- Email: [email protected]
The Effects of Loose Group versus Individual Stall Transport on Glucocorticosteriods and Dehydroepiandrosterone in Yearling Horses
ORIGINAL RESEARCH The Effects of Loose Group versus Individual Stall Transport on Glucocorticosteriods and Dehydroepiandrosterone in Yearling Horses Shannon M. Garey, MS,a Ted H. Friend, PhD,a Dennis H. Sigler, PhD,a and Luc R. Berghman, PhDb
ABSTRACT The European Union recently published regulations regarding the welfare of horses during transport, requiring that horses be transported in individual stalls. The objective of this study was to determine whether concentrations of cortisol, corticosterone, or dehydroepiandrosterone (DHEA) differed among horses transported in individual stalls versus in loose groups. A total of 20 yearlings that were regularly handled and accustomed to being tied, but were na€ıve to transport, were assigned to be transported for 6 hours in either individual stalls or a loose group. The experiment was replicated with a second trial 35 days later following a switchback design. Jugular blood samples were analyzed for plasma cortisol, corticosterone, and DHEA concentrations at pretransport, after 2, 4, and 6 hours of transport, and at 2 and 4 hours after unloading. The data were analyzed using a mixed model repeated measures analysis of variance for treatment effects, whereas differences between sample times within each trial, and pretransport concentrations between trials, were analyzed using paired T-tests. No significant differences were found between treatment groups in concentrations of cortisol (P ¼ .713), corticosterone (P ¼ .370), or DHEA (P ¼ .416). Cortisol and corticosterone concentrations increased significantly during transport, and returned to pretransport concentrations by 2 hours post-transport (P < .01). Changes in concentrations of cortisol and corticosterone indicated that transportation was a significant stressor; however, being transported in a loose group versus individual stalls was not different for these horses.
Keywords: Transport; Stress; Stall; Cortisol; Corticosterone
INTRODUCTION
From the Department of Animal Science, Texas A&M University, College Station, TXa; and Department of Poultry Science, Texas A&M University, College Station, TXb. Reprint requests: Ted H. Friend, PhD, Department of Animal Science, Texas A&M University, 2471 TAMU College Station, TX 77843. 0737-0806/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.jevs.2010.11.003
Regulation of the transport of horses for slaughter for human consumption in the United States is detailed in Chapter 88 of 9 Code of Federal Regulations (CFR), and is directed toward the group transport of commercial horses. Specifically, the Code bans the transport of commercial horses in double-deck trailers, which is common in the transport of other livestock species.1 It also details requirements for supplying adequate quality feed and potable water to horses within a minimum of 6 hours of transport, segregating stallions and other aggressive animals during transport, and ensuring adequate floor space for each animal. A recent set of regulations for horse transport is the European Union’s Council Regulation (EC) No. 1/ 2005, which mandates that all nonregistered horses should be transported in single deck trailers with a minimum of 75 cm of clearance above the withers and must be offered food and water every 8 hours during transport. The maximum transport period is 24 hours, and during long journeys, the horses are to be stalled individually unless they have a foal. Stalls must be constructed of adjustable partitions, and each horse should be provided with a space of 1 to 1.75 m2, depending on age.2 The basis for the European Union requiring that each horse be stalled individually is unclear. Horses are a very social species, and isolating them into individual stalls during a stressful event, such as transport, may have an additive effect on the overall stress of the animals if they are not accustomed to being restrained in stalls. In a 2002 study, researchers reported that horses that were cross-tied in individual stalls during transport showed greater stress, including higher cortisol concentrations, and took longer to return to pretransport conditions than loose horses when transported for 24 hours.3 However, restricting the space in which a group of horses are contained can cause the social structure of a herd to become unstable, thereby resulting in conflicts.4 In addition, studies on the optimal density for the transport of loose groups of horses5-7 and
696
Journal of Equine Veterinary Science Vol 30, No 12 (2010)
SM Garey et al Vol 30, No 12 (2010)
cattle8,9 indicate that lower density is preferable. Decreasing the amount of space that horses have in a loose group during transport prevents them from shifting to compensate for changes in speed and direction.10 Horses transported at a higher density were more likely to fall or be injured, and had a decreased likelihood of rising to their feet after falling.7 Ability of a horse to balance during transport is central to its behavior while in transit and may contribute to the amount of stress experienced by the animal. Several physiological indicators of stress have been used to identify and study transport stress in horses, with heart rates11-14 and cortisol concentrations3,6,11,14-17 having been most commonly used in recent studies. Plasma cortisol and corticosterone are known to increase in animals at the onset of a stressor, and stimulate gluconeogenesis to release energy stores that the animal can use to resist the stressor. However, long-term exposure to stressors, causing an extended elevation of the glucocorticoid response, can have a detrimental immunologic effect and cause the animal to become more susceptible to disease.18 In addition to glucocorticoids, dehydroepiandrosterone (DHEA), a neurosteroid, has recently been studied in the field of stress research and psychological disorders. Differences in DHEA and cortisol concentrations have been found between psychologically healthy patients and those diagnosed with chronic fatigue syndrome (CFS),19 post-traumatic stress disorder (PTSD),20 schizophrenia,21 attention-deficit hyperactivity disorder,22 and major depression.23 Similar to cortisol, DHEA secretion is initiated by adrenocorticotropic hormone (ACTH); however, several studies suggest that differences exist in the amount of time that DHEA and cortisol take to return to normal concentrations after the onset of an acute stressor or ACTH challenge, depending on the overall stress of the individual. A significant difference in DHEA: cortisol ratio over time was found between patients diagnosed with CFS and healthy control patients, where the ratio for CFS patients was significantly higher over time than control patients when administered the same dosage of ACTH.19 Conversely, schizophrenic patients were found to have higher cortisol:DHEA ratios than healthy patients under normal day-to-day conditions.21 Also, veterans with PTSD were found to have significantly higher DHEA concentrations than veteran patients without PTSD.20 However, patients with major depression showed significantly lower DHEA concentrations than healthy patients during normal activity over a 4-day period.23 These studies indicate that although DHEA may be lower during nonstressful activity in afflicted patients versus healthy patients, there may be a link between long-term stress disorders and elevated concentrations of DHEA or DHEA:cortisol ratios during a stressful event. The objectives of this study were to determine whether young horses that underwent extensive handling but had
697
not been transported show differences in glucocorticoids and DHEA when transported for 6 hours in individual stalls versus being transported in loose groups.
