- Email: [email protected]
Effects of housing and short-term transportation on hormone and lymphocyte receptor concentrations in beef cattle
Research in Veterinary Science 90 (2011) 341–345
Contents lists available at ScienceDirect
Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Effects of housing and short-term transportation on hormone and lymphocyte receptor concentrations in beef cattle Rosangela Odore a,*, Paola Badino a, Giovanni Re a, Raffaella Barbero a, Barbara Cuniberti a, Antonio D’Angelo b, Carlo Girardi a, Elena Fraccaro a, Martina Tarantola c a b c
Department of Animal Pathology, Division of Pharmacology and Toxicology, University of Turin, Italy Department of Animal Pathology, Division of Medicine, University of Turin, Italy Department of Animal Production, Epidemiology and Ecology, University of Turin, Italy
a r t i c l e
i n f o
Article history: Received 12 January 2010 Accepted 22 May 2010
Keywords: Beef cattle Hormones Receptors Lymphocytes Transportation Stress
a b s t r a c t The experiment was designed to evaluate the effects of housing system and short-term transportation on the pituitary and adrenal response and on blood progesterone concentrations of beef cattle. Since the use of steroid hormones in farm animals has been banned in the EU (Council Directive 96/22/EC), it seems important to study the possible modifications in serum progesterone concentrations induced by stress in cattle. Thirty-two, 6 months old male Piedmontese beef cattle (16 reared in a littered loose house, Group A, and 16 housed in a littered tying stall barn, Group B) were blood sampled at T1 (6 months old), T2 (12 months old), T3 (18 months old, before transportation to the slaughterhouse) and T4 (after transportation to the slaughterhouse) in order to measure hormonal concentrations and lymphocyte glucocorticoid (GR) and b-adrenergic (b-AR) receptor concentrations. Circulating hormone concentrations were measured using commercial radioimmunoassay kits, whereas lymphocyte receptor density was determined through binding assays. In beef cattle housed in tie stall barn a significant increase in serum cortisol concentration was observed at T3, whereas there was no effect of the housing system on blood progesterone concentrations. Short-term transportation caused a significant increase in blood cortisol and catecholamine concentrations in both groups, whereas lymphocyte GR and b-AR significantly decreased in Group A. Our data confirm the activation of the hypothalamic–pituitary–adrenal axis and the catecholaminergic system in short-term transportation and suggest that the stress-induced increase in circulating progesterone concentrations does not exceed the limit established by pending legislation. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction In recent years, animal welfare and optimization of producer and consumer expectations have lead to an increasing attention to the study of the effects of husbandry conditions and transportation on behavioural, biochemical, functional, endocrine and pathological variables in cattle (Fike and Spire, 2006; Broom, 2007; Gupta et al., 2007b). Despite the fact that animal welfare auditing programmes are now being conducted in many countries around the world in order to comply with OIE (World Organisation for Animal Health) animal welfare guidelines and to limit stress-induced meat quality problems, it is sometimes difficult to characterise reliable biomarkers of stress (Grandin, 2007). Among the indicators generally used to assess the welfare of animals being handled or transported, are the measurement of physiological variables such as hormones which may be quantified over repeated sampling * Corresponding author. Tel.: +39 011 6709018; fax: +39 011 6709017. E-mail address: [email protected] (R. Odore). 0034-5288/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2010.05.026
time points. In fact, stress, a physiological response to several exogenous or endogenous stimuli that normally cause neuroendocrine activation and response of the endocrine system, is mainly characterized by the concomitant release of catecholamines and glucocorticoids from the adrenal glands (Ehrhart-Bornstein and Bornstein, 2008). The hypothalamic corticotropin-releasing hormone (CRH) system and the sympathetic nervous system are postulated to be functionally related to each other and to regulate reciprocally their functions within the central nervous system (Ulrich-Lai and Engeland, 2005). In animal husbandry, stress occurs when animal experiences changes in the environment that stimulate body responses to restore homeostasis (Mumma et al., 2006). It is established that many stressors, such as immobilization, housing, handling, loading, unloading and transport increase the secretion of catecholamines from the adrenal medulla and sympathetic nerve terminals (Broom, 2007) and that such stressors also potentiate the secretion of the hypothalamic–pituitary–adrenocortical axis hormones (Jeong et al., 2000). Furthermore, the modifications of
342
R. Odore et al. / Research in Veterinary Science 90 (2011) 341–345
blood hormone concentrations (e.g. cortisol and catecholamines) due to stressful conditions are correlated with changes in lymphocyte receptor density (Odore et al., 2004). The correlation of circulating hormone concentrations with lymphocyte receptor density is considered as a more suitable indicator of stress than blood hormone levels alone. As stated by Knowles and Warriss (2007), the study of such indices related to immune function is useful to better characterise the response to stress in cattle and other animal species. In fact, these changes may have a significant effect on cattle welfare, reproductive performance, weight gain and meat quality (Villarroel et al., 2003). As far as cattle husbandry is concerned, the housing system and transportation represent two main stressful factors. Traditional rearing techniques often ensure the final product quality requested by the consumer, however in so doing, animal behaviour and welfare may be compromised (Xiccato et al., 2002). For example, it has been observed that insufficient space allowances induce a state of stress that alters the activity of the pituitary-adrenal axis, immune function, behaviour and growth rate (Gupta et al., 2007a). Moreover, Herskin et al. (2004) found significant changes in the acute stress response in cows kept in tie-stalls. A substantial amount of research has been devoted to the study of stress transportation in livestock (Murata et al., 1987; Odore et al., 2004; Gupta et al., 2007b; Buckham Sporer et al., 2007). Historically, significant increases in blood glucocorticoid hormones have been reported during the stress response following transportation. However, little is known about the effects of transportation on other steroid hormones, such as sex steroids. Buckham Sporer et al. (2008) found a significant increase in blood progesterone concentrations after truck transportation in beef bulls. Since the use of steroid hormones in farm animals has been banned in the EU (Council Directive 96/22/EC), it seems important to study the possible modifications in serum progesterone concentrations induced by stress in cattle. The finding of steroid hormones in the systemic circulation is not proof of illegal use since these hormones occur naturally in physiological concentrations in animals and humans. Limits in blood progesterone concentrations in cattle have been set in order to prevent consumers from ingesting meat from illegally treated animals, however; scientific evidence to support this decision is needed. The aim of the study was to measure blood cortisol, progesterone and catecholamine concentrations and lymphocyte glucocorticoid (GR) and b-adrenergic receptor (b-AR) concentrations in male beef cattle housed in a littered tying stall barn and in a littered loose house at different time points during the breeding period, before and after road transportation to the slaughterhouse.
iod, animals were transported from the fattening farm to the slaughterhouse using a double deck. The animals were loaded on the lower deck in the front compartment with spatial allowances per animal of 1.4 m2. The transporter was naturally ventilated. The journey duration was approximately 45 min. The truck travelled mainly on secondary roads at a speed of about 60 km/h. The mean final live weight was 640 ± (SEM) 18 kg and 630 ± (SEM) 20 kg for Groups A and B, respectively. At T3 and T4 additional blood samples were collected in order to separate the lymphocyte fraction and to measure circulating catecholamine concentrations.
2. Materials and methods
2.4. b-Adrenoceptor binding assay
2.1. Animals and sample collection
b-Adrenoceptors were measured through binding assay as described by Odore et al. (2006), but introducing some minor modifications. Briefly 100 lL of lymphocyte suspension were incubated for 1 h at 37 °C with increasing concentrations of the labelled antagonist ( )[3H]CGP 12177 (0.06–4 nM) in a total volume of 200 lL. The non-specific binding was assessed in the presence of 100 lM unlabelled isoproterenol. Incubation was stopped by adding 2 mL of ice-cold buffered saline (154 mM NaCl and 50 mM Tris–HCl, pH 7.4) and the incubation mixture was immediately filtered under vacuum using pre-soaked glass fibre filters (Whatman GF/C). Filters were then accurately washed with 3 3 mL of buffered saline and solubilised with 4 mL of scintillation fluid (Filter Count, Canberra Packard). The radioactivity retained on wet filters was measured by a Tri-Carb 1600 TR Canberra Packard scintillator spectrometer, at an efficiency of 60%. The quantitative parameters, number of sites/cell and Kd (nM), were determined with the aid of a computer program (GraphPad, Prism).
