichsel et al., 2015). Moreover, in stallions,
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mean salivary cortisol and plasma testosterone concentrations have been weakly correlated with an
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increase of cortisol concentrations in active sires during the breeding season (Aurich et al., 2015).
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Indeed, a blood cortisol increase has been observed in stallions in response to sexual arousal and
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semen collection (Rabb et al., 1989; Veronesi et al., 2010). Furthermore, the shift of energy
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metabolism in a catabolic or anabolic direction after exposure to stressful stimuli is characterized by
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a wide range of biochemical parameter changesthat generally mirror the metabolic state of an
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animal’s tissues, with respect to physical challenges and metabolic imbalances (Abeni et al., 2013;
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Noleto et al., 2016). The hypothesis was that the interaction between transport and the breeding
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season could affect the hormonal and biochemical homeostasis of breeding sires, with the
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expectation of a significant increase of stress markers, including muscle enzymes.
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On these bases, the aim of this study was to investigate the ACTH, cortisol, creatinine, CK, AST,
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ALT, urea and LDH changes of breeding stallions in response to road transport before and after the
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breeding season.
82 Materials and Methods
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Animals
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This study was approved by the Ethical Committee for the Care and Use of Animals of Messina
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University, in compliance with the guidelines of the Italian law (D.L. 4/3/2014 n. 26) and the EU
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Directive (Directive 2010/63) on the care and use of animals.
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Consent from the Equine Breeding Reproduction Centre of Catania was obtained for all horses to
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participate in the present study.
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This study was carried out on a total of 27 healthy pure Arabian and Anglo-Arabian breeding
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stallions, ranging in age 11.67 ± 5.02 years and with a mean weight of 435 ± 20 kg.
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The stallions were stabled at the Equine Breeding Reproduction Centre of Catania, Italy (latitude:
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37°30'4 68" N; longitude: 15°4'27 12" E), 380 m above sea level. All the animals were kept
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separate from other horses and were stabled in individual box stalls (4 x 4 m) on straw, under
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natural photoperiod and environmental temperature conditions. The stallions were individually fed
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with pelleted, complete supplement feed and vetch hay twice daily; fresh water and mineral
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supplements were freely available to maintain a physiological body condition. The stallions had
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been accustomed to transport and mating practice during the previous breeding seasons. All
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stallions were cooperative and showed an adequate sexual arousal and performance during the
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breeding routine. During breeding, all stallions achieved and maintained erection, covered the
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mares on the handler’s indication, and ejaculated within one or two mounts. The younger stallions,
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aged 4-5 years and with limited previous experiences of natural mounts, showed a slower and less
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consistent arousal and response for the first few coverings, but achieved ejaculation with one or two
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mounts. Two thirds of the older stallions (> 6 years) showed some precopulatory interaction,
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one mount. These stallions likely recognized routines, equipment or the breeding location. Training
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of both younger and mature breeding stallions involved operant conditioning to gradually encourage
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the stallions to cover a mare, according to the previous seasonal breeding experience.
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Specifically, the breeding activity in previous breeding season, represented by the number of mares
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covered per stallion ranged from 12 ± 2 mares for younger stallions and 31.50 ± 15.50 mares for
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older stallions. The reproductive history in previous years and the typical number of mares covered
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per stallion was in the range of 20 to 50 for older stallions.
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All subjects in this study had a normal sexual behavior, had no history of medical problems, had not
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received any pharmacological treatment for two weeks prior to the study and were healthy. The
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horses were transported from their previous state stud to various private stud farms between the last
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week of March and the last week of April, and returned in the first week of July.
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After the breeding season and on the basis of the mean number of mares successfully covered, paired
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to the mean number of mounts leading to ejaculation for each stallion per mare, the animals were
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assigned to two groups: 17 stallions with a poor score, mean: 7.71 ± 4.67 mares per stallion and
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breeding season (group A); 10 stallions with a good score, mean: 35.50 ± 10.66 mares per stallion
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and breeding season (group B). Stallions of group Awere 11.47 ± 5.01 years and stallions of group B
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were 12 ± 5.27 years.