MATERIALS AND METHODS A total of 20 yearling Quarter Horse mares (n ¼ 9) and geldings (n ¼ 11) that had regular handling and training experience, but no previous transport experience, were used. All the horses simultaneously participated in a nutrition study on the influence of dietary supplements on the incidence of gastric ulcers. All horses received 1.25% of body weight of grain and 1% of body weight of hay, with seven horses receiving a sulfated form of a trace mineral, six receiving a proteinated form of the same trace mineral, and the remaining seven receiving no supplement to their daily grain and hay diet. During feeding periods, horses were placed in assigned feeding stalls, and between feeding periods, the horses were housed in outside group paddocks adjacent to the feeding barn. Group paddocks were assigned by feed treatments, such that all horses within a feed treatment shared an assigned paddock throughout the study. Horses were exercised 3 days per week for 20 minutes per day and were accustomed to being individually tied. Each of the horses were assigned with a temperament score on a 1-5 scale, with 1 being least excitable and 5 being most excitable, about daily activities and exposure to new experiences. The scores were based on the experiences and interactions of a primary caretaker with each animal over the past year, and were relative to the scores of the other horses in the study. The horses were assigned to be transported in either individual stalls or loose groups for 6 hours. Gender was balanced, and temperament scores and dietary supplementation were equally represented in both treatments. Using a switchback design, the treatments were reversed during a second trial. To make sure the horses would safely walk up the loading ramp into the trailer and accept being stalled, each horse was individually walked into the trailer, held for <1 minute in a stall, and then exited the trailer 2 days before the start of trial 1. Each trial was completed over a 2-day period. The first trial was conducted in July and the second in August, with 35-day interval between trials. Five horses from each treatment group (stalled vs. loose group) were transported on the first day and the remaining five horses in each treatment group were transported on the second day. The horses were transported in a custom-built 16.2 2.4 2.62 m3 (length, width, height, respectively), single-deck, slat-sided trailer (Barrett Trailers, Purcell, Oklahoma) pulled by a semi-tractor. The trailer was divided into three sections as follows: individual stalls, a loose group compartment, and a small staging area for sample collection in the center (Fig. 1). Five individual stalls were constructed by diagonal placement of
SM Garey et al Vol 30, No 12 (2010)
698
Stalls
Group
Group
Stalls
Figure 1. Floor diagram of the trailer showing stall and loose group placement during the first (top) and second day (bottom) in trial 1. Configuration was reversed in trial 2, such that loose horses were in the front of the trailer on the first day (bottom) and in the rear on the second day (top). 1.57 3.05 m2 metal tubing panels (Priefert, Mount Pleasant, TX, USA). The panels were placed on the vertical support beams of the trailer that occurred at 0.91-m intervals to create rhomboid-shaped stalls that were 0.71 2.03 m2. Each horse was loosely tied to the center of the front of each stall, using a 0.76 m commercial elastic horse trailer tie. It was estimated that each horse had 1.44 m2 of floor space when tied at the front of the stall during transport because they could not back the full length of the stall. This resulted in an average of 263 kg/m2 per horse for the first trial, and 279 kg/m2 per horse for the second trial, given inter-trial growth of the horses. The group compartment was constructed using two 2.54-m solid swing gates. In trial 1, there was a total of 5.82 m2 of floor space, which was increased to 6.96 m2 in trial 2 by moving one swing gate 0.91 m to the next vertical support beam. This increase was made to allow for inter-trial growth of the horses. The loose horses averaged 325 kg/m2 and 284 kg/m2 of floor space for trials 1 and 2, respectively. The average floor space for both treatments was >50 kg/m2, which was less than the maximum recommended by Whiting,5 and consistent with common practice.5-7 At the end of each day’s trial, the placement of the stalls and group compartment was reversed within the trailer to mitigate confounding effects of traveling in the front versus the rear portion of the trailer (Fig. 1). In addition, the angle of the stalls was reversed to help control for any confounding effects of position or air flow when facing the passenger side versus the driver side of the trailer. Jugular blood samples were drawn 15 minute before loading at the Texas A&M Horse Center, College Station, Texas, with additional samples drawn after 2, 4, and 6 hours of transport over a standard route at highway speeds, and 2 and 4 hours after unloading. The truck returned to the Horse Center for the 20 to 24 minutes needed for blood sampling at 2-hour intervals. The stops required for blood sampling mimicked the breaks that routinely
occur during transport for fuel, driver rest stops, and weigh stations. These horses were accustomed to jugular venipuncture and were easily restrained by an assistant who held their halter during sample collections. Each of the stalled horses was untied from the trailer during ontrailer sampling, and backed toward the rear of their stall to provide room for the researchers to obtain jugular blood samples. An assistant held the halter of each horse and attempted to distract the horse by covering its eye. To allow sampling of the group horses, the gate was opened to the staging area to allow room for several assistants to catch and restrain each horse using their halter. The group horses remained in their group as blood samples were obtained from each horse, following the procedure used for the stalled horses. All horses were sampled in the same order at each 2-hour interval. Blood samples were collected using 20-gauge 1½ inch needles (Vacutainer Becton, Dickinson and Company, Franklin Lakes, NJ, USA) and 9 mL plastic evacuated collection tubes containing sodium heparin (Vacuette, Greiner Bio-One, New York, NY, USA). The tubes were centrifuged, and the plasma was collected and stored at 208 C. Plasma samples were analyzed by colorimetric enzyme-linked immunosorbent assay for total cortisol, corticosterone, and DHEA concentrations (Assay Designs, Ann Arbor, MI, USA). All samples were run in duplicate, and samples that had >10% variation between results were reanalyzed. Treatment effects on plasma concentrations of cortisol, corticosterone, and DHEA during trials 1 and 2 were analyzed using a mixed model repeated measures analysis of variance with each individual as the subject, with trial, treatment, sample time, and treatment-sample time interaction as factors. The model was an unstructured covariance (SAS 9.1, SAS Institute, Inc., Cary, NC, USA). Differences between sample times were analyzed using paired T-tests within each trial (SPSS 12.0.1, SPSS Inc., Chicago, IL, USA). Least squares means were presented with the data adjusted for covariance. Temperature– humidity index (THI) was calculated using the formula THI ¼ Temperature (0.550.55 Relative Humidity) (Temperature58), where temperature is in degrees Fahrenheit and relative humidity is in decimal form.24