A total of 32 6 months old male Piedmontese beef calves were monitored at different experimental time points during the breeding period at the fattening farm. Sixteen of them were reared in a littered loose house, Group A: mean live weight 180 ± (SEM) 15 kg, and 16 were housed in a tie stall, Group B: mean live weight 183 ± (SEM) 20 kg. Animals were blood sampled at T1 (6 months old), T2 (12 months old), T3 (18 months old, before transportation to the slaughterhouse) and T4 (after transportation to the slaughterhouse) in order to measure cortisol and progesterone concentrations. Animals of Group A were kept in a free stall housing system. They were housed in groups of nine animals per pen, each animal had a pen area allowance of 4.0 m2, with deep straw bedding. Group B cattle were tied by a tether fixed to the feed trough (space allowance 210 110 cm); animals were accommodated on concrete solid floor bedded with straw. At the end of the breeding per-
2.2. Lymphocyte separation Lymphocytes were isolated by a density gradient centrifugation method (Odore et al., 2004). Blood samples (130 mL) were collected in 10 mL tubes containing citric acid and dextrose (Becton Dickinson, Milan, Italy) via direct jugular venipuncture using mild restraint in a holding chute. Blood samples were immediately transported to the laboratory and centrifuged at 1300g for 15 min at room temperature. For each tube, the buffy-coat was collected, transferred to 5 mL tubes and diluted to 3 mL with Hanks’ balanced salt solution containing 1% sodium citrate. Diluted buffy-coats were carefully layered on 1.5 mL Histopaque-1077 and centrifuged at 1800g for 10 min at room temperature. After careful removal of plasma–platelet layers, the mononuclear cell layers were carefully collected, washed twice with Hanks’ solution and centrifuged at 3300g for 10 min at 4 °C. Pellets were then re-suspended in appropriate buffer (50 mM Tris-base, 1 mM EDTA, 12 mM thioglycerol, 250 mM glycerol and 10 mM sodium molybdate, pH 7.4). After counting the number of lymphocytes, cells were diluted in a specific buffer to a final concentration of 1 107/mL in order to measure b-AR and GR concentration. 2.3. Plasma catecholamine measurement Samples used for the determination of plasma catecholamine concentrations were collected in 5 mL tubes containing ethylene diaminotetraacetic acid (EDTA). Plasma was immediately separated by refrigerated centrifugation (2500g for 10 min at 4 °C) and then stored at 80 °C until analysis. Noradrenaline and adrenaline concentrations were measured using a commercial radioimmunoassay kit (KatCombi, IBL, Hamburg). The intra-assay and inter-assay coefficients of variation were 4.8 ± 0.9% and 5.7 ± 1.0%, respectively, whereas recovery was 94 ± 2% for noradrenaline and 89 ± 4% for adrenaline.
343
R. Odore et al. / Research in Veterinary Science 90 (2011) 341–345
2.5. Serum cortisol and progesterone measurement Blood samples for cortisol and progesterone concentration measurement were allowed to clot at room temperature, centrifuged (2500g for 10 min at 4 °C) and aliquots of obtained sera stored at 80 °C until assay. Commercial bovine cortisol (DSL 2000, Diagnostic Systems Laboratories, Inc.) and progesterone (DSL 3400 Diagnostic Systems Laboratories, Inc.) radioimmunoassay kits were used to measure serum hormone concentrations. The intra- and inter-assay coefficients of variation were 6.1 ± 1.4% and 8 ± 0.7% for cortisol and 4.4 ± 0.7% and 2.5 ± 0.5% for progesterone, respectively. The recovery was 95 ± 2.7% for cortisol and 92.3 ± 5% for progesterone. 2.6. Glucocorticoid receptor (GR) binding assay In order to measure lymphocyte GR density, aliquots of lymphocytes (200 lL) were incubated overnight at 4 °C with increasing concentrations (0.3–10 nM) of [3H]dexamethazone in a total volume of incubation of 250 lL. The non-specific binding was assessed in presence of 5 lM unlabelled dexamethazone. In order to separate free radioligand from the mixture, 250 lL of a dextran–charcoal solution (0.5% charcoal and 0.05% dextran) was added to each tube. After 15 min incubation, tubes were centrifuged for 15 min at 3200g at 4 °C, 200 lL of the supernatant were added to 4 mL of scintillation liquid (Pico Fluor, Canberra Packard), and the radioactivity was measured for 2 min by use of a b-counter (Tri-Carb 1600 TR Canberra Packard). The Kd and the number of sites/cell were calculated by use of GraphPad Prism.
were evaluated with ANOVA and Tuckey multiple comparison test with the significant limit set at P < 0.05. Inter-group differences for cortisol, progesterone and catecholamines at each experimental time point were analysed using Student’s t test, with the aid of a computer program (GraphPad Instat). 3. Results 3.1. Blood hormone concentrations In cattle reared in loose house system, no statistically significant differences were found among T1, T2 and T3 treatments (Table 1). In cattle reared in tie stall barn, blood cortisol concentrations increased significantly (P < 0.05) at T2 and T3 with respect to T1 (Table 1). Transport of animals to the slaughterhouse caused a significant increase in blood cortisol concentrations in Group A (P < 0.001) and in Group B (P < 0.001). Serum cortisol concentrations were higher (P < 0.05) in Group B than in Group A at T3 and T4 (Table 1). In Group A, serum progesterone concentrations were unchanged throughout the study (Table 1), whereas in Group B, an increase (P < 0.001) was detected at T4 (Table 1). At T4 serum progesterone concentration was significantly (P < 0.05) higher in Group B with respect to Group A. In both groups the transportation to the slaughterhouse caused a statistically significant (P < 0.05) increase in adrenaline and noradrenaline concentrations (Tables 2). At T3 blood catecholamine concentrations were significantly higher in Group B than in Group A (Table 2).
2.7. Drugs and radiochemicals ( )[3H]CGP 12177 (54 Ci/mmol) was obtained from Perkin Elmer and [3H]dexamethasone (89 Ci/mmol) was purchased from Amersham Pharmacia Biotech Europe. All other reagents were obtained from Sigma.
Table 2 Blood adrenaline and noradrenaline (ng/mL) concentrations in loose house (Group A) and tie stall (Group B) reared beef cattle at different experimental time points (T3 = 18 months old before transportation; T4 = 18 months old after transportation to the slaughterhouse); (mean values ± SEM; n = 16; Intra and inter-group differences Student’s t test).