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Experimental design
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Preliminary procedures (handling, loading, confinement and unloading) were carried out by the
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same staff. Six horses per load on four consecutive days (1st – 4th) and three horses on the last day
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(5th) of the same week were transported. The experimental design was the same for both trips to
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minimize effects of stabling time. Each stallion was always placed at the same location for both
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trips. Feed and water were provided before loading but not during transportation. The breeding
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season started on March 27 and ended on July 7. Covering of mares was performed once daily 5
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covered by the respective stallion. All stallions entered the various small state studs that did not
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keep a stallion specifically for this purpose, on March 25 and 26, and returned to the original state
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stud on July 8. The first covering after arrival regularly took place 24-48 h after transport. The time
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between the last covering and the return to the original state stud was 24 h.
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Natural cover in-hand involved the presentation of the stallion under halter to a mare that showed
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behavioral estrus, approximately 24-48 h prior to ovulation. To get maximal conception rates,
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mares were begun to be bred on the second or third day of receptivity to the stallion. Stallions and
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mares were allowed almost no contact other than brief and hand-directed precopulatory interaction
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immediately before the cover which was always performed in the morning between 7.00 and 9.00
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AM. Immediately before, the stallions were bridled and led into the covering barn, adjacent to their
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stable barn, by the same familiar person; immediately after entering, the stallion was exposed to the
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estrus mare. The stallion was allowed to sniff and touch the mare’s perineal and flank regions until
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the complete erection of the penis. Following successful erection, each stallion responded to the
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covered mare with repeated pelvic thrusts, until obtaining ejaculation. Ejaculation was
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demonstrated by: ragged tail of stallion (tail flagging), the display of rhythmic pulsations of the
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urethra on the ventral surface of the base of the penis. These behaviors were probably due to the
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ability of the stallions used in this study to respond to sub-optimal stimuli, either naturally or as a
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result of conditioning, as described by McDonnell (2000).
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Trailer design
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The commercial trailer (IVECO) used was 9.5 m long and 2.5 m wide, with a ceiling height of 2.5
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m. Six single compartments with swinging gates were available. Stocking density was about 2 m2/
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horse. Rubber padding lined the sides of the trailers from the floor up to an approximate height of
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1.2 m. The same trailer and the same driver were used in this study (one trip per day). The distance
156
travelled and the time between loading and unloading were 200 km and about 2 h, respectively. 6
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humidity were 19 °C and 62% (March/April), and 25 °C and 58% (July), respectively. A
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thermohygrometer (Model: Hygrothermograph ST-50, Sekonic Corporation, Tokyo, Japan) was
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used to monitor the temperature and relative humidity inside the trailer. On the first trip temperature
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and relative humidity inside the trailer were 22 °C and 80% after 1 h, and 23 °C and 81% after 2 h;
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when returning temperature and relative humidity were 28 °C and 65% after 1 h, and 29.5 °C and
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62% after 2 h.
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Processing of samples and analytical methods
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Blood sampling was always done by the same person. .
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The first blood samples were collected from the jugular vein immediately before loading, while
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horses were in their single box, at 07:00 AM (baseline samples). Transport started at 08:00 AM.
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The second blood samples were collected immediately after transport and unloading, on arrival at
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the different stud farms (post-transport samples), at 10:00 AM. Blood samples were collected
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immediately before loading (T0), after unloading of the first trip (T1) on arrival at the state stud;
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immediately before loading (T2) and after unloading when returning (T3) on arrival at the origin
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stud farm. This procedure took just few seconds for each horse. Horses were restrained with
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halters. The time points of the blood samples were the same for the first trip and when returning to
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minimize the time effects.
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Blood samples were collected using evacuated tubes (Venoject, Terumo®; Belgium) and were
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immediately refrigerated at 4°C after collection, and subsequently (within 1 h) centrifuged at 3,000g
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for 15 minutes at 0°C. Serum was harvested and stored in polystyrene tubes (Polyst test tube Sorvall
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CW1, Asti, Italy) at -20°C until assayed for cortisol and haematochemical parameters.
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Serum ACTH concentrations were analyzed in duplicate using a commercially available
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radioimmunoassay kit (ELSA-ACTH, CIS-Bio International, Gif-sur-Yvette, France), suitable for
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equine use (Ferlazzo et al., 1998). The hormone assay used has a range for the amount of ACTH
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interassay CVs were 6.0 % and 15.0 %, respectively.
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Total serum cortisol concentrations were analyzed in duplicate using a competitive enzyme-linked
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immunoassay (EIA, RADIM, Rome, Italy), suitable for equine use (Ferlazzo et al., 1998). During
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the first incubation, the cortisol sample competed with cortisol conjugated to horseradish peroxidase
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(HRPO) for the specific sites of the antiserum coated on the wells. After incubation, all unbound
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material was removed by aspiration and washing. The enzyme activity bound to the solid phase was
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inversely proportional to cortisol concentration in calibrators and samples and is made evident by
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incubating the wells with a chromogen solution (tetramethylbenzidine) in substrate buffer.