RESULTS
The temperature ranged from 318 C to 378 C during trial 1, and from 258 C to 348 C during trial 2. Although there were some slight differences in temperature, humidity fluctuated in the opposite direction between trials, such that the THI was relatively consistent between both trials ranging from 80 to 83 in trial 1, and from 76 to 83 in trial 2. Transport in individual stalls versus loose groups did not have a significant effect on cortisol (P ¼ .713), corticosterone (P ¼ .370), or DHEA (P ¼ .416) concentrations
SM Garey et al Vol 30, No 12 (2010)
699
Table 1. Least squares means of cortisol concentrations (ng/mL) in relation to time relative to start of transport for both treatment groups during trials 1 and 2 Treatment Time Pretransport Transport 2 hours 4 hours 6 hours Post-transport 2 hours 4 hours
Stall 2.74a
Group
SEM
2.81a
0.88
19.31b 20.25b 16.55b
16.98b 18.82b 15.01b
3.24 3.80 3.14
4.31a 3.87a
4.27a 2.45a
1.56 0.82
a,b,c
Means within a column without a common superscript differ (P < .01).
(Tables 1, 2, and 3). No significant interaction was reported between treatment and sample time as well as in cortisol, corticosterone, or DHEA concentrations between treatments (P > .370). Pretransport concentrations of cortisol and corticosterone were significantly lower than the concentrations after 2, 4, and 6 hours of transport, but did not differ significantly from concentrations at 2 and 4 hours post-transport (P < .01). However, sample-time comparisons of DHEA yielded no significant differences. There was a significant difference in DHEA concentrations between trials (P ¼ .036), but no significant differences in the cortisol (P ¼ .956) or corticosterone (P ¼ .230) concentrations between trials.
DISCUSSION AND CONCLUSIONS The findings of this study are inconsistent with a 2002 study3 which concluded that horses transported for 24 hours in loose pairs had decreased physiological measures of stress when compared with horses that were individually cross-tied. Stull and Rodiek3 reported a divergence of cortisol concentrations after 3 hours of transport, with stalled horses having higher cortisol concentrations than loose horses. However, the article does not indicate the sample times which were significantly different, the level of significance between treatment groups for each parameter measured, and whether any treatments by sample time interactions were found. Although Stull and Rodiek reported increasing differences in cortisol concentrations between treatment groups from 3 to 24 hours of transport,3 results obtained during this study found no significant differences between the treatment groups between 3 and 6 hours of transport. The horses used in this study loaded into the trailer, remained tied in the stalls, and unloaded from the trailer without incident. This may have been influenced by the
Table 2. Least squares means of corticosterone concentrations (ng/mL) in relation to time relative to start of transport for both treatment groups during trials 1 and 2 Treatment Time Pretransport Transport 2 hours 4 hours 6 hours Post-transport 2 hours 4 hours
Stall
Group
SEM
a
1.17
17.91b 14.11b 13.17b
13.74b 13.19b 10.37b
2.92 2.58 2.10
4.53a 3.29a
3.23a 2.56a
0.87 0.72
5.29
a
3.35
a,b
Means within a column without a common superscript differ (P < .01).
previous training and handling of the horses on a daily basis, and may not be reflective of how an untrained horse with minimal handling would react to being tied in a transport stall. In addition, the stalls were constructed out of metal tube panels that had large areas that the horses could see through, allowing the horses to have visual and a minimal amount of physical contact with the stalled horses next to them. This may have decreased the social isolation effect on the stress of the animals. Although there were no statistically significant differences in cortisol post-transport, behavioral differences were informally observed between trials 1 and 2. After transport in trial 1, the horses had droopy eyes and ears, stood in their paddocks after being turned out, and generally were not active throughout the 4-hour post-transport sample collection period. However, after transport in trial 2, the horses trotted out into their paddocks, many of them rolled, and several were observed biting, kicking, and generally at play during the post-transport sample collection period. This suggests that these horses were less fatigued and may have acclimated to the transport between trials 1 and 2, although they were 35 days apart. Because the THI was consistent between the 2 trials, the apparent differences in fatigue between trials were not likely because of the changes in temperature and humidity. The horses participating in this study were from the same University herd and were often maintained in one group since the time of birth. However, for 60 days before initial transport and during the 35 days between trial 1 and trial 2, they were maintained in groups that were determined by the diets they received in the nutrition study. In this study, because the treatments were balanced by diet, horses that were not recently housed together before transport were transported together in the loose groups. In both trials,
SM Garey et al Vol 30, No 12 (2010)
700
Table 3. Least squares means of DHEA concentrations (pg/mL) in relation to time relative to start of transport for both treatment groups during trials 1 and 2 Treatment Time Pretransport Transport 2 hours 4 hours 6 hours Post-transport 2 hours 4 hours
Stall
displacement, falls, injuries, and orientation during horse transportation. Appl Anim Behav Sci 2000;67:169–179. 8. Eldridge GA, Winfield CG, Cahill DJ. Responses of cattle to different space allowances, pen sizes and road conditions during transport.