2.8. Statistical analysis
Blood adrenaline concentrations Group A
Results are expressed as mean values ± SEM of triplicate measurements. Intra-group differences for cortisol and progesterone at different time points and lymphocyte receptor concentrations
T3 T4 Intra-group differences
0.10 ± 0.01ax 0.25 ± 0.03bx a vs. b P < 0.0001
Blood noradrenaline concentrations Group A Table 1 Blood cortisol and progesterone concentrations (ng/mL) in loose house (Group A) and tie stall (Group B) reared beef cattle at different experimental time points (T1 = 6 months old; T2 = 12 months old; T3 = 18 months old before transportation; T4 = 18 months old after transportation to the slaughterhouse); (mean values ± SEM; n = 16; Intra-group differences: ANOVA and Tukey Multiple Comparison Test; Intergroup differences Student’s t test). Blood cortisol concentrations Group A T1 T2 T3 T4 Intra-group differences
Group B
ax
ax
4.1 ± 0.4 4.5 ± 0.9ax 4.0 ± 0.7ax 9.2 ± 1.0bx a vs. b P < 0.001
3.0 ± 0.4 6.7 ± 0.9bx 6.3 ± 0.7by 20.6 ± 1.1cy a vs. b vs. c P < 0.05
Blood progesterone concentrations Group A T1 T2 T3 T4 Intra-group differences
Group B ax
0.12 ± 0.01 0.14 ± 0.02ax 0.12 ± 0.06ax 0.15 ± 0.04ax n.s.
ax
0.12 ± 0.05 0.12 ± 0.02ax 0.10 ± 0.01ax 0.39 ± 0.08by a vs. b P < 0.01
Inter-group differences n.s. n.s. x vs. y P < 0.05 x vs. y P < 0.0001
Inter-group differences n.s. n.s. n.s. x vs. y P < 0.05
T3 T4 Intra-group differences
0.26 ± 0.04ax 0.50 ± 0.05bx a vs. b P < 0.001
Group B 0.13 ± 0.01ay 0.35 ± 0.10bx a vs. b P < 0.05 Group B 0.42 ± 0.06ay 0.69 ± 0.07by a vs. b P < 0.01
Inter-group differences x vs. y P < 0.05 n.s.
Inter-group differences x vs. y P < 0.05 x vs. y P < 0.05
Table 3 Concentrations (sites/cell) of lymphocyte glucocorticoid and b-adrenergic receptors at T3 and T4 in loose house (Group A) and tie stall (Group B) beef cattle (mean values ± SEM; n = 16; ANOVA and Tukey Multiple Comparison Test). Lymphocyte glucocorticoid receptors Group A Group B T3 18,194 ± 4184 6775 ± 545 T4 6991 ± 1476 4065 ± 376 Statistical significance: T3 Group A vs. T3 Group B (P < 0.01); T3 Group A vs. T4 Group A (P < 0.01) Lymphocyte b-adrenoceptors Group A Group B T3 30,544 ± 3362 13,426 ± 935 T4 11,831 ± 1721 8432 ± 784 Statistical significance: T3 Group A vs. T4 Group A (P < 0.001); T3 Group A vs. T3 Group B (P < 0.001)
344
R. Odore et al. / Research in Veterinary Science 90 (2011) 341–345
3.2. Lymphocyte receptor concentrations Lymphocyte GR decreased in Group A (P < 0.01) and was not different in Group B after transport (Table 3). No significant differences were found among the Kd values in both groups (data not shown). Lymphocyte b-AR decreased after transport both in Group A (P < 0.001) and in Group B (Table 3). Statistically significant differences (P < 0.001) were observed between Group A and group B before transportation. The Kd values (nM) for b-AR demonstrated a high degree of the highly specific labelled ligand-receptor affinity. No differences (P > 0.05) were observed in both groups, either before or after transportation (data not shown). 4. Discussion In the present study, a significant increase in serum cortisol concentrations was detected in beef cattle reared in tie stall barns, whereas in the loose housing system no significant variation was observed during the breeding period. The effect of the rearing on circulating cortisol concentrations has been investigated in cattle (Veissier et al., 2001; Gupta et al., 2007a), sheep (Degabriele and Fell, 2001), pig (Gade, 2008) and horses (Visser et al., 2008). Gupta et al. (2007a) found that restricted space allowances increase acute plasma cortisol concentrations in bulls and Andrighetto et al. (1999) observed a favourable effect of group rearing on growth performance in the late period of growth. In our study, in tie stall barn beef cattle, blood cortisol concentrations remained unchanged between T2 and T3, suggesting the exposure to a continuously elevated stress due to the housing system. Chronic stress of housing is reported to modify the functioning of the pituitary gland and lead to either adaptation or regulatory changes in the pituitary and/or adrenal gland (Gupta et al., 2007a). By contrast, short-term transportation induced a significant increase in cortisol concentrations in both groups. Literature has extensively reviewed the journey aspects with potential effects on cattle, with duration of transport representing one of the main stressful factors (Knowles, 1999). In the case of long distance transport, animals usually suffer from long fasting periods and exhaustion, thus significant modifications of endocrine and biochemical variables may occur (Odore et al., 2004). However, Honkovaara et al. (2003) observed that animals transported for long distances express a lower stress profile than animals travelling over a short distance. In animals transported for long distances exhaustion of the adrenal gland may occur, whereas cattle transported for short distance journeys may express acute psychological stress to the novelty of the transport process. Van de Water et al. (2003) found that short-term transportation can elicit a wide range of physiological changes such an increase in cortisol, lactate and creatine kinase concentrations and identified loading and unloading as the main causes of stress. Also according to Eicher (2001), the greatest cortisol response is associated with shorter transports. The significant differences in blood cortisol concentrations observed at T3 and T4 between the two groups suggest that animals reared in tie stall barns were more stressed than the loose housed cattle. Mounier et al. (2006) found that bull calves kept in their social group during the pre-slaughter period had less increase in cortisol concentrations and that the effect was even more pronounced when animals had been reared together. Circulating progesterone concentrations increased significantly in Group B after transportation. Buckham Sporer et al. (2008) had already demonstrated that in young beef bulls transported by road for up to 9 h, serum progesterone concentrations significantly increased in association with the increase in cortisol. However, to our knowledge no data concerning the effects of the housing system and short-term transportation have been published to date. The stress-
induced increase in blood progesterone concentrations probably occurs as a result of the rate limiting enzymes responsible for the conversion of progesterone to cortisol (Cooper et al., 1995). This would suggest that stressful conditions such as the keeping animals in tie stall barns cause greater secretion of progesterone. By contrast, in our study, no significant differences in blood progesterone concentrations were found during the breeding period in both groups of animals. The increase observed after transportation in Group B cattle did not exceed the limit set by national legislation (1.5 ng/mL). This finding is consistent with mild blood progesterone concentration fluctuation in response to stress. In agreement with previous findings (Odore et al., 2004), a significant increase in adrenaline and noradrenaline concentrations was found post transport. As catecholamines have been shown to cause depletion in muscle glycogen in the pre-slaughter period, it is expected that sympathetic activation may influence meat quality (Muchenje et al., 2009). Indeed, sympathetic activation before slaughter increases muscle glycogenolysis and therefore reduces lactic acid production post-mortem and meat acidification (Foury et al., 2005). Nevertheless, the effect of rearing system on blood catecholamine levels should be further investigated. As previously demonstrated by Odore et al. (2004), bovine lymphocytes express measurable concentrations of b-adrenergic receptors (AR) and GR. Present data confirm the high affinity of [3H]CGP12177 and [3H]dexamethazone for b-AR and GR, respectively. The absence of statistically significant differences among the Kd values at any experimental time suggests that the housing system and short-term transportation do not modify the binding characteristics of lymphocyte b-AR and GR. Lymphocyte GR and b-AR concentrations were significantly decreased at T4 in Group A, whereas in Group B, the reduction was not statistically significant. This finding could be ascribed to the higher blood hormonal concentrations observed in Group B before transportation (T3). Lymphocyte receptors might be occupied by the high levels of circulating hormone that competes with the radioligand for specific receptor binding sites. At T3 both GR and b-AR were significantly lower in Group B than in Group A beef cattle. It has been shown that repeated stress reduces the density of cortical b-AR in rat cerebral cortex suggesting that the change in b-adrenoceptor concentration represents an important element of adaptation to stress (Stanford, 1996). Our results show that the rearing system and the short-term pre-slaughter transport can induce noticeable changes in stress response in cattle. As in goats and calves, pre-slaughter stress and the type of housing have been associated with significant changes in muscle metabolism and meat quality (Andrighetto et al., 1999; Kannan et al., 2003), consequently it is of interest to investigate fluctuations in hormonal concentrations from an animal welfare perspective. Under the conditions of the present study, blood progesterone concentrations increased after short-term transportation, whereas, unlike cortisol, progesterone concentrations were unaffected by the rearing system. As a consequence, the use of this hormone as a marker of stress in beef cattle should be evaluated carefully.
