J. Anim. Sci. 2005. 83:1948-1958
© 2005 American Society of Animal Science
Effect of repeated regrouping and relocation on the physiological, immunological, and hematological variables and performance of steers
S. Gupta*,
,
B. Earley*,
S. T. L. Ting*, and
M. A. Crowe,
,1
* Teagasc, Grange Research Center, Dunsany, Co. Meath, Ireland;
and
Faculty of Veterinary Medicine; and
and
Conway Institute of Biomedical and Biomolecular Research, University College Dublin, Belfield, Dublin-4, Ireland
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Abstract
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To investigate the effect of repeated regrouping and repenning (R&R) on the hypothalamic-pituitary-adrenal axis, immune function, blood biochemical and hematological variables, and ADG, 72 Holstein-Friesian (14-mo-old; 441 ± 3.2 kg) steers were assigned to either the control (C; n = 30) or regrouped (R; n = 42) treatments and housed six per pen in 12 pens. The R steers were exposed to six R&R over 84 d. New pen cohorts were allowed to stabilize for 14 d, and none of the R steers was allowed to share the same pen or penmates where or with whom they were previously housed. Control steers were housed in the same pen with the same penmates. Steers were blood sampled 2 h before and 2 h after the first, third, and sixth R&R. Steers were weighed the day before each R&R. Median area under the plasma cortisol curve (AUC) was greater (P < 0.05) in R than C steers after the first R&R. Following the first, third, and sixth R&R, the median ACTH AUC did not differ between the treatments. Cortisol AUC in R steers decreased (P < 0.001) following the third and sixth compared with the first R&R; however, cortisol AUC in response to exogenous ACTH (following administration of dexamethasone at –12 h) after the third R&R was greater in C than R steers (P < 0.05). Corticotropin-releasing hormone-induced cortisol and ACTH AUC were not different in C vs. R after the sixth R&R. There were no differences among treatments in haptoglobin, fibrinogen, and concanavalin A-induced interferon-
after the first, third, and sixth R&R. Albumin, urea, and NEFA were greater (P < 0.05) in R than C steers after the first R&R. ß-Hydroxy-butyrate and glucose concentrations were greater (P <0.05) in R than C, whereas no changes in the protein and globulin concentrations were found in C vs. R after the sixth R& R. White blood cell, differential and total count, red blood cell, and platelet numbers did not differ in C vs. R after the first and third R&R. Lymphocyte numbers and mean corpuscular volume were greater (P <0.05) in R than C steers after the sixth R&R. Monocyte numbers were greater (P <0.05) in R than C steers following first R&R. There was no difference in the overall ADG in C vs. R; however, there was a tendency (P = 0.10) for lesser ADG by R than C steers following second R& R. In conclusion, steers exposed to R&R responded with increased plasma cortisol, albumin, urea, and NEFA. Repeated R&R did not have a sustained detrimental effect on immune and production measurements.
Key Words: Hypothalamus-Pituitary-Adrenal Axis • Immunology • Physiology • Regrouping and Repenning • Steers • Stress
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Introduction
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Regrouping and repenning (R&R) of animals is a common husbandry practice to create homogeneous groups organized by age, BW, and production system (Bøe and Færevik et al., 2003
). Regrouping may occur by mixing grouped animals once or several times, and repenning may occur as a change in location of grouped animals once or several times. Studies (Hasegawa et al., 1997; Savi et al., 2003) on farm animals indicated that regrouping and repenning elicited physiological stress, suppression of immune function, and decreased performance. Veissier et al. (2001) found increased sensitivity of the adrenal cortex of regrouped calves to ACTH, whereas Hanlon et al. (1995) reported decreased lymphocyte response to keyhole limpet hemocynin (KLH) in regrouped deer. A short-term effect on production performance was reported in ewes to R&R (Sevi et al., 2001). In contrast, several studies (Kondo et al., 1990; Veissier et al., 2001) indicate that R&R of cattle does not have a detrimental effect on health, and that they adapt easily to grouping.
The aim in the present study was to evaluate the physiological, immunological, and performance responses of steers exposed first to an acute stress stimulus and then subsequently to repeated stress stimuli on their welfare. The hypothesis was that repeated R& R of beef steers would decrease sensitivity of the hypothalamus-pituitary-adrenal (HPA) axis to corticotropin-releasing hormone (CRH) and ACTH, decrease immune response, increase body metabolism, and decrease animal performance. Indices used to measure the function of the HPA axis were cortisol and ACTH response with and without exogenous ACTH and CRH administration; concanavalin A (Con A)-induced interferon (IFN)- production, and acute-phase proteins (haptoglobin and fibrinogen) for immune response; and blood biochemical and hematological variables for body metabolism.
