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Cortisol levels and leukocyte population values in transported and exercised horses after acupuncture needle stimulation
Journal of Veterinary Behavior 18 (2017) 56e61
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Equine Research
Cortisol levels and leukocyte population values in transported and exercised horses after acupuncture needle stimulation Maria Rizzo a, Francesca Arfuso a, Claudia Giannetto a, Elisabetta Giudice b, Francesco Longo c, Simona Di Pietro a, Giuseppe Piccione a, * a b c
Department of Veterinary Sciences, University of Messina, Polo Universitario dell’Annunziata, Messina, Italy Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, Messina, Italy Veterinary Practitioner, Bologna, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 October 2016 Received in revised form 28 November 2016 Accepted 16 December 2016 Available online 27 December 2016
Road transport and physical exercise represent stressful stimuli that can lead to homeostasis disruption with direct effect on health status, welfare, and physical performance of the athletic horse. Acupuncture is recognized as a practice modulating the physical well-being of athletes. In this study, the effect of acupuncture treatment on some hematochemical parameters was evaluated in 5 thoroughbred horses after road transport and exercise. Horses competed in 2 official races. For each race, animals were transported from their stables to the racetrack. Horses transported and competed in the first race represent the control group. Two weeks later, the same horses competed in the second race. Before road transport, they were treated with acupuncture (acupuncture group). From animals, blood samples were collected at rest (TPRE); after unloaded (TPOST); 30 minutes after unloaded (TPOST30); at rest in the transit stall (RPRE); at the end of the race (RPOST); and 30 minutes after the race (RPOST30). The effect of transport, exercise, and acupuncture was evaluated on cortisol concentration, white blood cell (WBC) count, and leukocytes population including lymphocytes, neutrophils, monocytes, eosinophils, and basophils. A significant effect of transport (P < 0.01) and exercise (P < 0.001) was found on cortisol, WBC, lymphocyte, and neutrophil values in both groups. Transported and exercised horses subjected to acupuncture treatment showed statistically significant lower cortisol, WBC, and lymphocyte values (P < 0.01). The results found in this study showed that transport and exercise are potential stressors for the athlete horse that may affect its welfare and physical performance. The data suggest that acupuncture practice influences animal’s psychological perception of a stressful condition, probably, by modulating the neural, immune, and endocrine control systems. Ó 2016 Elsevier Inc. All rights reserved.
Keywords: acupuncture horse stress exercise cortisol leukocytes road transport
Introduction Training, transport, and competition are the most important activities that sports horses undergo during their career and probably represent the major cause of injuries and health problems and of economic loss for horse breeding and industry. For that reason, a higher competence in this field could be useful for equine technicians. Effectively, transport and physical exercise represent
* Address for reprint requests and correspondence: Giuseppe Piccione, Department of Veterinary Sciences, University of Messina, Polo Universitario dell’Annunziata, 98168 Messina, Italy. Tel: þ39 090 3503584; Fax: þ39 090 3503975. E-mail address: [email protected] (G. Piccione). http://dx.doi.org/10.1016/j.jveb.2016.12.006 1558-7878/Ó 2016 Elsevier Inc. All rights reserved.
stressful stimuli that can lead to homeostasis disruption with direct effect on animal health status and physical performance of the athletic horse (Hinchcliff et al., 2004). Horses are transported for different reasons, including competitions, breeding, pleasure activities, sales, and slaughtering. Travel includes handling, loading, transport in self, unloading, and often adaptation to a new environment; each of these phases affects horse physiology and behavior in a different way (Hinchcliff et al., 2004). Athlete horses are accustomed to travel before competitions but, after transport, some of them show lower performance than usual. Physiological explanations and proper guidelines about this issue could help owners or trainer to better manage this kind of situation, avoiding related poor performance’s problems (Padalino, 2014). To limit health problems related to transport and exercise stress, it is
M. Rizzo et al. / Journal of Veterinary Behavior 18 (2017) 56e61
important to examine the health status of the horses before and after the physical effort to optimize the environmental conditions inside the truck or to provide them with electrolytes and antioxidants. It is recognized that stressors as transport and physical exercise evoke the activation of hypothalamic-pituitary-adrenal axis and the sympathetic nervous system leading to glucocorticoids and catecholamines release, which in turn modulate the inflammatory reaction. Because the immune and endocrine systems interact with each other under a stressful condition to re-establish homeostasis of the organism (Etim et al., 2013), it is important to monitor immunologic and endocrine markers for the assessment of the physical well-being of athletes (Akimoto et al., 2003). The scientific community is currently interested in studying innovative methods to help the athletes quickly recover from homeostasis disruption after transport, from muscle fatigue after exercise training or fierce competitions, and to help them achieve the best physiological situations, especially before competition. One of the practices that has been used to modulate the physical wellbeing of athletes is the acupuncture (Miyamoto, 1997), one of the traditional Chinese medicine techniques. Acupuncture has been used for treatment of injury, reduction of fatigue, and management of physical condition in athletic fields (Karvelas et al., 1996; Miyamoto, 1997). It has been demonstrated that acupuncture treatment leads to alleviation of muscle tension, improvement of local blood flow, increase of pain threshold, and modulation of autonomic nervous system (Knardahl et al., 1998; Barlas et al., 2000). However, only few studies have been actually published on the influence of acupuncture treatment on the physical wellbeing of transported athlete horses (Angeli et al., 2008; Godoi et al., 2014; Rizzo et al., 2017). To test the hypothesis that stimulation of acupuncture points may improve the recovery abilities of athletic horses by modulating the stress and inflammatory responses, we studied the changes in serum cortisol levels, white blood cell (WBC) count, and leukocyte populations in acupunctured thoroughbred horses after road transport and physical exercise. Materials and methods Animals and study design Five clinically healthy and regularly trained thoroughbred horses (5 gelding, mean age, 4 1 years; mean body weight, 437 15 kg) were enrolled in the study after the informed consent had been provided