Group
SEM
499.8
374.4
88.2
672.9 657.9 578.6
516.6 565.1 753.3
139.9 143.2 166.2
471.3 589.7
7. Collins MN, Friend TH, Jousan FD, Chen SC. Effects of density on
360.0 339.1
90.7 114.8
several of the loose horses showed agonistic behavior, including biting, kicking, and vocalizing when first loaded into the trailer. The group horses were visibly agitated by the agonistic behavior before the start of transport. When interpretating the results of this study, it is important to make the distinction that these horses were accustomed to being in individual stalls and being tied using a halter. Although horses that were being transported for sport underwent some training, bucking horses, feral horses, or horses being transported for slaughter may react violently to being placed in narrow stalls with their heads tied. However, obtaining blood samples from untrained horses while they are on transport vehicles is problematic. ACKNOWLEDGMENTS The authors thank USDA-APHIS-Veterinary Services for funding this research and Dr. Tim Cordes for assisting in study design.
Aust J Exp Agric 1988;28:155–159. 9. Tarrant PV, Kenny FJ, Harrington D, Murphy M. Long distance transportation of steers to slaughter: Effect of stocking density on physiology, behaviour and carcass quality. Livest Prod Sci 1992;30: 223–238. 10. Friend TH. A review of recent research on the transportation of horses. J Anim Sci 2001;79:E32–E40. 11. Clark DK, Friend TH, Dellmeier G. The effect of orientation during trailer transport on heart rate, cortisol and balance in horses. Appl Anim Behav Sci 1993;38:179–189. 12. Smith BL, Jones JH, Carlson GP, Pascoe JR. Effect of body direction on heart rate in trailered horses. Am J Vet Res 1994;55:1007–1011. 13. Smith BL, Jones JH, Hornof WJ, Miles JA, Longworth KE, Willits NH. Effects of road transport on indices of stress in horses. Equine Vet J 1996;28:446–454. 14. Waran NK, Cuddeford D. Effects of loading and transport on the heart rate and behaviour of horses. Appl Anim Behav Sci 1995;43: 71–81. 15. Friend TH. Dehydration, stress, and water consumption of horses during long-distance commercial transport. J Anim Sci 2000;78: 2568–2580. 16. Friend TH, Martin TM, Householder DD, Bushong DM. Stress responses of horses during a long period of transport in a commercial truck. J Am Vet Med Assoc 1998;212:838–844. 17. Stull CL, Rodiek AV. Physiological responses of horses to 24 hours of transportation using a commercial van during summer conditions. J Anim Sci 2000;78:1458–1466. 18. Munck A, Guyre PM, Holbrook NJ. Physiological functions of glucocorticoids in stress and their relation to pharmacological actions. Endocr Rev 1984;5:25–44. 19. Scott LV, Svec F, Dinan T. A preliminary study of dehydroepiandrosterone response to low-dose ACTH in chronic fatigue syndrome and in healthy subjects. Psychiat Res 2000;97:21–28.
REFERENCES 1. U.S. CFR. Title 9: Animals and animal products. Chapter 88: Commercial Transportation of Equines for Slaughter. Washington, DC: U.S. Govt. Printing Office; 2002.
20. Yehuda R, Brand SR, Golier JA, Yang R-K. Clinical correlates of DHEA associated with post-traumatic stress disorder. Acta Psychiat Scand 2006;114:187–193. 21. Ritsner M, Maayan R, Gibel A, Strous R, Modai I, Weizman A. Ele-
2. Council of the European Union. Council Regulation No. 1/2005. Official Journal of the European Union; 2005.
vation of the cortisol/dehydroepiandrosterone ratio in schizophrenia
3. Stull CL, Rodiek AV. Effects of cross-tying horses during 24 h of road
22. Strous R, Spivak B, Yoran-Hegesh R, Maayan R, Averbuch E,
transport. Equine Vet J 2002;34:550–555. 4. Goodwin D. Horse behaviour: evolution, domestication and feralisation. In: Waran N, ed. The welfare of horses. Dordrecht, The Netherlands: Kluwer Academic Publishers; 2002:1–18. 5. Whiting T. Maximum loading density of loose horses. Can J Anim Sci
patients. Eur Neuropsychopharmacol 2004;14:267–273. Kotler M, et al. Analysis of neurosteroid levels in attention deficit hyperactivity disorder. Int J Neuropsychopharmacol 2001;4:259–264. 23. Michael A, Jenaway A, Paykel E, Herbert J. Altered salivary dehydroepiandrosterone levels in major depression in adults. Biol Psychiat 2000;48:989–995.