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Materials and Methods
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All procedures in the study were conducted under experimental license from the Irish Department of Health in accordance with the Cruelty to Animals Act 1876 and the European Communities (Amendment of Cruelty to Animals Act 1876) Regulation, 1994.
Treatments
Seventy-two Holstein x Friesian (14-mo-old; mean BW = 441 ± 3.2 kg) steers were blocked by BW and assigned randomly to either control (C; n = 30) or regrouped (R; n = 42) treatments and housed six per pen in 12 pens alternatively at a space allowance of 2.8 m2/steer.
Animal Housing and Management
Before assignment to treatment, steers were weighed and housed from d –35 (day of treatment = d 0) to acclimatize them to handling and restraint. Steers were housed for 84 d on slatted-floor pen facilities, six pens facing each other. The dimension of each pen was 4.5 x 3.8 m, with a feed trough length of 4.5 m. A steel mesh with dimensions of 3.8 x 1.6 m separated the sides of each pen. The light was on 24 h in the housing facility. The steers had access to grass silage (a permanent grassland sward dominated by Lolium, Poa, and Agrostis species) with DM content (mean values) = 224 g/kg, in vitro DM digestibility = 887 g/kg of DM, pH = 4.2, and they were supplemented with 2.5 kg (asfed basis) of barley/soybean mix concentrate ration (mean values), with 41.9 g/kg of crude fiber, 155 g/kg of CP, 39 g/kg of acid-hydrolyzable oil, and 58.6 g/kg of ash per animal daily. Steers had free access to water in their pens.
Catheterization
To facilitate intensive blood collection, steers were fitted aseptically with indwelling jugular catheters on d –1 (day before the first R&R), 27, and 69 (1 d before the third and the sixth R&R; Table 1). The procedure was performed according to the method of Ting et al. (2003), with 12-gauge Anes spinal needles (Popper and Sons, Inc., New Hyde Park, NY) and polyvinyl tubing (approximately 1.47 mm i.d.; Ico Rally Corp., Palo Alto, CA; catalog No. SVL 105-18 CLR) attached to an 18-gauge needle at the blood collection end.
On d –1, four steers from each pen in both C and R treatments were catheterized. The remaining two steers from each pen in both C and R treatments were sham catheterized by aseptically piercing the left jugular vein with the 12-gauge spinal needle, and no catheters were fitted. On d 27, four steers were subjected to catheterization in each pen, two steers from the sham-catheterized and two others selected randomly from steers that were catheterized on d –1. The remaining two steers were sham operated in each pen. On d 69, all steers were catheterized. The difference in the numbers of steers catheterized at the third and sixth R&R was to avoid potential carryover effect on the pituitary and adrenal glands, where different steers were used for exogenous ACTH and CRH hormone administration. All of the catheters were exteriorized on the left side of the neck, flushed, and filled with sterile 3.5% sodium citrate solution, and plugged with a stopper. Catheters were secured in place in patches with the aid of an adhesive cement (Big Bull hip tag cement, Biguel Supply Co., Elysian, MN), Velcro, and zinc oxide wrapping bandages (Sterotape-Z.O., Steroplast Ltd., Bredbury, U.K; product code No. 4050). Sham-operated steers also were wrapped with zinc oxide bandages. After completion of the catheterization procedure, the steers were returned to their home pen and catheters were flushed with 2 mL of 3.5% (wt/vol) sodium citrate after each blood collection time.
Regrouping and Repenning
Regrouped steers were exposed to six R&R events from d 0 to 84 (Table 1
). Following each R&R new pen (n = 6 steers per pen) cohorts were allowed to stabilize for 14 d. In each R&R, none of the R steers was allowed to share the same pen or penmates where or with whom they were previously housed. Control steers were housed in the same pen with the same penmates from the beginning to the end of the experimental study. On the day of R&R, steers from R treatment were individually taken out from their pens and were regrouped and taken to their new pens. Each R&R of R steers was staggered and was carried out between 0800 and 0830. Immediately after R&R, the housing facility was closed and steers in the new pen cohorts were allowed to interact for 2 h without human interference. During this time, steers had access to water only. All of the steers were kept in their pens except at the time of R&R, weighing, and catheterization procedures.