by the owners. Animals were stabled in individual boxes (3.5 3.5 m) at the same training center located in Sicily, Italy (38 000 4900 N, 15 2501800 E, 80 m above sea level) under natural photoperiod (sunrise at 06.11 AM, sunset at 05.13 PM; mean temperature, 23 C; relative humidity, 70%). Horses were fed, twice a day (07.00 AM; 05.00 PM), a total food amount of about 2.5% of horse body weight (forage:concentrate ratio, 70:30), and water was available ad libitum. The horses competed in 2 official 1300-m races at Mediterraneo racetrack (Siracusa, Sicily, Italy). For each race, the 5 horses were transported from their stables to the racetrack in a 5-horse truck. Each animal traveled, tethered with a 50-cm rope on each side of the halter, in an individual tie stall (length, 2.3 m; width, 0.85 m), giving a total space of about 2 m2 and made 2 journeys of 145 km. The second journey was made 2 weeks after the first one. The driver and the route were always the same. Horses transported and competed in first official race represent the control group (CG). Two weeks later, the same horses competed in the second official race. Before road transport, they were subjected to acupuncture treatment (acupuncture group [AG]) and were loaded after 30 minutes onto the truck gently and without the use of force. Horses were subjected to acupuncture only once 30 minutes before being loaded on the truck. A stainless
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steel needle (diameter, 0.25 mm; length, 30 mm; Dongbang Acupuncture Inc., Chingdao, China) was inserted vertically into a depth of 2-3 cm, and stimulation was produced by bidirectional twisting of needles, as described in previous studies (Lee et al., 2014; Hana et al., 2015). In particular, Figure 1 shows the name and location of the selected acupoints received from AG. For acupuncture stimulation, stainless needles were inserted into the left side or right side of selected acupoints. The locations of acupoints were found according to the anatomical structures. Thermal and hygrometric records throughout the experimental period were carried out inside the truck for the whole study by means of a data logger (Gemini, UK). Temperatures inside the truck ranged between 24 and 21.5 C (maximum/minimum). The relative humidity inside the truck ranged between 68% and 75%. Blood sampling and laboratory analysis From each animal, blood samples were collected by the same operator at rest in their stall at 08:30 AM (TPRE); after being unloaded from the vehicle and housed in the transit stalls (TPOST); 30 minutes after unloaded (TPOST30); at rest in the transit stall (RPRE); at the end of the race (RPOST); and 30 minutes after the race (RPOST30). Blood samples were collected by jugular venipuncture into 8-mL vacutainer tubes with cloth activator (Terumo Co., Tokyo, Japan) and into 2-mL vacutainer tubes containing ethylenediaminetetraacetic acid (EDTA). Immediately after collection, blood samples were placed in refrigerated bags and transported to the laboratory for the analysis. Blood samples collected into tubes containing clot activator were centrifuged at 3000 rpm for 10 minutes, and the obtained sera were analyzed within 2 hours to estimate the concentration of cortisol using enzyme-linked immunosorbent assay (ELISA) kit (cortisol [Horse] ELISA kit; Abnova, Walnut, CA) by means of a microtiter plate reader (EZ Read 400 ELISA; Biochrom, Cambridge, UK). All calibrators and samples were run in duplicate, and samples exhibited parallel displacement to the standard curve for the ELISA analysis. The assay sensitivity was 1 ng/mL. The intra-assay and interassay of variation were 6% and 6.8%, respectively. EDTA whole blood samples were processed in the laboratory within 2 hours for the evaluation of WBC count and leukocyte identification and counting. The evaluation of WBC count was performed by means of an automated hematology analyzer (HeCo Vet C; SEAC, Florence, Italy). To leukocyte identification and counting, a manual analysis was performed on all whole blood samples. Two peripheral blood smears were performed for each sample and, after air drying, the obtained slides were stained through Dif-Stain kit (Titolchimica srl, Rome, Italy). The same laboratory professional has later performed the microscopic analysis of blood films by using an optical microscope (Nikon Eclipse e200; Nikon Instruments Europe BV, Amsterdam, The Netherlands). A manual 200-cell differential count was performed on each blood film. For each animal, the leukocyte differential count was calculated by averaging of the data recorded from each blood film of the same sample. Statistical analysis Data, expressed as mean values standard deviation, were tested for normality using the Shapiro-Wilk normality test. All data were normally distributed (P > 0.05), and the statistical analysis was performed. A general linear model for repeated measure was applied to assess significant effect of the experimental conditions (road transport, exercise, and acupuncture treatment) on studied parameters. When significant differences were found, Bonferroni post hoc comparison was applied. P values <0.05 were considered statistically significant.
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Figure 1. Name and location of the acupoints selected for the study with the schematic diagram of selected acupuncture points.
Statistical analysis was performed using the STATISTICA software package (STATISTICA 7; Stat Software Inc., Tulsa, OK). Results The mean values standard deviation of each parameter obtained from CG and AG throughout the monitoring period are reported in the Table. Statistical analysis revealed a statistically significant effect of transport (P < 0.01) and exercise (P < 0.001) on the values of
cortisol, lymphocytes, and neutrophils in both CG and AG as well as on the values of WBC in CG (Figure 2). No statistically significant effect of transport and exercise was found in monocytes, basophil, and eosinophil in both CG and AG (P > 0.05). Acupuncture treatment statistically influenced (P < 0.01) the values of cortisol, WBC, and lymphocytes (Figure 2). In particular, lower cortisol concentrations were found in transported and exercised horses of AG respect to CG at each sampling times, whereas lower WBC and lymphocyte values were found in transported and exercise horses of AG respect to CG at TPOST, TPOST30, RPOST, and RPOST30. No effect of
Table Mean values standard deviation of each parameter obtained from control (CG) and acupunctured horses (AG) during transport (at rest: TPRE; after unloading from the vehicle: TPOST; and 30 minutes after unloaded: TPOST30) and exercise (at rest: RPRE; at the end of the race: RPOST; and 30 minutes after the race: RPOST30) Parameters
Groups
Road transport
Cortisol (nmol/L)
AG CG AG CG AG CG AG CG AG CG AG CG AG CG
3.89 5.00 5.83 6.05 1.79 1.86 3.75 3.84 0.07 0.10 0.12 0.13 0.10 0.12
TPOST
TPRE
WBC (103/mL) Lymphocytes (103/mL) Neutrophils (103/mL) Monocytes (103/mL) Eosinophils (103/mL) Basophils (103/mL)
Exercise
0.14 0.19 0.38 0.57 0.22 0.09 0.34 0.56 0.02 0.03 0.05 0.03 0.04 0.03
CG, control group; AG, acupuncture group; WBC, white blood cell.
