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Teagasc, Grange Research Centre, Dunsany, Co. Meath, Ireland
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Abstract |
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production and neutrophil:lymphocyte ratio were measured. The effects of weaning, calf sex, time, and their respective interactions were described. Disruption of the established social group at 0 h increased (P < 0.001) the plasma cortisol concentration and neutrophil:lymphocyte ratio and decreased the leukocyte concentration (P < 0.001) and the in vitro interferon- response to the mitogen concanavalin-A (P < 0.001) and keyhole limpet hemocyanin (P < 0.001) for weaned and control animals compared with -168 h. There was no effect of weaning or sex on the behavioral reaction of calves to handling. Plasma cortisol and adrenaline concentrations were not affected by weaning or sex. Plasma noradrenaline concentration was influenced by weaning x sex (P < 0.05) and time x sex (P < 0.05). The response increased for male calves with weaning and increased with each sampling time after weaning. For heifers, the response was not affected by weaning and plasma concentrations decreased at 168 h after weaning. There was no effect of weaning or sex on leukocyte concentration. The neutrophil:lymphocyte ratio increased after weaning (P < 0.01) and was affected by sex (P < 0.05). Weaning decreased (P < 0.05) the in vitro interferon- response to the antigen keyhole limpet hemocyanin. There was a time x weaning x sex (P < 0.05) interaction for fibrinogen concentration but no effect of treatment on haptoglobin concentration. Abrupt weaning increased plasma cortisol and noradrenaline concentrations that were accompanied by attenuation of in vitro interferon- production to novel mitogen and antigen complexes up to 7 d after weaning.
Key Words: Calves • Catecholamines • Cortisol • Immunity • Stress • Weaning
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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), plasma acute-phase protein concentrations (Phillips et al., 1989), and neutrophil:lymphocyte (N:L) ratio (Church and Hudson, 1999). These results indicate that breaking the maternal bond is stressful to the calf. Stressful events have previously been associated with attenuation of immune function (Blecha et al., 1984; Sevi et al., 2001) and disease susceptibility in animals (Roth, 1982; Burton et al., 1995). MacKenzie et al. (1997) found no effect of abrupt weaning on the humoral immunity of weaned calves. Attenuation of the cell-mediated (Hickey et al., 2003), but not humoral, immunity (Fisher et al., 1997a) has been identified in situations of chronic stress. Pollock et al. (1991) also suggested that the cell-mediated, rather than humoral, immunity may be a more reliable indicator of the physiological status of calves older than 5 mo. The adrenal hormones are recognized indicators of stress in bovine models (Toates, 1995) and no work has been identified that describes the long-term effect of weaning on the mediators of stress. The objective of this study was to examine the effect of abrupt weaning (inclusive of social group disruption and maternal separation) on the physiological mediators of stress (cortisol, adrenaline, and noradrenaline) and measures of immune function (in vitro interferon- production, N:L ratio, and acute-phase protein concentrations). The hypothesis of this study was that the abrupt weaning of calves is a stressor that will increase the physiological measures of stress hormones, resulting in attenuation of the cell-mediated immunity. |
Materials and Methods |
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Animal Handling
Two weeks before the start of the study, calves were habituated to handling. During this time, on alternate days, each calf was placed in a crush adjacent to the area of grazing for a total duration of 15 min. Within this time allocation, the calf was haltered, restrained, and acclimatized to human interaction on three occasions.