Physiological Measurements
Basal Plasma Cortisol and ACTH Concentrations.
The cortisol and ACTH concentrations in the plasma of catheterized C and R steers were determined at the first (n = 20 for C; n = 28 for R), third (n = 10 for C; n = 14 for R), and sixth (n = 16 for C; n = 22 for R) R&R. The blood samples were collected at -2, 0, 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, and 3 h (0 h = first sample after 2 h of R& R). The tubes containing heparinized whole blood were centrifuged (1,600 x g at 8°C for 15 min), and then the plasma was collected and stored at –20°C until assayed for cortisol. The blood samples for ACTH determination were collected into iced tubes containing EDTA anticoagulants, the tubes were centrifuged at 2,000 x g, at 4°C for 15 min, and plasma was frozen and stored at –80°C until assayed. Commercial RIA kits were used to determine the plasma concentrations of cortisol (Corticote, ICN Pharmaceuticals, Orangeburg, NY; validated by Fisher et al., 1997) and ACTH (Diagnostic Products Corp., Los Angeles, CA) within 6 wk after the collection. The intraassay CV (n = 6) for samples containing 7.1, 16.5, and 55.8 ng of cortisol/mL were 10.7, 8.1, and 7.5%, respectively, and the interassay CV (n = 17) for the same samples were 22.6, 17.2, and 11.1%. The intraassay CV (n = four to six per assay) for sample containing 85.6 and 261.1 pg of ACTH/mL were 8.6 and 12.6%, respectively, and the interassay CV (n = 20) for the same samples were 9.7 and 11.3%, respectively.
ACTH Challenge.
The response of the adrenal cortex to ACTH (1.98 IU/kg BW0.75; Fisher et al., 1997a; Friend et al., 1977a) was tested following the third and sixth R&R on two steers from each pen (n = 14 for R; n = 10 for C treatments) that were chosen randomly at the start of the experiment. The same steers were used for the ACTH challenge at the third and sixth R&R occasions. Dexamethasone (20 µg/kg of BW; Faulding Pharmaceuticals Plc, U.K.) was administered (i.m.) at –12 h to all the steers undergoing ACTH challenge (Synacthen ampoules; Novartis Pharmaceutical Ltd., U.K.). Two steers from each pen (n = 14 for R; n = 10 for C treatments) received normal saline (5 mL, 0.9% [wt/vol] sterile) as a placebo at the time of both dexamethasone and ACTH injections. Immediately following the administration of dexamethasone, ACTH, and normal saline catheters were flushed with 2 mL of 3.5% (wt/vol) sterile sodium citrate solution. Blood samples were collected into the tubes containing EDTA (in an ice bath) for ACTH or heparin anticoagulants for cortisol through jugular catheters immediately before the administration of dexamethasone and saline and at –2, –0.25, 0, 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, and 3 h (–0.25 h = first sample after 2 h of R&R) relative to the time of ACTH and saline administration. Plasma storage and assays were as described above. Plasma samples collected for ACTH determination were above the range of the ACTH standard provided with the kits. These plasma samples were diluted by 1:20, 1:50, and 1:100 as required, and concentrations were corrected accordingly before analysis.
CRH Challenge.
Two steers from each pen (n = 10 for C; n = 14 for R) were administered CRH at 4 h following the sixth R&R. These steers were administered normal saline previously at the first and third R&R. In addition, steers from each C (n = 6) and R (n = 8) treatments were administered with normal saline (2 mL, 0.9% sterile). Two blood samples were collected at a 0.25-h interval each before the i.v. administration (through jugular catheter) of bovine CRH (bCRH; 0.3 µg/kg BW; American Peptide Co., Inc. Sunnyvale, CA; Gupta et al., 2004) and normal saline. Subsequent blood samples were collected through the jugular catheters at 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, and 3 h relative to the time of bCRH and saline administration for plasma cortisol and ACTH concentrations. Immediately following the administration of bCRH and normal saline, catheters were flushed with 2 mL of 3.5% sterile sodium citrate solution. The plasma storage and assay method for both cortisol and ACTH were as described previously.
Hematology and Biochemistry.