5.03 6.05 6.72 9.52 1.98 4.55 4.50 4.70 0.09 0.10 0.07 0.10 0.08 0.10
TPOST30 0.64 0.16 0.77 0.40 0.14 0.28 0.65 0.30 0.03 0.05 0.06 0.05 0.04 0.01
4.49 6.14 6.72 8.16 1.56 2.79 4.82 4.97 0.11 0.11 0.13 0.18 0.09 0.12
RPRE 0.39 0.66 0.22 0.98 0.10 0.32 0.26 0.63 0.02 0.06 0.07 0.09 0.03 0.02
3.99 5.11 7.09 7.59 2.13 2.51 4.71 4.76 0.07 0.08 0.08 0.12 0.11 0.13
RPOST
0.16 0.30 0.69 0.94 0.27 0.47 0.60 0.53 0.02 0.03 0.05 0.08 0.06 0.08
4.94 6.72 7.35 11.09 2.39 6.05 4.73 4.81 0.08 0.11 0.01 0.01 0.14 0.12
RPOST30
0.65 0.93 1.25 0.90 0.24 0.64 1.05 0.36 0.02 0.05 0.00 0.00 0.05 0.05
4.29 6.09 7.78 10.46 1.81 4.40 5.60 5.68 0.10 0.12 0.10 0.13 0.16 0.14
0.46 0.96 1.17 0.70 0.31 0.50 0.90 0.41 0.05 0.07 0.04 0.03 0.03 0.04
M. Rizzo et al. / Journal of Veterinary Behavior 18 (2017) 56e61
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Figure 2. Mean values standard deviation of white blood cell (WBC) count, lymphocytes, neutrophils, and cortisol obtained from acupuncture group (AG) and control group (CG) during transport (at rest: TPRE; after unloading from the vehicle: TPOST; and 30 minutes after unloaded: TPOST30) and exercise (at rest: RPRE; at the end of the race: RPOST; and 30 minutes after the race: RPOST30).
acupuncture stimulation was found on the values of neutrophils, monocytes, basophils, and eosinophils (P > 0.05). Discussion Road transport and physical exercise are considered physical and psychological stressors that challenge animal homeostasis. Under these stressful stimuli, the body must find a new dynamic equilibrium, and this requires, among other things, adaptive body responses of the nervous, endocrine, and immune systems. One physiological system that is extremely reactive to stress is neuroendocrine system whose activation leads to the release of hormones into the blood stream. Cortisol is one of the main stress response hormones (Etim et al., 2013). Although transport and competition of athletic horses are common practices, these events will always represent novel stressful stimuli for the horse (Cayado et al., 2006; Tadich et al., 2015). The results obtained in this study showed a significant increase of cortisol concentration after transport and physical exercise in both AG and CG. These findings agree with previous studies carried out on transported and exercised horses (Cayado et al., 2006; Padalino, 2014, 2015; Tadich et al., 2015) and correspond with the concept of an involvement of the psychological stress in the control of the hypothalamic-pituitaryadrenal axis activity resulting in hormonal changes indicative of acute stress (Stull and Rodiek, 2002; Etim et al., 2013). Cortisol is the central glucocorticoid in mammalian species, and its production and release from the cortex of the adrenal glands in response to stimuli/stressors plays a major role in mediating the physiological response. Particularly, cortisol has several effects, including the increase of blood pressure, the stimulation of glucose release from
body stores through glycolysis, and an increase in fat metabolism to provide the animal with extra energy (Etim et al., 2013). It is well recognized that, during stressful condition, the rise of cortisol concentration, together with catecholamine release, leads to the mobilization of cells from the marginal pool modifying the numbers of leukocytes in the circulating pool (Hinchcliff et al., 2004). According to previous studies carried out on equine athletes (Hodgson and Rose, 2014; Vazzana et al., 2014), our results showed higher WBC values in both AG and CG after transport and exercise. Although the WBC count increased, it remained within the reference range suggested for equine species (Kästner et al., 1999). Road transport and high-intensity exercise induced a moderate leukocytosis (Hodgson and Rose, 2014). After transport and highintensity exercise, changes in leukocyte count are likely because of catecholamine release and splenic contraction (Stull and Rodiek, 2002; Zobba et al., 2011). According to our results, the leukocytosis observed after road transport and acute intense exercise is biphasic, characterized by an initial increase in lymphocyte values followed by an increase in neutrophil levels (Benschop et al., 1996; Ceddia et al., 1999). In particular, the increase of lymphocyte values observed immediately after transport and exercise is followed by a decrease at TPOST30 and RPOST30 in both AG and CG; however, the values of these cells did not return to resting values. On the contrary, the rise of neutrophil values persisted until TPOST30 and RPOST30 in both AG and CG. This neutrophilia derives from the mobilization from the marginal pool, the reduced ability to migrate from blood to the peripheral tissues, and the increased mobilization of the population of bone marrow reserve (Satué et al., 2012). Lymphocytes have been shown to return
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to resting levels in as short as 1 hour after exercise, whereas neutrophils are slower in returning to resting values that could lead to the increased neutrophil to lymphocyte ratio reported in many studies (Benschop et al., 1996). Acupuncture treatment has been shown to affect cortisol, WBC, and lymphocyte values in both transported and exercised horses. In particular, AG showed lower cortisol levels compared with CG throughout the monitoring period as well as lower WBC and lymphocyte values than CG after stressful stimuli. The decreased cortisol values found in acupunctured subjects agree with previous studies carried out on humans (Kotani et al., 2001; Akimoto et al., 2003), and these findings could suggest, as previously stated, that the stimulation of acupoints was able to reduce stress-induced gene expression in the parvocellular portion of the paraventricular nucleus of the hypothalamus (Liao et al., 1990; Lee et al., 2004; Kim et al., 2009; Park et al., 2011). Moreover, it is widely accepted that acupuncture facilitates the release of certain neurotransmitters, especially opioids, in the central nervous system and activates either of sympathetic or parasympathetic nervous systems, which elicit profound psychophysical responses including potent analgesia and immune modulation (Mori et al., 2002; Sato et al., 2002; Han, 2003). Road transport and physical exercise cause the release of compounds, such as cytokines, prostaglandins, and acute phase protein, which are attributable to inflammation (Fazio et al., 2015; Tur1o et al., 2016). Release of inflammatory markers after physical effort has been proposed to result from the stress-induced damage to musculoskeletal system in horse studies (Tur1o et al., 2016). The lower values of WBC and lymphocytes found in horses that received acupuncture treatment compared with control horses suggest that acupuncture may be a feasible and effective treatment modality for decreasing subjective pain and inflammation. According to our findings, it has been reported that acupuncture can reduce inflammation (Kou et al., 2005; Nager et al., 2015) and could quantitatively and qualitatively regulate leukocytes and their subsets (Yamaguchi et al., 2007). A solid scientific framework is missing to analyze the precise mechanisms by which acupuncture affects peripheral immune parameters. It has been proposed that these mechanisms can be investigated through the psychoneuroimmunology paradigm, in which acupuncture may stimulate the afferent and efferent neuroendocrine pathways to and from the central nervous system, thus indirectly affecting immune functions, including leukocyte circulation (Pacheco-López et al., 2003). The immunomodulatory effects of acupuncture in mice were abolished by addition of badrenergic blockers, demonstrating that acupuncture can modulate some immune responses by activation of sympathetic nervous system (Lundeberg et al., 1991). All these suggesting data support the hypothesis that acupuncture may modulate leukocyte circulation, as observed in this study, through the neuroendocrineimmune network. Conclusion The results obtained in this study showed that thoroughbred horses display a classic physiological stress response to road transport and exercise and that acupuncture treatment affects serum cortisol and leukocyte circulation. In particular, acupunctured subjects displayed a reduction of cortisol, WBC, and lymphocyte values suggesting that acupuncture practice influences animal’s psychological perception of a stressful condition, probably, by modulating the neural, immune, and endocrine control systems. However, other acupuncture points and types of stimulation should be tested to better verify the beneficial effect of this therapy to reduce stress in horses. Future studies in these issues are necessary to investigate the underlying mechanism and its clinical relevance.