Acknowledgement The authors wish to thank Dr. L. Bertolotti for his support in the statistical analysis of data.
References Andrighetto, I., Gottardo, F., Andreoli, D., Cozzi, G., 1999. Effect of type of housing on veal calf growth performance, behaviour and meat quality. Livestock Production Science 57, 137–145.
R. Odore et al. / Research in Veterinary Science 90 (2011) 341–345 Broom, D.M., 2007. Causes of poor welfare and welfare assessment during handling and transportation. In: Grandin, T (Ed.), Livestock Handling and Transport, third ed. CABI, Oxfordshire, UK, pp. 30–43. Buckham Sporer, K.R., Burton, J.L., Earley, B., Crowe, M.A., 2007. Transportation stress in young bulls alters expression of neutrophil genes important for the regulation of apoptosis, tissue remodeling, margination and anti-bacterial function. Veterinary Immunology and Immunopathology 118, 19–29. Buckham Sporer, K.R., Weber, P.S., Burton, J.L., Earley, B., Crowe, M.A., 2008. Transportation of young beef bulls alters circulating physiological parameters that may be effective biomarkers of stress. Journal of Animal Science 86, 1325– 1334. Cooper, C., Evans, A.C.O., Cook, S., Rawlings, N.C., 1995. Cortisol, progesterone and bendorphin response to stress in calves. Canadian Journal of Animal Science 95, 197–201. Degabriele, R., Fell, L.R., 2001. Changes in behaviour, cortisol and lymphocyte types during isolation and group confinement of sheep. Immunology and Cell Biology 79, 583–589. Ehrhart-Bornstein, M., Bornstein, S.R., 2008. Cross-talk between adrenal medulla and adrenal cortex in stress. Annuals of the New York Academy of Sciences 1148, 112–117. Eicher, S.D., 2001. Transportation of cattle in the dairy industry: current research and future directions. Journal of Dairy Science 84 (Suppl E), E19–E24. Fike, K., Spire, M.F., 2006. Transportation of cattle. Veterinary Clinics of North America Food Animal Practice 22, 305–320. Foury, A., Devillers, N., Sanchez, M.-P., Griffon, H., Le Roy, P., Mormede, P., 2005. Stress hormones, carcass composition and meat quality in Large White Duroc pigs. Meat Science 69, 703–707. Gade, P.B., 2008. Effect of rearing system and mixing at loading on transport and lairage behaviour and meat quality: comparison of free range and conventionally raised pigs. Animal 2 (8), 1238–1246. Grandin, T., 2007. Introduction: effect of customer requirements, international standards and marketing structure on the handling and transport of livestock and poultry. In: Grandin, T (Ed.), third ed. Livestock Handling and Transport, CABI, Oxfordshire, UK, pp. 30–43. Gupta, S., Earley, B., Crowe, M.A., 2007a. Pituitary, adrenal, immune and performance responses of mature Holstein Friesian bulls housed on slatted floors at various space allowances. The Veterinary Journal 173, 594–604. Gupta, S., Earley, B., Crowe, M.A., 2007b. Effect of 12 h transportation on physiological, immunological and haematological parameters in bulls housed at different space allowances. The Veterinary Journal 173, 605–616. Herskin, M.S., Munksgaard, L., Ladewig, J., 2004. Effects of acute stressors on nociception, adrenocortical responses and behavior of dairy cows. Physiology and Behavior 83, 411–420. Honkavaara, M., Rintasalo, E., Ylonen, J., Pudas, T., 2003. Meat quality and transport stress in cattle. Deutsche Tierarztliche Wochenschrifte 110, 125–128. Jeong, K., Jacobson, L., Pacak, K., Widmaier, E.P., Goldstein, D.S., Majzoub, J.A., 2000. Impaired basal and restraint-induced epinephrine secretion in corticotropinreleasing-hormone deficient mice. Endocrinology 141, 1142–1150. Kannan, G., Kouakou, B., Terrill, T.H., Gelaye, S., 2003. Endocrine, blood metabolite, and meat quality changes in goats as influenced by short term, pre-slaughter stress. Journal of Animal Science 81, 1499–1507.
345
Knowles, T.G., 1999. A review of the road transport of cattle. The Veterinary Record 144, 197–201. Knowles, T.G., Warriss P., 2007. Stress physiology of animals during transport. In: Grandin, T (Ed.), third ed. Livestock Handling and Transport. CABI, Oxfordshire, UK, pp. 312–328. Mounier, L., Dubroeucq, H., Andanson, S., Veissier, I., 2006. Variations in meat pH of beef bulls in relation to conditions of transfer to slaughter and previous history of the animals. Journal of Animal Science 84, 1567–1576. Muchenje, V., Dzama, K., Chimonyo, M., Strydom, P.E., Raats, J.G., 2009. Relationship between pre-slaughter stress responsiveness and beef quality in three cattle breeds. Meat Science 81, 653–657. Mumma, J.O., Thaxton, J.P., Vizzer-Thaxton, Y., Dodson, W.L., 2006. Physiological stress in laying hens. Poultry Science 85, 761–769. Murata, H., Takahashi, H., Matsumoto, H., 1987. The effect of road transportation on peripheral blood lymphocyte subpopulations, lymphocyte blastogenesis, and neutrophil function in calves. British Veterinary Journal 143, 166– 174. Odore, R., D’Angelo, A., Badino, P., Bellino, C., Pagliasso, S., Re, G., 2004. Road transportation affects blood hormone levels and lymphocyte glucocorticoid and b-adrenergic receptor concentrations in calves. The Veterinary Journal 168, 297–303. Odore, R., Badino, P., Pagliasso, S., Nebbia, C., Cuniberti, B., Barbero, R., Re, G., 2006. Changes in lymphocyte glucocorticoid and b-adrenergic receptors in veal calves treated with clenbuterol and steroid hormones for growth promoting purposes. Journal of Veterinary Pharmacology and Therapeutics 29, 91– 97. Stanford, S.C., 1996. Stress: a major variable in the psychopharmacologic response. Pharmacology Biochemistry and Behavior 54, 211–217. Ulrich-Lai, Y.M., Engeland, W.C., 2005. Sympatho-adrenal activity and hypothalamic-pituitary-adrenal axis regulation. In: T. Steckler, T., Kalin, N.H., Reul, J.M.H.M. (Eds.), Handbook On stress, Immunology and Behaviour, Elsevier Science, Amsterdam, pp. 419–435. Van de Water, G., Verjans, F., Geers, R., 2003. The effect of short distance transport under commercial conditions on the physiology of veal calves; pH and colour profiles of veal. Livestock Production Science 82, 171–179. Villarroel, M., Maria, G., Sanudo, C., Garcia-Belenguer, S., Chacon, G., Gebre-Senbet, G., 2003. Effect of commercial transport in Spain on cattle welfare and meat quality. Deutsche Tierarztliche Wochenschrifte 110, 105–107. Veissier, I., Boissy, A., Passillé, A.M., de Rushen, J., Reenen, C.G., van Roussel, S., Andanson, S., Pradel, P., 2001. Calves’ responses to repeated social regrouping and relocation. Journal of Animal Science 79, 2580–2593. Visser, E.K., Ellis, A.D., Reenen, C.G., 2008. The effect of two different housing conditions on the welfare of young horses stabled for the first time. Applied Animal Behaviour Science 114, 521–533. Xiccato, G., Trocino, A., Queaque, P.I., Sartori, A., Carazzolo, A., 2002. Rearing veal calves with respect to animal welfare: effects of group housing and solid feed supplementation on growth performance and meat quality. Livestock Production Science 75, 269–280.