1999;79:115–118. 6. Stull CL. Responses of horses to trailer design, duration and floor area
24. National Oceanic and Atmospheric Administration. Livestock hot
during commercial transportation to slaughter. J Anim Sci 1999;77:
vice Central Registration, Registration Operations Manual. Washing-
2925–2933.
ton, DC: U.S: Govt. Printing Office; 1976:C-31–76.
weather stress. US Department Commerce, National Weather Ser-
ABSTRACT The European Union recently published regulations regarding the welfare of horses during transport, requiring that horses be transported in individual stalls. The objective of this study was to determine whether concentrations of cortisol, corticosterone, or dehydroepiandrosterone (DHEA) differed among horses transported in individual stalls versus in loose groups. A total of 20 yearlings that were regularly handled and accustomed to being tied, but were na€ıve to transport, were assigned to be transported for 6 hours in either individual stalls or a loose group. The experiment was replicated with a second trial 35 days later following a switchback design. Jugular blood samples were analyzed for plasma cortisol, corticosterone, and DHEA concentrations at pretransport, after 2, 4, and 6 hours of transport, and at 2 and 4 hours after unloading. The data were analyzed using a mixed model repeated measures analysis of variance for treatment effects, whereas differences between sample times within each trial, and pretransport concentrations between trials, were analyzed using paired T-tests. No significant differences were found between treatment groups in concentrations of cortisol (P ¼ .713), corticosterone (P ¼ .370), or DHEA (P ¼ .416). Cortisol and corticosterone concentrations increased significantly during transport, and returned to pretransport concentrations by 2 hours post-transport (P < .01). Changes in concentrations of cortisol and corticosterone indicated that transportation was a significant stressor; however, being transported in a loose group versus individual stalls was not different for these horses.
Keywords: Transport; Stress; Stall; Cortisol; Corticosterone
INTRODUCTION
From the Department of Animal Science, Texas A&M University, College Station, TXa; and Department of Poultry Science, Texas A&M University, College Station, TXb. Reprint requests: Ted H. Friend, PhD, Department of Animal Science, Texas A&M University, 2471 TAMU College Station, TX 77843. 0737-0806/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.jevs.2010.11.003
Regulation of the transport of horses for slaughter for human consumption in the United States is detailed in Chapter 88 of 9 Code of Federal Regulations (CFR), and is directed toward the group transport of commercial horses. Specifically, the Code bans the transport of commercial horses in double-deck trailers, which is common in the transport of other livestock species.1 It also details requirements for supplying adequate quality feed and potable water to horses within a minimum of 6 hours of transport, segregating stallions and other aggressive animals during transport, and ensuring adequate floor space for each animal. A recent set of regulations for horse transport is the European Union’s Council Regulation (EC) No. 1/ 2005, which mandates that all nonregistered horses should be transported in single deck trailers with a minimum of 75 cm of clearance above the withers and must be offered food and water every 8 hours during transport. The maximum transport period is 24 hours, and during long journeys, the horses are to be stalled individually unless they have a foal. Stalls must be constructed of adjustable partitions, and each horse should be provided with a space of 1 to 1.75 m2, depending on age.2 The basis for the European Union requiring that each horse be stalled individually is unclear. Horses are a very social species, and isolating them into individual stalls during a stressful event, such as transport, may have an additive effect on the overall stress of the animals if they are not accustomed to being restrained in stalls. In a 2002 study, researchers reported that horses that were cross-tied in individual stalls during transport showed greater stress, including higher cortisol concentrations, and took longer to return to pretransport conditions than loose horses when transported for 24 hours.3 However, restricting the space in which a group of horses are contained can cause the social structure of a herd to become unstable, thereby resulting in conflicts.4 In addition, studies on the optimal density for the transport of loose groups of horses5-7 and
696
Journal of Equine Veterinary Science Vol 30, No 12 (2010)
SM Garey et al Vol 30, No 12 (2010)
cattle8,9 indicate that lower density is preferable. Decreasing the amount of space that horses have in a loose group during transport prevents them from shifting to compensate for changes in speed and direction.10 Horses transported at a higher density were more likely to fall or be injured, and had a decreased likelihood of rising to their feet after falling.7 Ability of a horse to balance during transport is central to its behavior while in transit and may contribute to the amount of stress experienced by the animal. Several physiological indicators of stress have been used to identify and study transport stress in horses, with heart rates11-14 and cortisol concentrations3,6,11,14-17 having been most commonly used in recent studies. Plasma cortisol and corticosterone are known to increase in animals at the onset of a stressor, and stimulate gluconeogenesis to release energy stores that the animal can use to resist the stressor. However, long-term exposure to stressors, causing an extended elevation of the glucocorticoid response, can have a detrimental immunologic effect and cause the animal to become more susceptible to disease.18 In addition to glucocorticoids, dehydroepiandrosterone (DHEA), a neurosteroid, has recently been studied in the field of stress research and psychological disorders. Differences in DHEA and cortisol concentrations have been found between psychologically healthy patients and those diagnosed with chronic fatigue syndrome (CFS),19 post-traumatic stress disorder (PTSD),20 schizophrenia,21 attention-deficit hyperactivity disorder,22 and major depression.23 Similar to cortisol, DHEA secretion is initiated by adrenocorticotropic hormone (ACTH); however, several studies suggest that differences exist in the amount of time that DHEA and cortisol take to return to normal concentrations after the onset of an acute stressor or ACTH challenge, depending on the overall stress of the individual. A significant difference in DHEA: cortisol ratio over time was found between patients diagnosed with CFS and healthy control patients, where the ratio for CFS patients was significantly higher over time than control patients when administered the same dosage of ACTH.19 Conversely, schizophrenic patients were found to have higher cortisol:DHEA ratios than healthy patients under normal day-to-day conditions.21 Also, veterans with PTSD were found to have significantly higher DHEA concentrations than veteran patients without PTSD.20 However, patients with major depression showed significantly lower DHEA concentrations than healthy patients during normal activity over a 4-day period.23 These studies indicate that although DHEA may be lower during nonstressful activity in afflicted patients versus healthy patients, there may be a link between long-term stress disorders and elevated concentrations of DHEA or DHEA:cortisol ratios during a stressful event. The objectives of this study were to determine whether young horses that underwent extensive handling but had
697
not been transported show differences in glucocorticoids and DHEA when transported for 6 hours in individual stalls versus being transported in loose groups.