Acknowledgments Author contributions: All authors have made substantial contributions to each step of the experimental procedure and article preparation. In particular, Maria Rizzo has conceived the experiment design. Francesca Arfuso prepared the article and performed the laboratory analysis. Claudia Giannetto and Elisabetta Giudice performed the sampling and analyzed the data. Francesco Longo and Simona Di Pietro contributed to statistical elaboration and interpretation of obtained results. Giuseppe Piccione supervised all aspects of the experimental study including writing and editing of the article. Ethical considerations All treatments, housing, and animal care reported in the article were carried out in accordance with the standards recommended by the European Union Directive 2010/63/European Union for animal experiments. Conflict of interest The authors disclaim any financial support or relationships that may pose conflict of interest. References Akimoto, T., Nakahori, C., Aizawa, K., Kimura, F., Fukubayashi, T., Kono, I., 2003. Acupuncture and responses of immunologic and endocrine markers during competition. Med. Sci. Sports Exerc. 35, 1296e1302. Angeli, A.L., Pacca Loureiro Luna, S., 2008. Aquapuncture improves metabolic capacity in thoroughbred horses. J. Equine Vet. Sci. 28, 525e531. Barlas, P., Robinson, J., Allen, J., Baxter, G.D., 2000. Lack of effect of acupuncture upon signs and symptoms of delayed onset muscle soreness. Clin. Physiol. 20, 449e 456. Benschop, R.J., Rodriguez-Feuerhahn, M., Schedlowski, M., 1996. Catecholamineinduced leukocytosis: early observations, current research, and future directions. Brain Behav. Immun. 10, 77e91. Cayado, P., Munoz-Escassi, B., Domínguez, C., Manley, W., Olabarri, B., Sànchez De La Muelas, M., Castejon, F., Maranon, G., Vara, E., 2006. Hormone response to training and competition in athletic horses. Equine Vet. J. 36, 274e278. Ceddia, M.A., Price, E.A., Kohlmeier, C.K., Evans, J.K., Lu, Q., McAuley, E., Woods, J.A., 1999. Differential leukocytosis and lymphocyte mitogenic response to acute maximal exercise in the young and old. Med. Sci. Sports Exerc. 31, 829e836. Etim, N.N., Evans, E.I., Offiong, E.E.A., Williams, M.E., 2013. Stress and the neuroendocrine system: implications for animal well-being. Am. J. Biol. Life Sci. 1, 20e 26. Fazio, F., Ferrantelli, V., Cicero, A., Casella, S., Piccione, G., 2015. Utility of acute phase proteins as biomarkers of transport stress in ewes and beef cattle. Ital. J. Food Saf. 4, 4210. Godoi, T.L.O.S., Villas-Boas, J.D., dos Santos Almeida, N.A., Trigo, P.I., Queiroz de Almeida, F., de Medeiros, M.A., 2014. Pharmacopuncture versus acepromazine in stress responses of horses during road transport. J. Equine Vet. Sci. 34, 294e 301. Han, J.S., 2003. Acupuncture: neuropeptide release produced by electrical stimulation of different frequencies. Trends Neurosci. 26, 17e22. Hana, Y.J., Yi, S.Y., Lee, Y.J., Kim, K.H., Kim, E.J., Lee, S.D., 2015. Quantification of the parameters of twisting-rotating acupuncture manipulation using a needle force measurement system. Integr. Med. Res. 4, 57e65. Hinchcliff, K.W., Kaneps, A.J., Geor, R.J., 2004. Equine Sports Medicine and Surgery. Saunders, London, UK. Hodgson, D.R., Rose, R.J., 2014. The Athletic Horse. Principles and Practice of Equine Sport Medicine. W.B. Saunders Company, Philadelphia, PA. Karvelas, B.R., Hoffman, M.D., Zeni, A.I., 1996. Acute effects of acupuncture on physiological and psychological responses to cycle ergometry. Arch. Phys. Med. Rehabil. 77, 1256e1259. Kästner, S.B.R., Feige, K., Weishaupt, M.A., Auer, J.A., 1999. Heart rate and hematological responses of quarter horses to a reining competition. J. Equine Vet. Sci. 19, 127e131. Kim, H., Park, H.J., Han, S.M., Hahm, D.H., Lee, H.J., Kim, K.S., Shim, I., 2009. The effects of acupuncture stimulation at PC6 (Neiguan) on chronic mild stressinduced biochemical and behavioral responses. Neurosci. Lett. 460, 56e60. Knardahl, S., Elam, M., Olausson, B., Wallin, B.G., 1998. Sympathetic nerve activity after acupuncture in humans. Pain 75, 19e25. Kotani, N., Hashimoto, H., Sato, Y., Sessler, D.I., Yoshioka, H., Kitayama, M., Yasuda, T., Matsuki, A., 2001. Preoperative intradermal acupuncture reduces postoperative
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Journal of Veterinary Behavior journal homepage: www.journalvetbehavior.com
Equine Research
Cortisol levels and leukocyte population values in transported and exercised horses after acupuncture needle stimulation Maria Rizzo a, Francesca Arfuso a, Claudia Giannetto a, Elisabetta Giudice b, Francesco Longo c, Simona Di Pietro a, Giuseppe Piccione a, * a b c
Department of Veterinary Sciences, University of Messina, Polo Universitario dell’Annunziata, Messina, Italy Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, Messina, Italy Veterinary Practitioner, Bologna, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 October 2016 Received in revised form 28 November 2016 Accepted 16 December 2016 Available online 27 December 2016
Road transport and physical exercise represent stressful stimuli that can lead to homeostasis disruption with direct effect on health status, welfare, and physical performance of the athletic horse. Acupuncture is recognized as a practice modulating the physical well-being of athletes. In this study, the effect of acupuncture treatment on some hematochemical parameters was evaluated in 5 thoroughbred horses after road transport and exercise. Horses competed in 2 official races. For each race, animals were transported from their stables to the racetrack. Horses transported and competed in the first race represent the control group. Two weeks later, the same horses competed in the second race. Before road transport, they were treated with acupuncture (acupuncture group). From animals, blood samples were collected at rest (TPRE); after unloaded (TPOST); 30 minutes after unloaded (TPOST30); at rest in the transit stall (RPRE); at the end of the race (RPOST); and 30 minutes after the race (RPOST30). The effect of transport, exercise, and acupuncture was evaluated on cortisol concentration, white blood cell (WBC) count, and leukocytes population including lymphocytes, neutrophils, monocytes, eosinophils, and basophils. A significant effect of transport (P < 0.01) and exercise (P < 0.001) was found on cortisol, WBC, lymphocyte, and neutrophil values in both groups. Transported and exercised horses subjected to acupuncture treatment showed statistically significant lower cortisol, WBC, and lymphocyte values (P < 0.01). The results found in this study showed that transport and exercise are potential stressors for the athlete horse that may affect its welfare and physical performance. The data suggest that acupuncture practice influences animal’s psychological perception of a stressful condition, probably, by modulating the neural, immune, and endocrine control systems. Ó 2016 Elsevier Inc. All rights reserved.