Contents lists available at ScienceDirect
Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Effects of housing and short-term transportation on hormone and lymphocyte receptor concentrations in beef cattle Rosangela Odore a,*, Paola Badino a, Giovanni Re a, Raffaella Barbero a, Barbara Cuniberti a, Antonio D’Angelo b, Carlo Girardi a, Elena Fraccaro a, Martina Tarantola c a b c
Department of Animal Pathology, Division of Pharmacology and Toxicology, University of Turin, Italy Department of Animal Pathology, Division of Medicine, University of Turin, Italy Department of Animal Production, Epidemiology and Ecology, University of Turin, Italy
a r t i c l e
i n f o
Article history: Received 12 January 2010 Accepted 22 May 2010
Keywords: Beef cattle Hormones Receptors Lymphocytes Transportation Stress
a b s t r a c t The experiment was designed to evaluate the effects of housing system and short-term transportation on the pituitary and adrenal response and on blood progesterone concentrations of beef cattle. Since the use of steroid hormones in farm animals has been banned in the EU (Council Directive 96/22/EC), it seems important to study the possible modifications in serum progesterone concentrations induced by stress in cattle. Thirty-two, 6 months old male Piedmontese beef cattle (16 reared in a littered loose house, Group A, and 16 housed in a littered tying stall barn, Group B) were blood sampled at T1 (6 months old), T2 (12 months old), T3 (18 months old, before transportation to the slaughterhouse) and T4 (after transportation to the slaughterhouse) in order to measure hormonal concentrations and lymphocyte glucocorticoid (GR) and b-adrenergic (b-AR) receptor concentrations. Circulating hormone concentrations were measured using commercial radioimmunoassay kits, whereas lymphocyte receptor density was determined through binding assays. In beef cattle housed in tie stall barn a significant increase in serum cortisol concentration was observed at T3, whereas there was no effect of the housing system on blood progesterone concentrations. Short-term transportation caused a significant increase in blood cortisol and catecholamine concentrations in both groups, whereas lymphocyte GR and b-AR significantly decreased in Group A. Our data confirm the activation of the hypothalamic–pituitary–adrenal axis and the catecholaminergic system in short-term transportation and suggest that the stress-induced increase in circulating progesterone concentrations does not exceed the limit established by pending legislation. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction In recent years, animal welfare and optimization of producer and consumer expectations have lead to an increasing attention to the study of the effects of husbandry conditions and transportation on behavioural, biochemical, functional, endocrine and pathological variables in cattle (Fike and Spire, 2006; Broom, 2007; Gupta et al., 2007b). Despite the fact that animal welfare auditing programmes are now being conducted in many countries around the world in order to comply with OIE (World Organisation for Animal Health) animal welfare guidelines and to limit stress-induced meat quality problems, it is sometimes difficult to characterise reliable biomarkers of stress (Grandin, 2007). Among the indicators generally used to assess the welfare of animals being handled or transported, are the measurement of physiological variables such as hormones which may be quantified over repeated sampling * Corresponding author. Tel.: +39 011 6709018; fax: +39 011 6709017. E-mail address: [email protected] (R. Odore). 0034-5288/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2010.05.026
time points. In fact, stress, a physiological response to several exogenous or endogenous stimuli that normally cause neuroendocrine activation and response of the endocrine system, is mainly characterized by the concomitant release of catecholamines and glucocorticoids from the adrenal glands (Ehrhart-Bornstein and Bornstein, 2008). The hypothalamic corticotropin-releasing hormone (CRH) system and the sympathetic nervous system are postulated to be functionally related to each other and to regulate reciprocally their functions within the central nervous system (Ulrich-Lai and Engeland, 2005). In animal husbandry, stress occurs when animal experiences changes in the environment that stimulate body responses to restore homeostasis (Mumma et al., 2006). It is established that many stressors, such as immobilization, housing, handling, loading, unloading and transport increase the secretion of catecholamines from the adrenal medulla and sympathetic nerve terminals (Broom, 2007) and that such stressors also potentiate the secretion of the hypothalamic–pituitary–adrenocortical axis hormones (Jeong et al., 2000). Furthermore, the modifications of
342
R. Odore et al. / Research in Veterinary Science 90 (2011) 341–345
blood hormone concentrations (e.g. cortisol and catecholamines) due to stressful conditions are correlated with changes in lymphocyte receptor density (Odore et al., 2004). The correlation of circulating hormone concentrations with lymphocyte receptor density is considered as a more suitable indicator of stress than blood hormone levels alone. As stated by Knowles and Warriss (2007), the study of such indices related to immune function is useful to better characterise the response to stress in cattle and other animal species. In fact, these changes may have a significant effect on cattle welfare, reproductive performance, weight gain and meat quality (Villarroel et al., 2003). As far as cattle husbandry is concerned, the housing system and transportation represent two main stressful factors. Traditional rearing techniques often ensure the final product quality requested by the consumer, however in so doing, animal behaviour and welfare may be compromised (Xiccato et al., 2002). For example, it has been observed that insufficient space allowances induce a state of stress that alters the activity of the pituitary-adrenal axis, immune function, behaviour and growth rate (Gupta et al., 2007a). Moreover, Herskin et al. (2004) found significant changes in the acute stress response in cows kept in tie-stalls. A substantial amount of research has been devoted to the study of stress transportation in livestock (Murata et al., 1987; Odore et al., 2004; Gupta et al., 2007b; Buckham Sporer et al., 2007). Historically, significant increases in blood glucocorticoid hormones have been reported during the stress response following transportation. However, little is known about the effects of transportation on other steroid hormones, such as sex steroids. Buckham Sporer et al. (2008) found a significant increase in blood progesterone concentrations after truck transportation in beef bulls. Since the use of steroid hormones in farm animals has been banned in the EU (Council Directive 96/22/EC), it seems important to study the possible modifications in serum progesterone concentrations induced by stress in cattle. The finding of steroid hormones in the systemic circulation is not proof of illegal use since these hormones occur naturally in physiological concentrations in animals and humans. Limits in blood progesterone concentrations in cattle have been set in order to prevent consumers from ingesting meat from illegally treated animals, however; scientific evidence to support this decision is needed. The aim of the study was to measure blood cortisol, progesterone and catecholamine concentrations and lymphocyte glucocorticoid (GR) and b-adrenergic receptor (b-AR) concentrations in male beef cattle housed in a littered tying stall barn and in a littered loose house at different time points during the breeding period, before and after road transportation to the slaughterhouse.
iod, animals were transported from the fattening farm to the slaughterhouse using a double deck. The animals were loaded on the lower deck in the front compartment with spatial allowances per animal of 1.4 m2. The transporter was naturally ventilated. The journey duration was approximately 45 min. The truck travelled mainly on secondary roads at a speed of about 60 km/h. The mean final live weight was 640 ± (SEM) 18 kg and 630 ± (SEM) 20 kg for Groups A and B, respectively. At T3 and T4 additional blood samples were collected in order to separate the lymphocyte fraction and to measure circulating catecholamine concentrations.
2. Materials and methods
2.4. b-Adrenoceptor binding assay
2.1. Animals and sample collection
b-Adrenoceptors were measured through binding assay as described by Odore et al. (2006), but introducing some minor modifications. Briefly 100 lL of lymphocyte suspension were incubated for 1 h at 37 °C with increasing concentrations of the labelled antagonist ( )[3H]CGP 12177 (0.06–4 nM) in a total volume of 200 lL. The non-specific binding was assessed in the presence of 100 lM unlabelled isoproterenol. Incubation was stopped by adding 2 mL of ice-cold buffered saline (154 mM NaCl and 50 mM Tris–HCl, pH 7.4) and the incubation mixture was immediately filtered under vacuum using pre-soaked glass fibre filters (Whatman GF/C). Filters were then accurately washed with 3 3 mL of buffered saline and solubilised with 4 mL of scintillation fluid (Filter Count, Canberra Packard). The radioactivity retained on wet filters was measured by a Tri-Carb 1600 TR Canberra Packard scintillator spectrometer, at an efficiency of 60%. The quantitative parameters, number of sites/cell and Kd (nM), were determined with the aid of a computer program (GraphPad, Prism).