Unclotted (EDTA) whole blood samples were collected from catheterized C and R steers, 2 h before and after the first (d 0; n = 20 for C; n = 28 for R), the third (d 28; n = 20 for C; n = 28 for R), and the sixth (d 70; n = 26 for C; n = 36 for R) R&R. They were analyzed for total white blood cell numbers (WBC), red blood cell (RBC), monocyte, lymphocyte numbers, mean corpuscular volume (MCV), hemoglobin (Hb), and platelet numbers using an automated electronic particle analyzer (Celltac, MEK-6108K, Nihon-Kohdon, Tokyo, Japan) within 1 h of blood sampling. Further blood samples containing heparin as anticoagulant were collected for determination of ß-hydroxy butyrate (ßHB), albumin, globulin, total protein, and urea, and containing fluoride anticoagulant for NEFA and glucose determination. In all cases, plasma was separated by centrifugation (1,600 x g at 8° C for 15 min) and stored at –20°C until assayed using commercial biochemical assay kits (Boehringer Mannheim, Mannheim, Germany, and Randox Private Ltd., Crumlin, U.K.) on an automated biochemical (SPACE; Schiapperelli Biosynthesis Inc., Alfa Wassermann BV, Woerden, The Netherlands) analyzer.
Immunological Measurements
Stimulated Lymphocyte Production of Interferon- .
Catheterized steers were blood sampled at 2 h before and after the first (n = 20 for C; n = 28 for R steers), third (n = 20 for C; n = 28 for R steers), and sixth (n = 26 for C; n = 36 for R steers) R&R. The stimulated lymphocyte production of IFN- following whole blood culture of heparinized plasma was determined with a modification (Fisher et al., 1997a; Ting et al., 2003) of the procedure described by Wood et al. (1990).
Haptoglobin and Fibrinogen.
The whole blood samples collected into heparinized tubes were centrifuged at 3,000 x g, 8°C for 10 min and plasma stored at –20°C until assayed for haptoglobin. Plasma haptoglobin concentrations were determined as the hemoglobin binding capacity using a biochemical assay kit (Tridelta Development Ltd., Greystones, Ireland; catalog No. TP801) previously validated for bovine plasma by Skinner et al. (1991). Blood samples collected into sodium citrate tubes were centrifuged at 3,000 x g, at 8°C for 10 min and plasma stored at –20°C until assayed for fibrinogen. Fibrinogen, the circulating precursor of fibrin in the blood-clotting cascade, was determined by using a commercial kit (Roche Diagnostics GmbH, Mannheim, Germany; catalog No. 524484) adapted for bovine plasma (Earley and Crowe, 2002).
Environmental Conditions and Production
The mean daily air temperature and relative humidity in the housing facility were recorded continuously using Tiny Talk data loggers (Radionics, Dublin, Ireland). The BW of steers was recorded on eight different occasions, on d -7 and on d 13, 27, 41, 55, 69, 83, and 97 to determine the ADG.
Statistical Analyses
All statistical analyses were performed using SAS (SAS Inst., Inc., Cary, NC). The probability plot of the residuals (Shapiro-Wilk test; P <0.005) using the UNI-VARIATE procedure was used to determine the normality of the data. The data that showed lack of normality and heterogeneity of variance were analyzed by non-parametric analysis using Kruskall-Wallis and Mann Whitney procedures based on the rank transformation (Zar, 1999). For each steer, the median values for area under the cortisol and ACTH vs. time curves (AUC cortisol and AUC ACTH) were analyzed after rank transformation by Kruskall-Wallis one-way ANOVA (Zar, 1999). The area (ng•mL–1•min–1) under the cortisol and ACTH vs. time curve was calculated from the time of treatment either R&R or ACTH/CRH/saline administration until the final sample of the day following the first, third, and sixth R&R using a linear trapezoidal rule:
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where Ct is the concentration of a plasma cortisol sample in nanograms per milliliter of an animal at time t, and for the next samples Ct+1, with a time interval of 
I in hours between them, and 
is the sum of the responses from Ct to n-1 total number of concentration time points (Veissier et al., 2001). Hematological variables (WBC, RBC, platelet, monocyte, lymphocyte numbers, MCV, and Hb), globulin, NEFA, glucose, IFN-, haptoglobin, and fibrinogen were analyzed after rank transformation by ANOVA for the effect of treatment with the pre-treatment values (values before R&R at 2 h) included as a significant covariate. The statistical differences between the treatments were determined by Wilcoxon sign rank test. To evaluate the adaptation over time for cortisol and ACTH responses data were analyzed after rank transformation with a Friedman test for repeated measures, and the statistical differences were determined by Tukey’s studentized range test (Zar, 1999). Data relating to mean albumin, ßHB, protein, Hb, urea, and ADG were analyzed parametrically by ANOVA for the main effect of treatment and taking pretreatment values as a covariate. During the ACTH challenge at both the third (C steer) and sixth (C steer) R&R, and CRH challenge at the sixth R&R (R steers) the catheter, from one steer at each time was lost after the fourth, fifth, and sixth blood collection time; therefore, the complete data from these animals were excluded from the statistical analyses.