Keywords: acupuncture horse stress exercise cortisol leukocytes road transport
Introduction Training, transport, and competition are the most important activities that sports horses undergo during their career and probably represent the major cause of injuries and health problems and of economic loss for horse breeding and industry. For that reason, a higher competence in this field could be useful for equine technicians. Effectively, transport and physical exercise represent
* Address for reprint requests and correspondence: Giuseppe Piccione, Department of Veterinary Sciences, University of Messina, Polo Universitario dell’Annunziata, 98168 Messina, Italy. Tel: þ39 090 3503584; Fax: þ39 090 3503975. E-mail address: [email protected] (G. Piccione). http://dx.doi.org/10.1016/j.jveb.2016.12.006 1558-7878/Ó 2016 Elsevier Inc. All rights reserved.
stressful stimuli that can lead to homeostasis disruption with direct effect on animal health status and physical performance of the athletic horse (Hinchcliff et al., 2004). Horses are transported for different reasons, including competitions, breeding, pleasure activities, sales, and slaughtering. Travel includes handling, loading, transport in self, unloading, and often adaptation to a new environment; each of these phases affects horse physiology and behavior in a different way (Hinchcliff et al., 2004). Athlete horses are accustomed to travel before competitions but, after transport, some of them show lower performance than usual. Physiological explanations and proper guidelines about this issue could help owners or trainer to better manage this kind of situation, avoiding related poor performance’s problems (Padalino, 2014). To limit health problems related to transport and exercise stress, it is
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important to examine the health status of the horses before and after the physical effort to optimize the environmental conditions inside the truck or to provide them with electrolytes and antioxidants. It is recognized that stressors as transport and physical exercise evoke the activation of hypothalamic-pituitary-adrenal axis and the sympathetic nervous system leading to glucocorticoids and catecholamines release, which in turn modulate the inflammatory reaction. Because the immune and endocrine systems interact with each other under a stressful condition to re-establish homeostasis of the organism (Etim et al., 2013), it is important to monitor immunologic and endocrine markers for the assessment of the physical well-being of athletes (Akimoto et al., 2003). The scientific community is currently interested in studying innovative methods to help the athletes quickly recover from homeostasis disruption after transport, from muscle fatigue after exercise training or fierce competitions, and to help them achieve the best physiological situations, especially before competition. One of the practices that has been used to modulate the physical wellbeing of athletes is the acupuncture (Miyamoto, 1997), one of the traditional Chinese medicine techniques. Acupuncture has been used for treatment of injury, reduction of fatigue, and management of physical condition in athletic fields (Karvelas et al., 1996; Miyamoto, 1997). It has been demonstrated that acupuncture treatment leads to alleviation of muscle tension, improvement of local blood flow, increase of pain threshold, and modulation of autonomic nervous system (Knardahl et al., 1998; Barlas et al., 2000). However, only few studies have been actually published on the influence of acupuncture treatment on the physical wellbeing of transported athlete horses (Angeli et al., 2008; Godoi et al., 2014; Rizzo et al., 2017). To test the hypothesis that stimulation of acupuncture points may improve the recovery abilities of athletic horses by modulating the stress and inflammatory responses, we studied the changes in serum cortisol levels, white blood cell (WBC) count, and leukocyte populations in acupunctured thoroughbred horses after road transport and physical exercise. Materials and methods Animals and study design Five clinically healthy and regularly trained thoroughbred horses (5 gelding, mean age, 4 1 years; mean body weight, 437 15 kg) were enrolled in the study after the informed consent had been provided by the owners. Animals were stabled in individual boxes (3.5 3.5 m) at the same training center located in Sicily, Italy (38 000 4900 N, 15 2501800 E, 80 m above sea level) under natural photoperiod (sunrise at 06.11 AM, sunset at 05.13 PM; mean temperature, 23 C; relative humidity, 70%). Horses were fed, twice a day (07.00 AM; 05.00 PM), a total food amount of about 2.5% of horse body weight (forage:concentrate ratio, 70:30), and water was available ad libitum. The horses competed in 2 official 1300-m races at Mediterraneo racetrack (Siracusa, Sicily, Italy). For each race, the 5 horses were transported from their stables to the racetrack in a 5-horse truck. Each animal traveled, tethered with a 50-cm rope on each side of the halter, in an individual tie stall (length, 2.3 m; width, 0.85 m), giving a total space of about 2 m2 and made 2 journeys of 145 km. The second journey was made 2 weeks after the first one. The driver and the route were always the same. Horses transported and competed in first official race represent the control group (CG). Two weeks later, the same horses competed in the second official race. Before road transport, they were subjected to acupuncture treatment (acupuncture group [AG]) and were loaded after 30 minutes onto the truck gently and without the use of force. Horses were subjected to acupuncture only once 30 minutes before being loaded on the truck. A stainless