A total of 32 6 months old male Piedmontese beef calves were monitored at different experimental time points during the breeding period at the fattening farm. Sixteen of them were reared in a littered loose house, Group A: mean live weight 180 ± (SEM) 15 kg, and 16 were housed in a tie stall, Group B: mean live weight 183 ± (SEM) 20 kg. Animals were blood sampled at T1 (6 months old), T2 (12 months old), T3 (18 months old, before transportation to the slaughterhouse) and T4 (after transportation to the slaughterhouse) in order to measure cortisol and progesterone concentrations. Animals of Group A were kept in a free stall housing system. They were housed in groups of nine animals per pen, each animal had a pen area allowance of 4.0 m2, with deep straw bedding. Group B cattle were tied by a tether fixed to the feed trough (space allowance 210 110 cm); animals were accommodated on concrete solid floor bedded with straw. At the end of the breeding per-
2.2. Lymphocyte separation Lymphocytes were isolated by a density gradient centrifugation method (Odore et al., 2004). Blood samples (130 mL) were collected in 10 mL tubes containing citric acid and dextrose (Becton Dickinson, Milan, Italy) via direct jugular venipuncture using mild restraint in a holding chute. Blood samples were immediately transported to the laboratory and centrifuged at 1300g for 15 min at room temperature. For each tube, the buffy-coat was collected, transferred to 5 mL tubes and diluted to 3 mL with Hanks’ balanced salt solution containing 1% sodium citrate. Diluted buffy-coats were carefully layered on 1.5 mL Histopaque-1077 and centrifuged at 1800g for 10 min at room temperature. After careful removal of plasma–platelet layers, the mononuclear cell layers were carefully collected, washed twice with Hanks’ solution and centrifuged at 3300g for 10 min at 4 °C. Pellets were then re-suspended in appropriate buffer (50 mM Tris-base, 1 mM EDTA, 12 mM thioglycerol, 250 mM glycerol and 10 mM sodium molybdate, pH 7.4). After counting the number of lymphocytes, cells were diluted in a specific buffer to a final concentration of 1 107/mL in order to measure b-AR and GR concentration. 2.3. Plasma catecholamine measurement Samples used for the determination of plasma catecholamine concentrations were collected in 5 mL tubes containing ethylene diaminotetraacetic acid (EDTA). Plasma was immediately separated by refrigerated centrifugation (2500g for 10 min at 4 °C) and then stored at 80 °C until analysis. Noradrenaline and adrenaline concentrations were measured using a commercial radioimmunoassay kit (KatCombi, IBL, Hamburg). The intra-assay and inter-assay coefficients of variation were 4.8 ± 0.9% and 5.7 ± 1.0%, respectively, whereas recovery was 94 ± 2% for noradrenaline and 89 ± 4% for adrenaline.
343
R. Odore et al. / Research in Veterinary Science 90 (2011) 341–345
2.5. Serum cortisol and progesterone measurement Blood samples for cortisol and progesterone concentration measurement were allowed to clot at room temperature, centrifuged (2500g for 10 min at 4 °C) and aliquots of obtained sera stored at 80 °C until assay. Commercial bovine cortisol (DSL 2000, Diagnostic Systems Laboratories, Inc.) and progesterone (DSL 3400 Diagnostic Systems Laboratories, Inc.) radioimmunoassay kits were used to measure serum hormone concentrations. The intra- and inter-assay coefficients of variation were 6.1 ± 1.4% and 8 ± 0.7% for cortisol and 4.4 ± 0.7% and 2.5 ± 0.5% for progesterone, respectively. The recovery was 95 ± 2.7% for cortisol and 92.3 ± 5% for progesterone. 2.6. Glucocorticoid receptor (GR) binding assay In order to measure lymphocyte GR density, aliquots of lymphocytes (200 lL) were incubated overnight at 4 °C with increasing concentrations (0.3–10 nM) of [3H]dexamethazone in a total volume of incubation of 250 lL. The non-specific binding was assessed in presence of 5 lM unlabelled dexamethazone. In order to separate free radioligand from the mixture, 250 lL of a dextran–charcoal solution (0.5% charcoal and 0.05% dextran) was added to each tube. After 15 min incubation, tubes were centrifuged for 15 min at 3200g at 4 °C, 200 lL of the supernatant were added to 4 mL of scintillation liquid (Pico Fluor, Canberra Packard), and the radioactivity was measured for 2 min by use of a b-counter (Tri-Carb 1600 TR Canberra Packard). The Kd and the number of sites/cell were calculated by use of GraphPad Prism.
were evaluated with ANOVA and Tuckey multiple comparison test with the significant limit set at P < 0.05. Inter-group differences for cortisol, progesterone and catecholamines at each experimental time point were analysed using Student’s t test, with the aid of a computer program (GraphPad Instat). 3. Results 3.1. Blood hormone concentrations In cattle reared in loose house system, no statistically significant differences were found among T1, T2 and T3 treatments (Table 1). In cattle reared in tie stall barn, blood cortisol concentrations increased significantly (P < 0.05) at T2 and T3 with respect to T1 (Table 1). Transport of animals to the slaughterhouse caused a significant increase in blood cortisol concentrations in Group A (P < 0.001) and in Group B (P < 0.001). Serum cortisol concentrations were higher (P < 0.05) in Group B than in Group A at T3 and T4 (Table 1). In Group A, serum progesterone concentrations were unchanged throughout the study (Table 1), whereas in Group B, an increase (P < 0.001) was detected at T4 (Table 1). At T4 serum progesterone concentration was significantly (P < 0.05) higher in Group B with respect to Group A. In both groups the transportation to the slaughterhouse caused a statistically significant (P < 0.05) increase in adrenaline and noradrenaline concentrations (Tables 2). At T3 blood catecholamine concentrations were significantly higher in Group B than in Group A (Table 2).
2.7. Drugs and radiochemicals ( )[3H]CGP 12177 (54 Ci/mmol) was obtained from Perkin Elmer and [3H]dexamethasone (89 Ci/mmol) was purchased from Amersham Pharmacia Biotech Europe. All other reagents were obtained from Sigma.
Table 2 Blood adrenaline and noradrenaline (ng/mL) concentrations in loose house (Group A) and tie stall (Group B) reared beef cattle at different experimental time points (T3 = 18 months old before transportation; T4 = 18 months old after transportation to the slaughterhouse); (mean values ± SEM; n = 16; Intra and inter-group differences Student’s t test).