MATERIALS AND METHODS A total of 20 yearling Quarter Horse mares (n ¼ 9) and geldings (n ¼ 11) that had regular handling and training experience, but no previous transport experience, were used. All the horses simultaneously participated in a nutrition study on the influence of dietary supplements on the incidence of gastric ulcers. All horses received 1.25% of body weight of grain and 1% of body weight of hay, with seven horses receiving a sulfated form of a trace mineral, six receiving a proteinated form of the same trace mineral, and the remaining seven receiving no supplement to their daily grain and hay diet. During feeding periods, horses were placed in assigned feeding stalls, and between feeding periods, the horses were housed in outside group paddocks adjacent to the feeding barn. Group paddocks were assigned by feed treatments, such that all horses within a feed treatment shared an assigned paddock throughout the study. Horses were exercised 3 days per week for 20 minutes per day and were accustomed to being individually tied. Each of the horses were assigned with a temperament score on a 1-5 scale, with 1 being least excitable and 5 being most excitable, about daily activities and exposure to new experiences. The scores were based on the experiences and interactions of a primary caretaker with each animal over the past year, and were relative to the scores of the other horses in the study. The horses were assigned to be transported in either individual stalls or loose groups for 6 hours. Gender was balanced, and temperament scores and dietary supplementation were equally represented in both treatments. Using a switchback design, the treatments were reversed during a second trial. To make sure the horses would safely walk up the loading ramp into the trailer and accept being stalled, each horse was individually walked into the trailer, held for <1 minute in a stall, and then exited the trailer 2 days before the start of trial 1. Each trial was completed over a 2-day period. The first trial was conducted in July and the second in August, with 35-day interval between trials. Five horses from each treatment group (stalled vs. loose group) were transported on the first day and the remaining five horses in each treatment group were transported on the second day. The horses were transported in a custom-built 16.2 2.4 2.62 m3 (length, width, height, respectively), single-deck, slat-sided trailer (Barrett Trailers, Purcell, Oklahoma) pulled by a semi-tractor. The trailer was divided into three sections as follows: individual stalls, a loose group compartment, and a small staging area for sample collection in the center (Fig. 1). Five individual stalls were constructed by diagonal placement of
SM Garey et al Vol 30, No 12 (2010)
698
Stalls
Group
Group
Stalls
Figure 1. Floor diagram of the trailer showing stall and loose group placement during the first (top) and second day (bottom) in trial 1. Configuration was reversed in trial 2, such that loose horses were in the front of the trailer on the first day (bottom) and in the rear on the second day (top). 1.57 3.05 m2 metal tubing panels (Priefert, Mount Pleasant, TX, USA). The panels were placed on the vertical support beams of the trailer that occurred at 0.91-m intervals to create rhomboid-shaped stalls that were 0.71 2.03 m2. Each horse was loosely tied to the center of the front of each stall, using a 0.76 m commercial elastic horse trailer tie. It was estimated that each horse had 1.44 m2 of floor space when tied at the front of the stall during transport because they could not back the full length of the stall. This resulted in an average of 263 kg/m2 per horse for the first trial, and 279 kg/m2 per horse for the second trial, given inter-trial growth of the horses. The group compartment was constructed using two 2.54-m solid swing gates. In trial 1, there was a total of 5.82 m2 of floor space, which was increased to 6.96 m2 in trial 2 by moving one swing gate 0.91 m to the next vertical support beam. This increase was made to allow for inter-trial growth of the horses. The loose horses averaged 325 kg/m2 and 284 kg/m2 of floor space for trials 1 and 2, respectively. The average floor space for both treatments was >50 kg/m2, which was less than the maximum recommended by Whiting,5 and consistent with common practice.5-7 At the end of each day’s trial, the placement of the stalls and group compartment was reversed within the trailer to mitigate confounding effects of traveling in the front versus the rear portion of the trailer (Fig. 1). In addition, the angle of the stalls was reversed to help control for any confounding effects of position or air flow when facing the passenger side versus the driver side of the trailer. Jugular blood samples were drawn 15 minute before loading at the Texas A&M Horse Center, College Station, Texas, with additional samples drawn after 2, 4, and 6 hours of transport over a standard route at highway speeds, and 2 and 4 hours after unloading. The truck returned to the Horse Center for the 20 to 24 minutes needed for blood sampling at 2-hour intervals. The stops required for blood sampling mimicked the breaks that routinely
occur during transport for fuel, driver rest stops, and weigh stations. These horses were accustomed to jugular venipuncture and were easily restrained by an assistant who held their halter during sample collections. Each of the stalled horses was untied from the trailer during ontrailer sampling, and backed toward the rear of their stall to provide room for the researchers to obtain jugular blood samples. An assistant held the halter of each horse and attempted to distract the horse by covering its eye. To allow sampling of the group horses, the gate was opened to the staging area to allow room for several assistants to catch and restrain each horse using their halter. The group horses remained in their group as blood samples were obtained from each horse, following the procedure used for the stalled horses. All horses were sampled in the same order at each 2-hour interval. Blood samples were collected using 20-gauge 1½ inch needles (Vacutainer Becton, Dickinson and Company, Franklin Lakes, NJ, USA) and 9 mL plastic evacuated collection tubes containing sodium heparin (Vacuette, Greiner Bio-One, New York, NY, USA). The tubes were centrifuged, and the plasma was collected and stored at 208 C. Plasma samples were analyzed by colorimetric enzyme-linked immunosorbent assay for total cortisol, corticosterone, and DHEA concentrations (Assay Designs, Ann Arbor, MI, USA). All samples were run in duplicate, and samples that had >10% variation between results were reanalyzed. Treatment effects on plasma concentrations of cortisol, corticosterone, and DHEA during trials 1 and 2 were analyzed using a mixed model repeated measures analysis of variance with each individual as the subject, with trial, treatment, sample time, and treatment-sample time interaction as factors. The model was an unstructured covariance (SAS 9.1, SAS Institute, Inc., Cary, NC, USA). Differences between sample times were analyzed using paired T-tests within each trial (SPSS 12.0.1, SPSS Inc., Chicago, IL, USA). Least squares means were presented with the data adjusted for covariance. Temperature– humidity index (THI) was calculated using the formula THI ¼ Temperature (0.550.55 Relative Humidity) (Temperature58), where temperature is in degrees Fahrenheit and relative humidity is in decimal form.24