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Colorimetric reading was carried out using a spectrophotometer at 405 nm wave length (Sirio S,
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SEAC, Florence, Italy). Assay sensitivity was 5 ng/mL. The intraassay and interassay CVs were
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4.0% and 6.9%, respectively.
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Serum metabolic parameters (creatinine, CK, urea, AST, ALT, LDH) were assessed using a kinetic
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method by a biochemistry autoanalyzer (SLIM, SEAC, Florence, Italy), using commercially
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available tests of SEAC/Radim kits (Pomezia, Rome, Italy).
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198 Statistics
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Data are presented as mean ± standard deviation (SD). A two-way analysis of variance with
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repeated measures (two way RM-ANOVA) was applied to test for the effects of the different types
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of stimuli: transport, different number of covered mares (Group A and Group B) and sampling time
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(T0, T1, T2 and T3), as well as for the interactions between them, on hormonal concentrations and
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biochemical values. When the F value was significant (P < 0.05), the differences between
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individual means over time were assessed using a post-hoc multiple comparison test (Bonferroni).
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All calculations were performed using the PRISM package (GraphPad Software Inc., San Diego,
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CA, USA).
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Results
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Pituitary-adrenocortical concentrations and metabolic values are presented as mean ( ± SD) in
211
Figures 1, 2 and Table 1, respectively.
212 ACTH
214
Compared to T0, significant increases in circulating ACTH concentrations at T1 in group A
215
(P<0.001), and at T1 (P<0.05) and T3 (P<0.01) in group B were observed. No significant differences
216
in ACTH concentrations between the two Groups A and B were observed.
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217 Cortisol
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Compared to T0, significant increases of cortisol concentrations in both groups A and B at T2 (P<0.01)
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and at T3 (P<0.001) were observed.
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The two-way RM-ANOVA indicated that there was a significant interaction between the Groups A and
222
B and time on cortisol changes (F= 4.45; P=0.020).
223
Specifically, Group A showed a significant interaction between the first trip and when returning and
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time for cortisol (F= 63.35; P=0.00), with higher serum cortisol concentrations when returning at T2
225
(P=0.05) and T3 (P=0.01) than at T0 and T1 of the first trip, respectively. Similarly, Group B
226
showed a significant interaction between both trips and time for cortisol (F= 120.95; P=0.000), with
227
higher serum cortisol concentrations when returning at T2 (P=0.05) and T3 (P=0.01) than at T0 and
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T1 of the first trip, respectively.
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Metabolic parameters
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Compared to T0, significant increases of CK in both groups A and B at T1 (P<0.001), T2 (P<0.01) and
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T3 (P<0.001) were observed. The two way RM-ANOVA indicated that there was a significant
233
interaction between the Groups A and B and time on CK changes (F= 3.70; P=0.01).
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ACCEPTED MANUSCRIPT Group A showed a significant interaction between the first trip and when returning and time on CK
235
changes (F= 72.75; P=0.001), with higher serum CK concentrations when returning at T2 (P<0.05)
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and T3 (P<0.001) compared to T0 and T1 of the first trip, respectively. Similarly, Group B showed a
237
significant interaction between the first trip and when returning and time on CK changes (F= 190.95;
238
P=0.0001), with higher serum CK concentrations when returning at T2 (P<0.05) and T3 (P<0.001)
239
than at T0 and T1 of the first trip, respectively.
240
Also, there was a significant effect of different number of mares covered per stallion and time on CK
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changes (F= 9.33; P<0.005), with higher mean concentrations of CK in Group B than Group A at T2
242
(P<0.05).
243
Compared to T0, increases were observed at T1 and T3 in creatinine concentrations and ALT
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activities in both Groups A (P < 0.05) and B (P < 0.01); in LDH activities at T1 in both Groups A
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and B (P < 0.001); in urea concentrations in both Groups A and B (P < 0.05); in AST activities at
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T1 (P < 0.01) and T2 (P < 0.001) in both Groups A and B.
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Discussion
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The comparisons of the hormonal results obtained in the current study with published data reported
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for horses did not reveal any marked discrepancies for baseline circulating ACTH (Donaldson et
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al., 2005; Niinistő et al., 2010; Ayala et al., 2012) and cortisol (Waran, 1993; Stull and Rodiek,
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2002; Smiet et al., 2014) concentrations.