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steel needle (diameter, 0.25 mm; length, 30 mm; Dongbang Acupuncture Inc., Chingdao, China) was inserted vertically into a depth of 2-3 cm, and stimulation was produced by bidirectional twisting of needles, as described in previous studies (Lee et al., 2014; Hana et al., 2015). In particular, Figure 1 shows the name and location of the selected acupoints received from AG. For acupuncture stimulation, stainless needles were inserted into the left side or right side of selected acupoints. The locations of acupoints were found according to the anatomical structures. Thermal and hygrometric records throughout the experimental period were carried out inside the truck for the whole study by means of a data logger (Gemini, UK). Temperatures inside the truck ranged between 24 and 21.5 C (maximum/minimum). The relative humidity inside the truck ranged between 68% and 75%. Blood sampling and laboratory analysis From each animal, blood samples were collected by the same operator at rest in their stall at 08:30 AM (TPRE); after being unloaded from the vehicle and housed in the transit stalls (TPOST); 30 minutes after unloaded (TPOST30); at rest in the transit stall (RPRE); at the end of the race (RPOST); and 30 minutes after the race (RPOST30). Blood samples were collected by jugular venipuncture into 8-mL vacutainer tubes with cloth activator (Terumo Co., Tokyo, Japan) and into 2-mL vacutainer tubes containing ethylenediaminetetraacetic acid (EDTA). Immediately after collection, blood samples were placed in refrigerated bags and transported to the laboratory for the analysis. Blood samples collected into tubes containing clot activator were centrifuged at 3000 rpm for 10 minutes, and the obtained sera were analyzed within 2 hours to estimate the concentration of cortisol using enzyme-linked immunosorbent assay (ELISA) kit (cortisol [Horse] ELISA kit; Abnova, Walnut, CA) by means of a microtiter plate reader (EZ Read 400 ELISA; Biochrom, Cambridge, UK). All calibrators and samples were run in duplicate, and samples exhibited parallel displacement to the standard curve for the ELISA analysis. The assay sensitivity was 1 ng/mL. The intra-assay and interassay of variation were 6% and 6.8%, respectively. EDTA whole blood samples were processed in the laboratory within 2 hours for the evaluation of WBC count and leukocyte identification and counting. The evaluation of WBC count was performed by means of an automated hematology analyzer (HeCo Vet C; SEAC, Florence, Italy). To leukocyte identification and counting, a manual analysis was performed on all whole blood samples. Two peripheral blood smears were performed for each sample and, after air drying, the obtained slides were stained through Dif-Stain kit (Titolchimica srl, Rome, Italy). The same laboratory professional has later performed the microscopic analysis of blood films by using an optical microscope (Nikon Eclipse e200; Nikon Instruments Europe BV, Amsterdam, The Netherlands). A manual 200-cell differential count was performed on each blood film. For each animal, the leukocyte differential count was calculated by averaging of the data recorded from each blood film of the same sample. Statistical analysis Data, expressed as mean values standard deviation, were tested for normality using the Shapiro-Wilk normality test. All data were normally distributed (P > 0.05), and the statistical analysis was performed. A general linear model for repeated measure was applied to assess significant effect of the experimental conditions (road transport, exercise, and acupuncture treatment) on studied parameters. When significant differences were found, Bonferroni post hoc comparison was applied. P values <0.05 were considered statistically significant.
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Figure 1. Name and location of the acupoints selected for the study with the schematic diagram of selected acupuncture points.
Statistical analysis was performed using the STATISTICA software package (STATISTICA 7; Stat Software Inc., Tulsa, OK). Results The mean values standard deviation of each parameter obtained from CG and AG throughout the monitoring period are reported in the Table. Statistical analysis revealed a statistically significant effect of transport (P < 0.01) and exercise (P < 0.001) on the values of
cortisol, lymphocytes, and neutrophils in both CG and AG as well as on the values of WBC in CG (Figure 2). No statistically significant effect of transport and exercise was found in monocytes, basophil, and eosinophil in both CG and AG (P > 0.05). Acupuncture treatment statistically influenced (P < 0.01) the values of cortisol, WBC, and lymphocytes (Figure 2). In particular, lower cortisol concentrations were found in transported and exercised horses of AG respect to CG at each sampling times, whereas lower WBC and lymphocyte values were found in transported and exercise horses of AG respect to CG at TPOST, TPOST30, RPOST, and RPOST30. No effect of
Table Mean values standard deviation of each parameter obtained from control (CG) and acupunctured horses (AG) during transport (at rest: TPRE; after unloading from the vehicle: TPOST; and 30 minutes after unloaded: TPOST30) and exercise (at rest: RPRE; at the end of the race: RPOST; and 30 minutes after the race: RPOST30) Parameters
Groups
Road transport
Cortisol (nmol/L)
AG CG AG CG AG CG AG CG AG CG AG CG AG CG
3.89 5.00 5.83 6.05 1.79 1.86 3.75 3.84 0.07 0.10 0.12 0.13 0.10 0.12
TPOST
TPRE
WBC (103/mL) Lymphocytes (103/mL) Neutrophils (103/mL) Monocytes (103/mL) Eosinophils (103/mL) Basophils (103/mL)
Exercise
0.14 0.19 0.38 0.57 0.22 0.09 0.34 0.56 0.02 0.03 0.05 0.03 0.04 0.03
CG, control group; AG, acupuncture group; WBC, white blood cell.