RESULTS
The temperature ranged from 318 C to 378 C during trial 1, and from 258 C to 348 C during trial 2. Although there were some slight differences in temperature, humidity fluctuated in the opposite direction between trials, such that the THI was relatively consistent between both trials ranging from 80 to 83 in trial 1, and from 76 to 83 in trial 2. Transport in individual stalls versus loose groups did not have a significant effect on cortisol (P ¼ .713), corticosterone (P ¼ .370), or DHEA (P ¼ .416) concentrations
SM Garey et al Vol 30, No 12 (2010)
699
Table 1. Least squares means of cortisol concentrations (ng/mL) in relation to time relative to start of transport for both treatment groups during trials 1 and 2 Treatment Time Pretransport Transport 2 hours 4 hours 6 hours Post-transport 2 hours 4 hours
Stall 2.74a
Group
SEM
2.81a
0.88
19.31b 20.25b 16.55b
16.98b 18.82b 15.01b
3.24 3.80 3.14
4.31a 3.87a
4.27a 2.45a
1.56 0.82
a,b,c
Means within a column without a common superscript differ (P < .01).
(Tables 1, 2, and 3). No significant interaction was reported between treatment and sample time as well as in cortisol, corticosterone, or DHEA concentrations between treatments (P > .370). Pretransport concentrations of cortisol and corticosterone were significantly lower than the concentrations after 2, 4, and 6 hours of transport, but did not differ significantly from concentrations at 2 and 4 hours post-transport (P < .01). However, sample-time comparisons of DHEA yielded no significant differences. There was a significant difference in DHEA concentrations between trials (P ¼ .036), but no significant differences in the cortisol (P ¼ .956) or corticosterone (P ¼ .230) concentrations between trials.
DISCUSSION AND CONCLUSIONS The findings of this study are inconsistent with a 2002 study3 which concluded that horses transported for 24 hours in loose pairs had decreased physiological measures of stress when compared with horses that were individually cross-tied. Stull and Rodiek3 reported a divergence of cortisol concentrations after 3 hours of transport, with stalled horses having higher cortisol concentrations than loose horses. However, the article does not indicate the sample times which were significantly different, the level of significance between treatment groups for each parameter measured, and whether any treatments by sample time interactions were found. Although Stull and Rodiek reported increasing differences in cortisol concentrations between treatment groups from 3 to 24 hours of transport,3 results obtained during this study found no significant differences between the treatment groups between 3 and 6 hours of transport. The horses used in this study loaded into the trailer, remained tied in the stalls, and unloaded from the trailer without incident. This may have been influenced by the
Table 2. Least squares means of corticosterone concentrations (ng/mL) in relation to time relative to start of transport for both treatment groups during trials 1 and 2 Treatment Time Pretransport Transport 2 hours 4 hours 6 hours Post-transport 2 hours 4 hours
Stall
Group
SEM
a
1.17
17.91b 14.11b 13.17b
13.74b 13.19b 10.37b
2.92 2.58 2.10
4.53a 3.29a
3.23a 2.56a
0.87 0.72
5.29
a
3.35
a,b
Means within a column without a common superscript differ (P < .01).
previous training and handling of the horses on a daily basis, and may not be reflective of how an untrained horse with minimal handling would react to being tied in a transport stall. In addition, the stalls were constructed out of metal tube panels that had large areas that the horses could see through, allowing the horses to have visual and a minimal amount of physical contact with the stalled horses next to them. This may have decreased the social isolation effect on the stress of the animals. Although there were no statistically significant differences in cortisol post-transport, behavioral differences were informally observed between trials 1 and 2. After transport in trial 1, the horses had droopy eyes and ears, stood in their paddocks after being turned out, and generally were not active throughout the 4-hour post-transport sample collection period. However, after transport in trial 2, the horses trotted out into their paddocks, many of them rolled, and several were observed biting, kicking, and generally at play during the post-transport sample collection period. This suggests that these horses were less fatigued and may have acclimated to the transport between trials 1 and 2, although they were 35 days apart. Because the THI was consistent between the 2 trials, the apparent differences in fatigue between trials were not likely because of the changes in temperature and humidity. The horses participating in this study were from the same University herd and were often maintained in one group since the time of birth. However, for 60 days before initial transport and during the 35 days between trial 1 and trial 2, they were maintained in groups that were determined by the diets they received in the nutrition study. In this study, because the treatments were balanced by diet, horses that were not recently housed together before transport were transported together in the loose groups. In both trials,
SM Garey et al Vol 30, No 12 (2010)
700
Table 3. Least squares means of DHEA concentrations (pg/mL) in relation to time relative to start of transport for both treatment groups during trials 1 and 2 Treatment Time Pretransport Transport 2 hours 4 hours 6 hours Post-transport 2 hours 4 hours
Stall
displacement, falls, injuries, and orientation during horse transportation. Appl Anim Behav Sci 2000;67:169–179. 8. Eldridge GA, Winfield CG, Cahill DJ. Responses of cattle to different space allowances, pen sizes and road conditions during transport.