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Results
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Physiological Measurements
Plasma Cortisol and ACTH.
The median basal plasma cortisol (minimum and maximum) and ACTH (minimum and maximum) concentrations in the C steers was 5.13 (0.92 and 13.54) ng/mL and 68.85 (40.73 and 129.56) pg/mL, respectively, with no difference between C and R steers before the pretreatment period at –2 h. Following the first R&R, the median area under the plasma cortisol curve from 0 to 180 min, was greater (P <0.005) in R than C steers (Figure 1), with no difference observed among treatments following the third and sixth R&R. No differences were found following the first, third, and sixth R&R from 0 to 180 min for median area under the plasma ACTH curve. Overall, the change in median values for cortisol AUC over time in R steers decreased (P = 0.001) following the first vs. third and sixth R&R (median 1225.5 vs. 737.7 and 966.9 ng•mL–1•min–1); however, cortisol AUC in R steers following the sixth R&R was greater (P <0.001) than the third R&R. No differences were observed following the first, third, and sixth R&R in the control steers (data not shown). In contrast, the median values for the ACTH AUC over time in C and R steers, following the third compared with first and sixth R&R (17,362.2 vs. 12,479.2 and 11,014.1 for C, P = 0.001; 17,455.8 vs. 12,247.9 and 11689.1 ng•mL–1•min–1 for R, P = 0.003) was greater.
ACTH Challenge.
Plasma Cortisol and ACTH responses to exogenous ACTH and saline for steers are shown in Figure 2. Immediately before the administration of dexamethasone at –12 and at –2 h before the third and sixth R&R the median cortisol and ACTH concentrations were not different (data not shown) among C vs. R steers. Following the 14 h of dexamethasone administration at the third and sixth R&R, the cortisol and ACTH concentrations decreased (0.05 ng/mL and 41.57 pg/mL, respectively; P <0.05). There was a response to exogenous ACTH compared with saline for median plasma concentrations of AUC for cortisol and ACTH in C and R steers following the third (R, P = 0.001; C, P = 0.001 for both cortisol and ACTH) and sixth (R, P = 0.001; C, P = 0.001 for both cortisol and ACTH) R&R occasions. Following the administration of ACTH after the third R&R, less median plasma AUC for cortisol (P = 0.048) and ACTH (P = 0.049) were observed in R than C steers; however, no difference in median plasma AUC for cortisol and ACTH were observed among treatments at the sixth R&R.
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Figure 2. Box-whisker plots of area under the curve (AUC) for plasma cortisol (upper panel) and ACTH (lower panel) following exogenous treatment with ACTH (1.98 IU/kg BW0.75) or saline (5 mL, 0.9% sterile) to control (C) and regrouped (R) steers after the third (n = 10 for C, and n = 14 for R) and sixth (n = 10 for C, and n = 14 for R) regrouping and repenning (R&R). The boxes show the median (—), upper ( ), and lower (•) quartiles. The box-whisker plot shows the 25th ( ) and 75th (x) percentiles of plasma cortisol and ACTH concentrations. Within each panel, for each regrouping and repenning, bars with a, b for C and c, d for R steers differ, P <0.05.
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CRH Challenge.
The administration of exogenous bCRH increased cortisol (C, P = 0.001; R, P = 0.001) and ACTH (C, P = 0.001; R, P = 0.002) concentrations within each C and R steers (Figure 3) compared with saline administration. There was no effect of exogenous administration of bCRH on the median plasma concentrations of AUC for cortisol and AUC for ACTH among C vs. R steers after the sixth R&R.
Hematology and Biochemistry.