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The present study is in line with previous studies, demonstrating that previous travelling experience,
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with the regular transportation, does not reduce the early adrenocortical response to transport,
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perceived as a stressful challenge (Fazio et al., 2008a; 2008b; Schmidt et al., 2010a; 2010b). The
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concomitant increase of ACTH and cortisol concentrations after the 200 km journey, compared to
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the baseline values, confirmed that a pituitary-adrenocortical system response to travel with a
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substantial release of both hormones (Ferlazzo et al., 2012). These increases suggest that the
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ACCEPTED MANUSCRIPT animals’ reactions to transport stress were probably markedly influenced by the end of breeding
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season, as suggested by the highest cortisol concentrations observed at T2 and T3 in both Groups A
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and B. Although the cortisol concentrations were clearly elevated in response to transport, the higher
263
increase after returning could be due to the additional effects of breeding season, confirming that the
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HPA axis is responsive to horses’ and donkeys’ sexual excitement (Villani et al., 2006; Veronesi et
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al., 2011).
266
Nevertheless, no significant differences were observed between Groups A and B, independently of
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the number of mares covered in the breeding season per stallion, nor between different ages of
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stallions. With respect to age effects, it is possible to presume that the breeding season was
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comparable to moderate exercise training that attenuated the effect of age on the cortisol response, as
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observed in horses after acute exertion (Malinowski et al., 2006). No correlations between the
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stallions’ age and fertility were observed. The qualitative and quantitative intra- and inter-stallion
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variations in sperm morphology and fertility showed no consistent differences. Indeed, in both
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Groups A and B the stallions showed an excellent fertility and very high conception rate.
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It is possible to conclude that transport stress induced the greatest pituitary-adrenocortical response,
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which suggests that the transported stallions continuously activated their various, aspecific,
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biological responses regardless of stressful physical and mental stimuli. These data confirm the
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recent effective influence of equine resistant mechanisms to short-term stress, and support the
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hypothesis that breeding stallions can be transported without negatively affecting their fertility
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(Deichsel et al., 2015), as previously reported also for fertility of mares (Berghold et al., 2007). In
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any case, the higher release of cortisol after returning (T3) than on arrival (T1) can be used as a
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reliable marker of stress, confirming that horses are a prey species and respond to stressful
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situations as such as physical or mental stimuli with increased cortisol release (Hada et al., 2003;
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Forkman et al., 2007). Finally, our data confirm the observation of AboEl-Maaty (2011), showing
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that horses held in small state studs, had higher cortisol levels compared to those of the Equine
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Breeding Reproduction Centre, where the welfare conditions are probably better monitored.
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circulating creatinine, urea, ALT and LDH values (Lindner and Hatzipanagiotou, 1998; Wessely-
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Szponder et al., 2015; Noleto et al., 2016; Padalino et al., 2017).
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The metabolic demand increases after workload and exercise, according to the intensity, duration
290
and/or distance, as well as the training state, and may induce changes in bloodstream biochemical
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and endocrinological parameters that can affect the horse’s performance (Wanderly et al., 2015)
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The fact that the highest serum CK, AST, ALT and LDH, associated with creatinine and urea
293
increases after transport, can be attributed to the skeletal muscle stress, generated during transport
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by the physical activity of posture, and the consequences of the effort made during breeding
295
activity plus postural support. The highest increase of creatinine concentrations and CK activities
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showed that transport is comparable to physical exercise, since it involves muscle activity. These
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increases can be evaluated on the basis of the training state of horses (Overgaard et al., 2004;
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Wessely-Szponder et al., 2015). Moreover, it is well known that under routine physiological
299
conditions skeletal muscle fibres release a significant CK even in the absence of muscle damage
300
(Muñoz et al., 2002). The CK pattern confirmed that there was a rapid increase in its activity
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within 4-12 h from the damaging stimulus (Valberg et al., 1993), and may reflect the muscular
302
effort of standing up during transport. Moreover, the highest CK, AST and LDH activities
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observed after transport could be attributed to the challenges of membrane permeability, or to the
304
increases of their synthesis or decreases of their clearance, according to physical workload (Boyd,
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1985; Wessely-Szponder et al., 2015).
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It is therefore feasible that the adrenergic system, involved during the first stages of transport,
307
induced a consequent increase of enzyme activities, representing a stronger stress-causing factor
308
than others (Tischner and Niezgoda, 2000; Thomassian et al., 2007).