5.03 6.05 6.72 9.52 1.98 4.55 4.50 4.70 0.09 0.10 0.07 0.10 0.08 0.10
TPOST30 0.64 0.16 0.77 0.40 0.14 0.28 0.65 0.30 0.03 0.05 0.06 0.05 0.04 0.01
4.49 6.14 6.72 8.16 1.56 2.79 4.82 4.97 0.11 0.11 0.13 0.18 0.09 0.12
RPRE 0.39 0.66 0.22 0.98 0.10 0.32 0.26 0.63 0.02 0.06 0.07 0.09 0.03 0.02
3.99 5.11 7.09 7.59 2.13 2.51 4.71 4.76 0.07 0.08 0.08 0.12 0.11 0.13
RPOST
0.16 0.30 0.69 0.94 0.27 0.47 0.60 0.53 0.02 0.03 0.05 0.08 0.06 0.08
4.94 6.72 7.35 11.09 2.39 6.05 4.73 4.81 0.08 0.11 0.01 0.01 0.14 0.12
RPOST30
0.65 0.93 1.25 0.90 0.24 0.64 1.05 0.36 0.02 0.05 0.00 0.00 0.05 0.05
4.29 6.09 7.78 10.46 1.81 4.40 5.60 5.68 0.10 0.12 0.10 0.13 0.16 0.14
0.46 0.96 1.17 0.70 0.31 0.50 0.90 0.41 0.05 0.07 0.04 0.03 0.03 0.04
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Figure 2. Mean values standard deviation of white blood cell (WBC) count, lymphocytes, neutrophils, and cortisol obtained from acupuncture group (AG) and control group (CG) during transport (at rest: TPRE; after unloading from the vehicle: TPOST; and 30 minutes after unloaded: TPOST30) and exercise (at rest: RPRE; at the end of the race: RPOST; and 30 minutes after the race: RPOST30).
acupuncture stimulation was found on the values of neutrophils, monocytes, basophils, and eosinophils (P > 0.05). Discussion Road transport and physical exercise are considered physical and psychological stressors that challenge animal homeostasis. Under these stressful stimuli, the body must find a new dynamic equilibrium, and this requires, among other things, adaptive body responses of the nervous, endocrine, and immune systems. One physiological system that is extremely reactive to stress is neuroendocrine system whose activation leads to the release of hormones into the blood stream. Cortisol is one of the main stress response hormones (Etim et al., 2013). Although transport and competition of athletic horses are common practices, these events will always represent novel stressful stimuli for the horse (Cayado et al., 2006; Tadich et al., 2015). The results obtained in this study showed a significant increase of cortisol concentration after transport and physical exercise in both AG and CG. These findings agree with previous studies carried out on transported and exercised horses (Cayado et al., 2006; Padalino, 2014, 2015; Tadich et al., 2015) and correspond with the concept of an involvement of the psychological stress in the control of the hypothalamic-pituitaryadrenal axis activity resulting in hormonal changes indicative of acute stress (Stull and Rodiek, 2002; Etim et al., 2013). Cortisol is the central glucocorticoid in mammalian species, and its production and release from the cortex of the adrenal glands in response to stimuli/stressors plays a major role in mediating the physiological response. Particularly, cortisol has several effects, including the increase of blood pressure, the stimulation of glucose release from
body stores through glycolysis, and an increase in fat metabolism to provide the animal with extra energy (Etim et al., 2013). It is well recognized that, during stressful condition, the rise of cortisol concentration, together with catecholamine release, leads to the mobilization of cells from the marginal pool modifying the numbers of leukocytes in the circulating pool (Hinchcliff et al., 2004). According to previous studies carried out on equine athletes (Hodgson and Rose, 2014; Vazzana et al., 2014), our results showed higher WBC values in both AG and CG after transport and exercise. Although the WBC count increased, it remained within the reference range suggested for equine species (Kästner et al., 1999). Road transport and high-intensity exercise induced a moderate leukocytosis (Hodgson and Rose, 2014). After transport and highintensity exercise, changes in leukocyte count are likely because of catecholamine release and splenic contraction (Stull and Rodiek, 2002; Zobba et al., 2011). According to our results, the leukocytosis observed after road transport and acute intense exercise is biphasic, characterized by an initial increase in lymphocyte values followed by an increase in neutrophil levels (Benschop et al., 1996; Ceddia et al., 1999). In particular, the increase of lymphocyte values observed immediately after transport and exercise is followed by a decrease at TPOST30 and RPOST30 in both AG and CG; however, the values of these cells did not return to resting values. On the contrary, the rise of neutrophil values persisted until TPOST30 and RPOST30 in both AG and CG. This neutrophilia derives from the mobilization from the marginal pool, the reduced ability to migrate from blood to the peripheral tissues, and the increased mobilization of the population of bone marrow reserve (Satué et al., 2012). Lymphocytes have been shown to return
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to resting levels in as short as 1 hour after exercise, whereas neutrophils are slower in returning to resting values that could lead to the increased neutrophil to lymphocyte ratio reported in many studies (Benschop et al., 1996). Acupuncture treatment has been shown to affect cortisol, WBC, and lymphocyte values in both transported and exercised horses. In particular, AG showed lower cortisol levels compared with CG throughout the monitoring period as well as lower WBC and lymphocyte values than CG after stressful stimuli. The decreased cortisol values found in acupunctured subjects agree with previous studies carried out on humans (Kotani et al., 2001; Akimoto et al., 2003), and these findings could suggest, as previously stated, that the stimulation of acupoints was able to reduce stress-induced gene expression in the parvocellular portion of the paraventricular nucleus of the hypothalamus (Liao et al., 1990; Lee et al., 2004; Kim et al., 2009; Park et al., 2011). Moreover, it is widely accepted that acupuncture facilitates the release of certain neurotransmitters, especially opioids, in the central nervous system and activates either of sympathetic or parasympathetic nervous systems, which elicit profound psychophysical responses including potent analgesia and immune modulation (Mori et al., 2002; Sato et al., 2002; Han, 2003). Road transport and physical exercise cause the release of compounds, such as cytokines, prostaglandins, and acute phase protein, which are attributable to inflammation (Fazio et al., 2015; Tur1o et al., 2016). Release of inflammatory markers after physical effort has been proposed to result from the stress-induced damage to musculoskeletal system in horse studies (Tur1o et al., 2016). The lower values of WBC and lymphocytes found in horses that received acupuncture treatment compared with control horses suggest that acupuncture may be a feasible and effective treatment modality for decreasing subjective pain and inflammation. According to our findings, it has been reported that acupuncture can reduce inflammation (Kou et al., 2005; Nager et al., 2015) and could quantitatively and qualitatively regulate leukocytes and their subsets (Yamaguchi et al., 2007). A solid scientific framework is missing to analyze the precise mechanisms by which acupuncture affects peripheral immune parameters. It has been proposed that these mechanisms can be investigated through the psychoneuroimmunology paradigm, in which acupuncture may stimulate the afferent and efferent neuroendocrine pathways to and from the central nervous system, thus indirectly affecting immune functions, including leukocyte circulation (Pacheco-López et al., 2003). The immunomodulatory effects of acupuncture in mice were abolished by addition of badrenergic blockers, demonstrating that acupuncture can modulate some immune responses by activation of sympathetic nervous system (Lundeberg et al., 1991). All these suggesting data support the hypothesis that acupuncture may modulate leukocyte circulation, as observed in this study, through the neuroendocrineimmune network. Conclusion The results obtained in this study showed that thoroughbred horses display a classic physiological stress response to road transport and exercise and that acupuncture treatment affects serum cortisol and leukocyte circulation. In particular, acupunctured subjects displayed a reduction of cortisol, WBC, and lymphocyte values suggesting that acupuncture practice influences animal’s psychological perception of a stressful condition, probably, by modulating the neural, immune, and endocrine control systems. However, other acupuncture points and types of stimulation should be tested to better verify the beneficial effect of this therapy to reduce stress in horses. Future studies in these issues are necessary to investigate the underlying mechanism and its clinical relevance.