The median WBC, RBC and platelet numbers, monocyte numbers, and Hb did not differ in C vs. R steers after the first, third, and sixth R&R (Table 2). Median MCV and lymphocyte numbers were greater (P <0.05) in R than C steers after the sixth R&R. Median monocyte numbers were greater (P <0.05) in R than C steers following the first R&R. Mean albumin (35.2 ± 0.38 vs. 34.4 ±0.44 g/L), urea (4.1 ±0.11 vs. 2.7 ±0.14 mmol/L) and median NEFA (0.15 vs. 0.11 mmol/L) were greater (P <0.05) in R than in C steers following the first R&R, whereas mean ßHB (0.31 ± 0.013 vs. 0.28 ±0.012 mmol/L) and median glucose (4.2 vs. 4.1 mmol/L) were greater (P <0.05) in R than in C steers following the sixth R&R. Mean ßHB, mean protein, median globulin, and median glucose did not differ in C and R steers after the first and third R&R (data not shown). No differences were observed in the mean albumin, mean urea, and median NEFA following the third and sixth R&R. Mean protein and median globulin were not different following the sixth R&R in C vs. R steers (data not shown).
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Table 2. Effect of the first, third, and sixth regrouping and repenning (R&R) on total white blood cell (WBC), red blood cell (RBC), and platelet numbers, mean corpuscular volume (MCV), lymphocyte and monocyte numbers, and hemoglobin of control and regrouped steers
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Immunological Measurements
Stimulated Lymphocyte Production of Interferon-, Haptoglobin, and Fibrinogen.
No differences were observed between C vs. R steers in their responses to Con A-induced in vitro IFN- production following the first, third, and sixth R&R (Figure 4). There were no differences among treatments in plasma haptoglobin and fibrinogen concentrations following the first, third, and sixth R&R (Figure 5).
Environmental Conditions, ADG, and General Health
The mean daily air temperature and relative humidity in the housing facility were 16.9°C (range 5.8 to 25.9°C) and 81.98% (range 38.4 to 100.0%) during the experimental period from April to June. Overall there was no effect of repeated R&R on the ADG between R and C steers (Table 3); however, there was a tendency (P <0.10) for a decreased ADG by R compared with C steers following the second R&R.
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Table 3. Effect of repeated regrouping and repenning (R& R) on the ADG (kg) by 14-mo-old control and regrouped steers during an 83-d experimental perioda
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Discussion
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Regrouping and repenning is a conventional management practice of farm animals. Animals respond to the brupt or repeated breakage of social bonds either by adaptation within normal physiological measurements or by decreased productivity. In the present study, the acute exposure of steers to the first R&R resulted in a specific endocrine response characterized by increased plasma cortisol response in R compared with C steers; however, the first R&R did not affect the release of ACTH from the pituitary gland. The increase in cortisol may have occurred due to the mixing of unfamiliar animals at different locations and was in contrast to the work of Veissier et al. (2001) in calves and Sevi et al. (2003) in ewes, where both authors reported no difference in the basal plasma cortisol concentrations following regrouping and repenning treatments, and was not consistent with Hanlon et al. (1995), who reported lower basal cortisol concentrations in mixed vs. control deer. This result is in agreement with Broom and Johnson (1993), who reported that plasma ACTH concentrations could decrease rapidly after the application of the acute stressor, which could account for our inability to demonstrate change in ACTH. The lack of change in plasma cortisol and ACTH concentrations at the third and sixth R&R indicated that the steers were likely accustomed to being moved and grouped with strangers. The hormonal adaptations indicated by decreased responsiveness of the HPA axis to the R&R stressor on d 28 onward and until d 84 suggest that habituation occurred with the possible involvement of the central nervous system, which is the first organ to perceive stress stimuli and control the secretory activity of the HPA-axis (Garcia-Belenguer et al., 1993). Furthermore, these findings also suggest that cortisol might be a better indicator than ACTH to study acute stress responses.
There was an increased integrated cortisol response in C compared with R steers following exogenous ACTH administration at the third R&R. This finding is in contrast with previous studies (Hanlon et al., 1995; Veissier et al., 2001) of social regrouping and relocation induced by a weekly exposure of change in animal partners and their locations. The difference with the previous studies could be attributed to the 2-wk interval between each R&R, species variation, age, and BW of the animals in the present investigation, which might have lowered the magnitude of the stress required to stimulate an adrenal response in R steers. Unlike basal cortisol concentrations, cortisol response induced by exogenous ACTH administration may provide an independent index of adrenocortical sensitivity (Moberg and Mench, 2000). There are inconsistencies in the interpretation of increased (Friend et al., 1985; Janssens et al., 1994) or decreased (Ladewig and Smidt, 1989; Fisher et al., 1997a) cortisol response following ACTH administration under stressful situations among animals. The present findings suggest that decreased sensitivity of the adrenal to ACTH following acute stress stimuli (first R&R), coupled with the chronic effect of subsequent repeated R&R (first, second, and third), downregulated the pituitary-adrenal axis or increased the sensitivity of the pituitary to cortisol negative feedback. Furthermore, following repeated exposure (i.e., after the sixth R&R), there was no change in cortisol response to ACTH challenge, which indicates a decrease in the adrenal responsiveness to ACTH. The effect could be occurring either via ACTH receptor concentrations in the adrenal gland, or in the synthesis, release, or clearance of cortisol. When considering an adaptive response, the negative effect of R&R seems to be restricted to only a short period. Interestingly, the induced plasma concentrations of ACTH by exogenous ACTH administration in R steers were less than C steers, indicating rapid clearance of ACTH in the R&R animals and warrants further investigation. The difference in the concentrations of induced cortisol and ACTH following ACTH challenge compared with saline injection within the same treatments was a drug effect.