Group
SEM
499.8
374.4
88.2
672.9 657.9 578.6
516.6 565.1 753.3
139.9 143.2 166.2
471.3 589.7
7. Collins MN, Friend TH, Jousan FD, Chen SC. Effects of density on
360.0 339.1
90.7 114.8
several of the loose horses showed agonistic behavior, including biting, kicking, and vocalizing when first loaded into the trailer. The group horses were visibly agitated by the agonistic behavior before the start of transport. When interpretating the results of this study, it is important to make the distinction that these horses were accustomed to being in individual stalls and being tied using a halter. Although horses that were being transported for sport underwent some training, bucking horses, feral horses, or horses being transported for slaughter may react violently to being placed in narrow stalls with their heads tied. However, obtaining blood samples from untrained horses while they are on transport vehicles is problematic. ACKNOWLEDGMENTS The authors thank USDA-APHIS-Veterinary Services for funding this research and Dr. Tim Cordes for assisting in study design.
Aust J Exp Agric 1988;28:155–159. 9. Tarrant PV, Kenny FJ, Harrington D, Murphy M. Long distance transportation of steers to slaughter: Effect of stocking density on physiology, behaviour and carcass quality. Livest Prod Sci 1992;30: 223–238. 10. Friend TH. A review of recent research on the transportation of horses. J Anim Sci 2001;79:E32–E40. 11. Clark DK, Friend TH, Dellmeier G. The effect of orientation during trailer transport on heart rate, cortisol and balance in horses. Appl Anim Behav Sci 1993;38:179–189. 12. Smith BL, Jones JH, Carlson GP, Pascoe JR. Effect of body direction on heart rate in trailered horses. Am J Vet Res 1994;55:1007–1011. 13. Smith BL, Jones JH, Hornof WJ, Miles JA, Longworth KE, Willits NH. Effects of road transport on indices of stress in horses. Equine Vet J 1996;28:446–454. 14. Waran NK, Cuddeford D. Effects of loading and transport on the heart rate and behaviour of horses. Appl Anim Behav Sci 1995;43: 71–81. 15. Friend TH. Dehydration, stress, and water consumption of horses during long-distance commercial transport. J Anim Sci 2000;78: 2568–2580. 16. Friend TH, Martin TM, Householder DD, Bushong DM. Stress responses of horses during a long period of transport in a commercial truck. J Am Vet Med Assoc 1998;212:838–844. 17. Stull CL, Rodiek AV. Physiological responses of horses to 24 hours of transportation using a commercial van during summer conditions. J Anim Sci 2000;78:1458–1466. 18. Munck A, Guyre PM, Holbrook NJ. Physiological functions of glucocorticoids in stress and their relation to pharmacological actions. Endocr Rev 1984;5:25–44. 19. Scott LV, Svec F, Dinan T. A preliminary study of dehydroepiandrosterone response to low-dose ACTH in chronic fatigue syndrome and in healthy subjects. Psychiat Res 2000;97:21–28.
REFERENCES 1. U.S. CFR. Title 9: Animals and animal products. Chapter 88: Commercial Transportation of Equines for Slaughter. Washington, DC: U.S. Govt. Printing Office; 2002.
20. Yehuda R, Brand SR, Golier JA, Yang R-K. Clinical correlates of DHEA associated with post-traumatic stress disorder. Acta Psychiat Scand 2006;114:187–193. 21. Ritsner M, Maayan R, Gibel A, Strous R, Modai I, Weizman A. Ele-
2. Council of the European Union. Council Regulation No. 1/2005. Official Journal of the European Union; 2005.
vation of the cortisol/dehydroepiandrosterone ratio in schizophrenia
3. Stull CL, Rodiek AV. Effects of cross-tying horses during 24 h of road
22. Strous R, Spivak B, Yoran-Hegesh R, Maayan R, Averbuch E,
transport. Equine Vet J 2002;34:550–555. 4. Goodwin D. Horse behaviour: evolution, domestication and feralisation. In: Waran N, ed. The welfare of horses. Dordrecht, The Netherlands: Kluwer Academic Publishers; 2002:1–18. 5. Whiting T. Maximum loading density of loose horses. Can J Anim Sci
patients. Eur Neuropsychopharmacol 2004;14:267–273. Kotler M, et al. Analysis of neurosteroid levels in attention deficit hyperactivity disorder. Int J Neuropsychopharmacol 2001;4:259–264. 23. Michael A, Jenaway A, Paykel E, Herbert J. Altered salivary dehydroepiandrosterone levels in major depression in adults. Biol Psychiat 2000;48:989–995.
1999;79:115–118. 6. Stull CL. Responses of horses to trailer design, duration and floor area
24. National Oceanic and Atmospheric Administration. Livestock hot
during commercial transportation to slaughter. J Anim Sci 1999;77:
vice Central Registration, Registration Operations Manual. Washing-
2925–2933.
ton, DC: U.S: Govt. Printing Office; 1976:C-31–76.
weather stress. US Department Commerce, National Weather Ser-