2.8. Statistical analysis
Blood adrenaline concentrations Group A
Results are expressed as mean values ± SEM of triplicate measurements. Intra-group differences for cortisol and progesterone at different time points and lymphocyte receptor concentrations
T3 T4 Intra-group differences
0.10 ± 0.01ax 0.25 ± 0.03bx a vs. b P < 0.0001
Blood noradrenaline concentrations Group A Table 1 Blood cortisol and progesterone concentrations (ng/mL) in loose house (Group A) and tie stall (Group B) reared beef cattle at different experimental time points (T1 = 6 months old; T2 = 12 months old; T3 = 18 months old before transportation; T4 = 18 months old after transportation to the slaughterhouse); (mean values ± SEM; n = 16; Intra-group differences: ANOVA and Tukey Multiple Comparison Test; Intergroup differences Student’s t test). Blood cortisol concentrations Group A T1 T2 T3 T4 Intra-group differences
Group B
ax
ax
4.1 ± 0.4 4.5 ± 0.9ax 4.0 ± 0.7ax 9.2 ± 1.0bx a vs. b P < 0.001
3.0 ± 0.4 6.7 ± 0.9bx 6.3 ± 0.7by 20.6 ± 1.1cy a vs. b vs. c P < 0.05
Blood progesterone concentrations Group A T1 T2 T3 T4 Intra-group differences
Group B ax
0.12 ± 0.01 0.14 ± 0.02ax 0.12 ± 0.06ax 0.15 ± 0.04ax n.s.
ax
0.12 ± 0.05 0.12 ± 0.02ax 0.10 ± 0.01ax 0.39 ± 0.08by a vs. b P < 0.01
Inter-group differences n.s. n.s. x vs. y P < 0.05 x vs. y P < 0.0001
Inter-group differences n.s. n.s. n.s. x vs. y P < 0.05
T3 T4 Intra-group differences
0.26 ± 0.04ax 0.50 ± 0.05bx a vs. b P < 0.001
Group B 0.13 ± 0.01ay 0.35 ± 0.10bx a vs. b P < 0.05 Group B 0.42 ± 0.06ay 0.69 ± 0.07by a vs. b P < 0.01
Inter-group differences x vs. y P < 0.05 n.s.
Inter-group differences x vs. y P < 0.05 x vs. y P < 0.05
Table 3 Concentrations (sites/cell) of lymphocyte glucocorticoid and b-adrenergic receptors at T3 and T4 in loose house (Group A) and tie stall (Group B) beef cattle (mean values ± SEM; n = 16; ANOVA and Tukey Multiple Comparison Test). Lymphocyte glucocorticoid receptors Group A Group B T3 18,194 ± 4184 6775 ± 545 T4 6991 ± 1476 4065 ± 376 Statistical significance: T3 Group A vs. T3 Group B (P < 0.01); T3 Group A vs. T4 Group A (P < 0.01) Lymphocyte b-adrenoceptors Group A Group B T3 30,544 ± 3362 13,426 ± 935 T4 11,831 ± 1721 8432 ± 784 Statistical significance: T3 Group A vs. T4 Group A (P < 0.001); T3 Group A vs. T3 Group B (P < 0.001)
344
R. Odore et al. / Research in Veterinary Science 90 (2011) 341–345
3.2. Lymphocyte receptor concentrations Lymphocyte GR decreased in Group A (P < 0.01) and was not different in Group B after transport (Table 3). No significant differences were found among the Kd values in both groups (data not shown). Lymphocyte b-AR decreased after transport both in Group A (P < 0.001) and in Group B (Table 3). Statistically significant differences (P < 0.001) were observed between Group A and group B before transportation. The Kd values (nM) for b-AR demonstrated a high degree of the highly specific labelled ligand-receptor affinity. No differences (P > 0.05) were observed in both groups, either before or after transportation (data not shown). 4. Discussion In the present study, a significant increase in serum cortisol concentrations was detected in beef cattle reared in tie stall barns, whereas in the loose housing system no significant variation was observed during the breeding period. The effect of the rearing on circulating cortisol concentrations has been investigated in cattle (Veissier et al., 2001; Gupta et al., 2007a), sheep (Degabriele and Fell, 2001), pig (Gade, 2008) and horses (Visser et al., 2008). Gupta et al. (2007a) found that restricted space allowances increase acute plasma cortisol concentrations in bulls and Andrighetto et al. (1999) observed a favourable effect of group rearing on growth performance in the late period of growth. In our study, in tie stall barn beef cattle, blood cortisol concentrations remained unchanged between T2 and T3, suggesting the exposure to a continuously elevated stress due to the housing system. Chronic stress of housing is reported to modify the functioning of the pituitary gland and lead to either adaptation or regulatory changes in the pituitary and/or adrenal gland (Gupta et al., 2007a). By contrast, short-term transportation induced a significant increase in cortisol concentrations in both groups. Literature has extensively reviewed the journey aspects with potential effects on cattle, with duration of transport representing one of the main stressful factors (Knowles, 1999). In the case of long distance transport, animals usually suffer from long fasting periods and exhaustion, thus significant modifications of endocrine and biochemical variables may occur (Odore et al., 2004). However, Honkovaara et al. (2003) observed that animals transported for long distances express a lower stress profile than animals travelling over a short distance. In animals transported for long distances exhaustion of the adrenal gland may occur, whereas cattle transported for short distance journeys may express acute psychological stress to the novelty of the transport process. Van de Water et al. (2003) found that short-term transportation can elicit a wide range of physiological changes such an increase in cortisol, lactate and creatine kinase concentrations and identified loading and unloading as the main causes of stress. Also according to Eicher (2001), the greatest cortisol response is associated with shorter transports. The significant differences in blood cortisol concentrations observed at T3 and T4 between the two groups suggest that animals reared in tie stall barns were more stressed than the loose housed cattle. Mounier et al. (2006) found that bull calves kept in their social group during the pre-slaughter period had less increase in cortisol concentrations and that the effect was even more pronounced when animals had been reared together. Circulating progesterone concentrations increased significantly in Group B after transportation. Buckham Sporer et al. (2008) had already demonstrated that in young beef bulls transported by road for up to 9 h, serum progesterone concentrations significantly increased in association with the increase in cortisol. However, to our knowledge no data concerning the effects of the housing system and short-term transportation have been published to date. The stress-
induced increase in blood progesterone concentrations probably occurs as a result of the rate limiting enzymes responsible for the conversion of progesterone to cortisol (Cooper et al., 1995). This would suggest that stressful conditions such as the keeping animals in tie stall barns cause greater secretion of progesterone. By contrast, in our study, no significant differences in blood progesterone concentrations were found during the breeding period in both groups of animals. The increase observed after transportation in Group B cattle did not exceed the limit set by national legislation (1.5 ng/mL). This finding is consistent with mild blood progesterone concentration fluctuation in response to stress. In agreement with previous findings (Odore et al., 2004), a significant increase in adrenaline and noradrenaline concentrations was found post transport. As catecholamines have been shown to cause depletion in muscle glycogen in the pre-slaughter period, it is expected that sympathetic activation may influence meat quality (Muchenje et al., 2009). Indeed, sympathetic activation before slaughter increases muscle glycogenolysis and therefore reduces lactic acid production post-mortem and meat acidification (Foury et al., 2005). Nevertheless, the effect of rearing system on blood catecholamine levels should be further investigated. As previously demonstrated by Odore et al. (2004), bovine lymphocytes express measurable concentrations of b-adrenergic receptors (AR) and GR. Present data confirm the high affinity of [3H]CGP12177 and [3H]dexamethazone for b-AR and GR, respectively. The absence of statistically significant differences among the Kd values at any experimental time suggests that the housing system and short-term transportation do not modify the binding characteristics of lymphocyte b-AR and GR. Lymphocyte GR and b-AR concentrations were significantly decreased at T4 in Group A, whereas in Group B, the reduction was not statistically significant. This finding could be ascribed to the higher blood hormonal concentrations observed in Group B before transportation (T3). Lymphocyte receptors might be occupied by the high levels of circulating hormone that competes with the radioligand for specific receptor binding sites. At T3 both GR and b-AR were significantly lower in Group B than in Group A beef cattle. It has been shown that repeated stress reduces the density of cortical b-AR in rat cerebral cortex suggesting that the change in b-adrenoceptor concentration represents an important element of adaptation to stress (Stanford, 1996). Our results show that the rearing system and the short-term pre-slaughter transport can induce noticeable changes in stress response in cattle. As in goats and calves, pre-slaughter stress and the type of housing have been associated with significant changes in muscle metabolism and meat quality (Andrighetto et al., 1999; Kannan et al., 2003), consequently it is of interest to investigate fluctuations in hormonal concentrations from an animal welfare perspective. Under the conditions of the present study, blood progesterone concentrations increased after short-term transportation, whereas, unlike cortisol, progesterone concentrations were unaffected by the rearing system. As a consequence, the use of this hormone as a marker of stress in beef cattle should be evaluated carefully.