In humans, CRH challenge is used as a diagnostic tool to monitor the course of chronic stress due to Cushing’s syndrome, adrenal insufficiency, and major depressive disorders (O’Connor, 2000). In humans and domestic animals, the duration or concentrations of stress-induced increase in circulating ACTH and cortisol concentrations may be controlled by downregulating the sensitivity of the HPA axis. Therefore, the use of CRH administration in stressed farm animals for testing the sensitivity of pituitary to ACTH release above physiological threshold concentrations is of benefit. In the present study, steers subjected to repeated R&R did not differ from controls in CRH-induced ACTH and cortisol responses. The lack of change on the functioning of the HPA axis following the administration of CRH between stressed and unstressed animals is in agreement with studies in pigs (Janssens et al., 1994) and in calves (Veissier et al., 2001). In contrast, the release of ACTH following CRH challenge has been reported in food-restricted rats (Hauger et al., 1990). Fisher et al. (2002b) showed a decrease in ACTH and cortisol response to CRH in cows that were restricted from lying. The present findings confirm that the longer stressor duration modified the functioning of the HPA axis, and following the presence of repeated R&R stressor, there are either adaptation or regulatory changes in the hypothalamus and/or pituitary to minimize the effects of the stress imposed.
In vitro-stimulated lymphocyte IFN- is an important cytokine that induces a variety of physiologically significant responses that contribute to immunity (Shtrichman and Samuel, 2001). Interferon- production has been used as a reliable measure of immune activation to common husbandry stressors such as castration (Fisher et al., 1997a; Earley and Crowe, 2002; Ting et al., 2003) and weaning (Hickey et al., 2003) in cattle. In the present study, repeated R&R did not induce an immunosuppressive response in the production of IFN- following lymphocyte stimulation in response to a novel mitogen, Con A. These findings are not in complete agreement with those of Hanlon et al. (1995), who reported decreased lymphocyte responses to KLH (a novel antigen) and no effect on the lymphocyte response to Con A (a mitogen), following repeated introduction of deer to a new group. Given that the increased concentrations of CRH and glucocorticoids are associated with decreased immune responses (Anisman, 2002), the present study showed that IFN- production did not seem to be associated with increased concentrations of cortisol following the first R&R. Furthermore, the findings of the present study indicate that neither initial acute stress exposure (first R&R) nor later acute stress exposure (third and sixth R&R) coupled with the chronic effect of repeated R&R has affected the IFN- production from cultured lymphocytes in response to Con A in steers. Previous studies (Fisher et al., 1997a; Earley and Crowe, 2002; Ting et al., 2003) have shown the suppression of IFN- following d 1 and 3 of castration stress in calves, where there was a peak cortisol concentration within 30 min of castration. On the basis of these results, it is suggested that daily sampling at least up to 2 to 3 d of treatment would be beneficial to predict changes in IFN- against any stressor.
Initial (first) and repeated (third and sixth) R&R of R steers did not alter the concentrations of plasma haptoglobin and fibrinogen compared with C steers. Other cattle (castration, Earley and Crowe, 2002; Ting et al., 2003; and transportation, Phillips et al., 1989) stress models have reported increased plasma concentrations of acute-phase proteins such as haptoglobin and fibrinogen (Anisman, 2002). This increased acute-phase protein response is the reaction of an animal to disturbance in their homeostasis caused by infection, tissue injury, neoplastic growth, or immunological disorder. In the present investigation, the stress induced by repeated R&R was insufficient to induce changes in haptoglobin and fibrinogen plasma concentrations. The correlation of increased acute-phase proteins with increased activated T-cell proliferation and glucocorticoid in animals (Anisman, 2002) highlights the need for further research on plasma haptoglobin and fibrinogen and their interaction with the neuroendocrine system during noninflammatory stressful circumstances.