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The CK pathway related to skeletal muscle metabolism proved to be a sensitive marker in relation to
310
muscle workload, as shown by the higher values after the return. On the other hand, the increases of
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AST, ALT and LDH activities are not in line with changes observed in horses during transport,
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usinga different orientation (Padalino et al., 2012).
313 Conclusion
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The hypothesis that there is a temporal interaction between transport and the breeding season of
316
stallions was confirmed by the significant increase of stress markers, including muscle enzymes.
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This study showed that the pituitary-adrenocortical axis is efficiently stimulated by transport and that
318
the breeding season strengthens the increase of cortisol and CK values of stallions as an effect of
319
mental and physical arousal.
320
From a practical point of view, the evaluation of the reference values and related changes of
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endocrine and biochemical variables of stallions at the start of breeding season represents an
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additional requirement for future implementation of reliable markers for the seasonal breeders’
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status. The potential positive effectof normal behavior on reproductive success remains an important
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matter for discussion.
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325 Acknowledgments
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The authors thank the team of Equine Breeding Reproduction Centre of Catania, Italy, veterinary
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practitioners and breeding farm managers who collaborated with this study. This research did not
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receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
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Ethical considerations
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This study was approved by the Ethical Committee for the Care and Use of Animals of
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University of Messina, in compliance with the guidelines of the Italian law on the care and
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use of animals (D.L. 4/3/2014 n. 26) and the EU Directive (Directive 2010/63).
335 336
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induced
inflammatory
responses
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horses.
Polish
J.
Vet.
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407-413
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Table 1 - Mean values (mean ± SD) of the bloodstream biochemical parameters of stallions before and after transports
Groups
Returning Before T2
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Parameters
The first trip Before T0 After T1
After T3
325.66 ± 40.23c 328.96 ± 24.85c
243.10 ± 33.15 bA 303.10 ± 32.17 bAα
creatinine (mg/dL) A B
1.34 ± 0.40 1.39 ± 0.46
2.07 ± 0.68a 2.13 ± 0.56b
1.38 ± 0.62 2.13 ± 0.56b
2.10 ± 0.17 a 2.44 ± 0.33b
urea (mg/dL)
A B
21.27 ± 5.22 20.42 ± 6.59
27.54 ± 8.29a 27.21 ± 5.39a
20.93 ± 5.18 20.81 ± 3.11
28.08 ± 4.15 a 28.19 ± 4.08 a
AST (U/L)
A B
107.64 ± 25.03 103.56 ± 35.51
287.62 ± 55.51b 272.33 ± 55.50b
111.05 ± 38.05 114.09 ± 33.20
383.02 ± 22.13 c 339.03 ± 23.69 c
ALT (U/L)
A B
20.70 ± 6.12 20.73 ± 8.38
30.10 ± 6.39a 29.23 ± 6.23b
22.33 ± 3.34 24.22 ± 4.14
27.10 ± 3.24a 30.39 ± 3.27b
LDH (U/L)
A B
164.79 ± 45.47 170.91 ± 38.05
289.95 ± 42.49c 192.82 ± 50.02c
155.23 ± 20.04 181.33 ± 23.13
275.45 ± 30.15c 301.25 ± 30.44c
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433.10 ± 43.18cB 452.10 ± 43.12cB
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A B
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121.33 ± 20.51 131.11 ± 31.59
CK (U/L)
Superscripts indicate significant (aP < 0.05; bP < 0.01; cP < 0.001) differences vs T0. Superscripts indicate significant (AP < 0.05; BP < 0.01) differences vs the first trip. Superscript indicates significant (αP < 0.05) difference vs Group A.
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Figures
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Figure 1 - Circulating ACTH concentrations (mean ± SD) of stallions before and after transports
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Asterisks indicate significant (*P < 0.05; **P < 0.01; ***P < 0.001)) differences vs T0.
Figure 2 - Circulating cortisol concentrations (mean ± SD) of stallions before and after transports Asterisks indicate significant (*P < 0.01; **P < 0.001) differences vs T0
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Symbols indicate significant (●P < 0.05; ●●P < 0.01) differences vs the first trip
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ACCEPTED MANUSCRIPT HIGHLIGHTS ● The horses’ pituitary-adrenocortical responses to transport were evaluated ● The exploration of biochemical parameters was also considered. ● The concomitant increases of ACTH and cortisol after transport were observed.
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● Significant increases of all biochemical parameters after transport were described