Acknowledgement The authors wish to thank Dr. L. Bertolotti for his support in the statistical analysis of data.
References Andrighetto, I., Gottardo, F., Andreoli, D., Cozzi, G., 1999. Effect of type of housing on veal calf growth performance, behaviour and meat quality. Livestock Production Science 57, 137–145.
R. Odore et al. / Research in Veterinary Science 90 (2011) 341–345 Broom, D.M., 2007. Causes of poor welfare and welfare assessment during handling and transportation. In: Grandin, T (Ed.), Livestock Handling and Transport, third ed. CABI, Oxfordshire, UK, pp. 30–43. Buckham Sporer, K.R., Burton, J.L., Earley, B., Crowe, M.A., 2007. Transportation stress in young bulls alters expression of neutrophil genes important for the regulation of apoptosis, tissue remodeling, margination and anti-bacterial function. Veterinary Immunology and Immunopathology 118, 19–29. Buckham Sporer, K.R., Weber, P.S., Burton, J.L., Earley, B., Crowe, M.A., 2008. Transportation of young beef bulls alters circulating physiological parameters that may be effective biomarkers of stress. Journal of Animal Science 86, 1325– 1334. Cooper, C., Evans, A.C.O., Cook, S., Rawlings, N.C., 1995. Cortisol, progesterone and bendorphin response to stress in calves. Canadian Journal of Animal Science 95, 197–201. Degabriele, R., Fell, L.R., 2001. Changes in behaviour, cortisol and lymphocyte types during isolation and group confinement of sheep. Immunology and Cell Biology 79, 583–589. Ehrhart-Bornstein, M., Bornstein, S.R., 2008. Cross-talk between adrenal medulla and adrenal cortex in stress. Annuals of the New York Academy of Sciences 1148, 112–117. Eicher, S.D., 2001. Transportation of cattle in the dairy industry: current research and future directions. Journal of Dairy Science 84 (Suppl E), E19–E24. Fike, K., Spire, M.F., 2006. Transportation of cattle. Veterinary Clinics of North America Food Animal Practice 22, 305–320. Foury, A., Devillers, N., Sanchez, M.-P., Griffon, H., Le Roy, P., Mormede, P., 2005. Stress hormones, carcass composition and meat quality in Large White Duroc pigs. Meat Science 69, 703–707. Gade, P.B., 2008. Effect of rearing system and mixing at loading on transport and lairage behaviour and meat quality: comparison of free range and conventionally raised pigs. Animal 2 (8), 1238–1246. Grandin, T., 2007. Introduction: effect of customer requirements, international standards and marketing structure on the handling and transport of livestock and poultry. In: Grandin, T (Ed.), third ed. Livestock Handling and Transport, CABI, Oxfordshire, UK, pp. 30–43. Gupta, S., Earley, B., Crowe, M.A., 2007a. Pituitary, adrenal, immune and performance responses of mature Holstein Friesian bulls housed on slatted floors at various space allowances. The Veterinary Journal 173, 594–604. Gupta, S., Earley, B., Crowe, M.A., 2007b. Effect of 12 h transportation on physiological, immunological and haematological parameters in bulls housed at different space allowances. The Veterinary Journal 173, 605–616. Herskin, M.S., Munksgaard, L., Ladewig, J., 2004. Effects of acute stressors on nociception, adrenocortical responses and behavior of dairy cows. Physiology and Behavior 83, 411–420. Honkavaara, M., Rintasalo, E., Ylonen, J., Pudas, T., 2003. Meat quality and transport stress in cattle. Deutsche Tierarztliche Wochenschrifte 110, 125–128. Jeong, K., Jacobson, L., Pacak, K., Widmaier, E.P., Goldstein, D.S., Majzoub, J.A., 2000. Impaired basal and restraint-induced epinephrine secretion in corticotropinreleasing-hormone deficient mice. Endocrinology 141, 1142–1150. Kannan, G., Kouakou, B., Terrill, T.H., Gelaye, S., 2003. Endocrine, blood metabolite, and meat quality changes in goats as influenced by short term, pre-slaughter stress. Journal of Animal Science 81, 1499–1507.
345
Knowles, T.G., 1999. A review of the road transport of cattle. The Veterinary Record 144, 197–201. Knowles, T.G., Warriss P., 2007. Stress physiology of animals during transport. In: Grandin, T (Ed.), third ed. Livestock Handling and Transport. CABI, Oxfordshire, UK, pp. 312–328. Mounier, L., Dubroeucq, H., Andanson, S., Veissier, I., 2006. Variations in meat pH of beef bulls in relation to conditions of transfer to slaughter and previous history of the animals. Journal of Animal Science 84, 1567–1576. Muchenje, V., Dzama, K., Chimonyo, M., Strydom, P.E., Raats, J.G., 2009. Relationship between pre-slaughter stress responsiveness and beef quality in three cattle breeds. Meat Science 81, 653–657. Mumma, J.O., Thaxton, J.P., Vizzer-Thaxton, Y., Dodson, W.L., 2006. Physiological stress in laying hens. Poultry Science 85, 761–769. Murata, H., Takahashi, H., Matsumoto, H., 1987. The effect of road transportation on peripheral blood lymphocyte subpopulations, lymphocyte blastogenesis, and neutrophil function in calves. British Veterinary Journal 143, 166– 174. Odore, R., D’Angelo, A., Badino, P., Bellino, C., Pagliasso, S., Re, G., 2004. Road transportation affects blood hormone levels and lymphocyte glucocorticoid and b-adrenergic receptor concentrations in calves. The Veterinary Journal 168, 297–303. Odore, R., Badino, P., Pagliasso, S., Nebbia, C., Cuniberti, B., Barbero, R., Re, G., 2006. Changes in lymphocyte glucocorticoid and b-adrenergic receptors in veal calves treated with clenbuterol and steroid hormones for growth promoting purposes. Journal of Veterinary Pharmacology and Therapeutics 29, 91– 97. Stanford, S.C., 1996. Stress: a major variable in the psychopharmacologic response. Pharmacology Biochemistry and Behavior 54, 211–217. Ulrich-Lai, Y.M., Engeland, W.C., 2005. Sympatho-adrenal activity and hypothalamic-pituitary-adrenal axis regulation. In: T. Steckler, T., Kalin, N.H., Reul, J.M.H.M. (Eds.), Handbook On stress, Immunology and Behaviour, Elsevier Science, Amsterdam, pp. 419–435. Van de Water, G., Verjans, F., Geers, R., 2003. The effect of short distance transport under commercial conditions on the physiology of veal calves; pH and colour profiles of veal. Livestock Production Science 82, 171–179. Villarroel, M., Maria, G., Sanudo, C., Garcia-Belenguer, S., Chacon, G., Gebre-Senbet, G., 2003. Effect of commercial transport in Spain on cattle welfare and meat quality. Deutsche Tierarztliche Wochenschrifte 110, 105–107. Veissier, I., Boissy, A., Passillé, A.M., de Rushen, J., Reenen, C.G., van Roussel, S., Andanson, S., Pradel, P., 2001. Calves’ responses to repeated social regrouping and relocation. Journal of Animal Science 79, 2580–2593. Visser, E.K., Ellis, A.D., Reenen, C.G., 2008. The effect of two different housing conditions on the welfare of young horses stabled for the first time. Applied Animal Behaviour Science 114, 521–533. Xiccato, G., Trocino, A., Queaque, P.I., Sartori, A., Carazzolo, A., 2002. Rearing veal calves with respect to animal welfare: effects of group housing and solid feed supplementation on growth performance and meat quality. Livestock Production Science 75, 269–280.