Blood cells are sensitive indicators of physiological and pathophysiological responses in animals. A change in blood cell composition indicates a response to restore animal homeostasis when exposed to abrupt physical conditions (Radostits et al., 1994). The present study showed that initial and repeated (up to the third) R&R did not affect the WBC, RBC, platelet, lymphocyte, MCV, and Hb of R steers; however, increased lymphocyte numbers and MCV following the sixth R&R in R compared with C steers are indicative of a slight increase in sensitivity of the R steers during the recovery process associated with changes in the physical environment. These results could be attributed to the fact that the involvement of nonadrenal catecholamine hormone might be boosting blood cell composition to increase the number of nongranulocyte subpopulations in blood (Dhabhar et al., 1996). Corticosterone, acting at the type II adrenal steroid receptor, is a major mediator of the stress induced changes in blood lymphocyte and monocyte distribution (Dhabhar et al., 1996), but results of this study did not completely concur with these findings. Nonetheless, monocytosis observed following the first R&R may be initiated by increased cortisol concentrations. The overall picture of hematological variables indicates that the health of the steers in this study was not compromised with the repeated R&R stress, and these findings suggest that the regrouping and repenning may not necessarily be a sufficiently potent stressor to disrupt the homeostasis of steers in the current study.
Changes in blood metabolites are indicative of energy mobilization, a mechanism necessary to maintain the homeostasis (Moberg and Mench, 2000). Stressful events are typically associated with increased energy demands as well as decreased appetite and this leads to depletion of energy stores, in particular liver glycogens and body fat (Balm, 1999). The increase in the albumin, urea, and NEFA following first R&R may indicate alterations in metabolism. Given that glucocorticoids are glycogenolytic factors during stress and stimulate gluconeogenesis, to the detriment of body protein, and, together with neuropeptides (CRH), they are central switches for reallocation of energy streams from body growth towards functions promoting immediate survival (Wendelaar and Balm, 1999). Increased plasma glucose and ßHB, a ketone body in blood, in R steers at the sixth R&R suggest a greater energy demand during recovery from the chronic effects of repeating R&R to restore homeostasis. The other possibility for increases in glucose and ßHB could be consumption of a starchy diet (Steffens and Boer, 1999); however, there was no change in the diet of the steers throughout the study hence time of blood sampling is more likely to be a factor.
Grouping has reported negative effects on production performance (Sevi et al., 2001). Repeated R&R of steers did not, however, decrease overall ADG in the present study.
In conclusion, steers responded to regrouping and repenning with increased basal plasma cortisol and changes in metabolic activity. Repeated R&R decreased the sensitivity of the adrenal to ACTH with no effect on the pituitary following CRH challenge compared with controls. There was adaptation, however, of the metabolic system over time among steers repeatedly exposed to regrouping and repenning, such that regrouping and repenning was without any detrimental effects on the performance, immune status, or health of the animals.
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Implications
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In addition to conventional management practices in beef cattle husbandry, regrouping and relocation of animals is a common practice. The results of this study suggest that the exposure and duration of stressors (such as regrouping and repenning) play an important role in modulating the regulation and function of the hypothalamic-pituitary-adrenal axis. The first regrouping and repenning (an acute stress stimulus) resulted in increased activity of the pituitary-adrenal axis, with a failure to restore concentrations of cortisol and metabolic functions. However, the prolonged effects of mixing and relocation (chronic stress) coupled with repeated additional mixings (acute stress stimuli) limited the effect of regrouping and repenning, and steers seemingly became accustomed to these procedures Therefore, steers can be repeatedly regrouped and relocated without detrimental effects on immune and production measurements; however, in commercial conditions, apart from the regrouping and repenning stressor, beef cattle are exposed to transport, handling, and housing stressors, so further studies are required to investigate the duration and stabilization of adaptive changes in the hypothalamic-pituitary-adrenal axis, to ascertain the absence of stress in cattle.
1 Correspondence: Faculty of Veterinary Medicine, University College Dublin (phone: +353-1-716-6255; fax: 353-1-716-6253); e-mail: mark.crowe{at}ucd.ie.
Received for publication September 13, 2004.
Accepted for publication May 20, 2005.
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Abstract
Introduction