Acknowledgments Author contributions: All authors have made substantial contributions to each step of the experimental procedure and article preparation. In particular, Maria Rizzo has conceived the experiment design. Francesca Arfuso prepared the article and performed the laboratory analysis. Claudia Giannetto and Elisabetta Giudice performed the sampling and analyzed the data. Francesco Longo and Simona Di Pietro contributed to statistical elaboration and interpretation of obtained results. Giuseppe Piccione supervised all aspects of the experimental study including writing and editing of the article. Ethical considerations All treatments, housing, and animal care reported in the article were carried out in accordance with the standards recommended by the European Union Directive 2010/63/European Union for animal experiments. Conflict of interest The authors disclaim any financial support or relationships that may pose conflict of interest. References Akimoto, T., Nakahori, C., Aizawa, K., Kimura, F., Fukubayashi, T., Kono, I., 2003. Acupuncture and responses of immunologic and endocrine markers during competition. Med. Sci. Sports Exerc. 35, 1296e1302. Angeli, A.L., Pacca Loureiro Luna, S., 2008. Aquapuncture improves metabolic capacity in thoroughbred horses. J. Equine Vet. Sci. 28, 525e531. Barlas, P., Robinson, J., Allen, J., Baxter, G.D., 2000. Lack of effect of acupuncture upon signs and symptoms of delayed onset muscle soreness. Clin. Physiol. 20, 449e 456. Benschop, R.J., Rodriguez-Feuerhahn, M., Schedlowski, M., 1996. Catecholamineinduced leukocytosis: early observations, current research, and future directions. Brain Behav. Immun. 10, 77e91. Cayado, P., Munoz-Escassi, B., Domínguez, C., Manley, W., Olabarri, B., Sànchez De La Muelas, M., Castejon, F., Maranon, G., Vara, E., 2006. Hormone response to training and competition in athletic horses. Equine Vet. J. 36, 274e278. Ceddia, M.A., Price, E.A., Kohlmeier, C.K., Evans, J.K., Lu, Q., McAuley, E., Woods, J.A., 1999. Differential leukocytosis and lymphocyte mitogenic response to acute maximal exercise in the young and old. Med. Sci. Sports Exerc. 31, 829e836. Etim, N.N., Evans, E.I., Offiong, E.E.A., Williams, M.E., 2013. Stress and the neuroendocrine system: implications for animal well-being. Am. J. Biol. Life Sci. 1, 20e 26. Fazio, F., Ferrantelli, V., Cicero, A., Casella, S., Piccione, G., 2015. Utility of acute phase proteins as biomarkers of transport stress in ewes and beef cattle. Ital. J. Food Saf. 4, 4210. Godoi, T.L.O.S., Villas-Boas, J.D., dos Santos Almeida, N.A., Trigo, P.I., Queiroz de Almeida, F., de Medeiros, M.A., 2014. Pharmacopuncture versus acepromazine in stress responses of horses during road transport. J. Equine Vet. Sci. 34, 294e 301. Han, J.S., 2003. Acupuncture: neuropeptide release produced by electrical stimulation of different frequencies. Trends Neurosci. 26, 17e22. Hana, Y.J., Yi, S.Y., Lee, Y.J., Kim, K.H., Kim, E.J., Lee, S.D., 2015. Quantification of the parameters of twisting-rotating acupuncture manipulation using a needle force measurement system. Integr. Med. Res. 4, 57e65. Hinchcliff, K.W., Kaneps, A.J., Geor, R.J., 2004. Equine Sports Medicine and Surgery. Saunders, London, UK. Hodgson, D.R., Rose, R.J., 2014. The Athletic Horse. Principles and Practice of Equine Sport Medicine. W.B. Saunders Company, Philadelphia, PA. Karvelas, B.R., Hoffman, M.D., Zeni, A.I., 1996. Acute effects of acupuncture on physiological and psychological responses to cycle ergometry. Arch. Phys. Med. Rehabil. 77, 1256e1259. Kästner, S.B.R., Feige, K., Weishaupt, M.A., Auer, J.A., 1999. Heart rate and hematological responses of quarter horses to a reining competition. J. Equine Vet. Sci. 19, 127e131. Kim, H., Park, H.J., Han, S.M., Hahm, D.H., Lee, H.J., Kim, K.S., Shim, I., 2009. The effects of acupuncture stimulation at PC6 (Neiguan) on chronic mild stressinduced biochemical and behavioral responses. Neurosci. Lett. 460, 56e60. Knardahl, S., Elam, M., Olausson, B., Wallin, B.G., 1998. Sympathetic nerve activity after acupuncture in humans. Pain 75, 19e25. Kotani, N., Hashimoto, H., Sato, Y., Sessler, D.I., Yoshioka, H., Kitayama, M., Yasuda, T., Matsuki, A., 2001. Preoperative intradermal acupuncture reduces postoperative
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