Behavioral Score
At each blood sampling time (described below), an observer scored the reaction of calves to handling at three distinct stages while in the crush. Stages were separated by a 2-min time lapse. These stages were identified as the animal’s reaction to restraint by a halter, to jugular clamping, and to jugular venipuncture. An animal was given a score of 1 if it did not react to the presence and actions of the human at any stage, or in the case of stage three, once the needle was inserted. An animal scored 2 if it continued to react at any one of the identified stages but subsequently calmed during the intervening 2 min before the procedure moved to the next stage. An animal scored 3 if it continued to react at any two of the identified stages but subsequently calmed during the intervening 2 min before the procedure moved to the third stage. An animal scored 4 if it reacted during all stages. The intensity of the reaction for stages 2 to 4 was also scored. The reaction scored 1 (mild) if the animal stepped in any direction but never pulled against the restraining rope. The reaction scored 2 (intense) if the animal pulled against the restraining rope while moving or stationary. The reaction scored 3 (severe) if the animal attempted to turn while restrained or went down on fore and/or hind legs. The final score attributed to each animal at each bleed was calculated as score of stage x score of reaction (for unresponsive cows, the score was automatically 1).
Physiological Measurements
All calves were blood sampled by jugular venipuncture at -168, 24, 48, and 168 h after weaning. Due to management constraints (lack of daylight at outfield bleeding pens), only the bull calves were blood sampled at 6 h after weaning. Samples were collected into lithium heparinized tubes, the plasma harvested after centrifugation at 3,000 x g for 10 min, and then frozen. Plasma was subsequently analyzed for cortisol concentration using a RIA (Corti-Cote, Orangeburg, NY). This assay was adapted and validated for bovine plasma (Fisher et al., 1996), with intraassay CV for samples containing 5.5, 18.7, and 70.7 ng of cortisol/mL of 13.3, 10.1, and 9.1%, respectively, and interassay CV for the same samples of 17.5, 11.3, and 10.8%. For catecholamine analysis, five animals were randomly selected from each treatment group, within each herd. These calves were blood sampled by jugular venipuncture into heparinized tubes at 6 (males only), 24, 48, and 168 h after weaning. Samples were immediately transferred to heparinized tubes containing 5 mg of sodium metabisulfite and transferred to the laboratory on ice. Plasma was harvested within 1 h of collection and frozen at -70°C. Catecholamine concentrations were quantified using a HPLC assay based on the method of Lefcourt and Elasser (1995).
Immune Measures
Blood samples were collected at -168, 6 (males only), 24, 48, and 168 h after weaning. Blood samples collected into lithium heparinized tubes were centrifuged at 3,000 x g for 10 min and the plasma was frozen. Plasma was analyzed for haptoglobin concentration (validated for bovine plasma by Skinner et al., 1991) using an appropriate assay kit (TP801, supplied by Tridelta Development Ltd., Bray, Ireland). Blood samples collected into sodium citrate tubes were centrifuged at 3,000 x g for 10 min and the plasma frozen. Plasma fibrinogen was measured using an appropriate assay kit (524484, Roche-Boerhinger, Mannheim, Germany). Blood samples were collected into K3-EDTA tubes and the leukocyte profile (total white blood cell concentration and lymphocyte and neutrophil proportions) for each animal was described using the May-Grunwals-Giemsa stain of eosine-methylene blue, modified for microscope and conducted by the National Equine Centre of Ireland. The stimulated lymphocyte production of interferon- was determined following whole blood culture of heparinized plasma in the presence of keyhole limpet hemocyanin (KLH) antigen and Concanavalin A (Con-A) mitogen (Wood et al., 1990). The interferon- production responses were quantified in vitro using an ELISA procedure (Rothel et al., 1990; CSL Biosciences, Parksville, Victoria, Australia). In brief, this procedure required that duplicate 1.48-mL aliquots of blood were cultured in 24-well culture plates (Costar Corp., Cambridge, MA) with 20 µL of PBS containing 1 mg/mL of KLH, 1.0 mg/mL of Con-A, or no additive, for 16 h at 37°C and in an atmosphere of 5% CO2 in air. The culture plates were then centrifuged and the supernatant harvested and frozen at -20°C until it was assayed for interferon- production using an ELISA procedure (BOVIGAM, Biocor Animal Health). In vitro KLH- and Con-A-stimulated interferon- production was calculated by subtracting the absorbency at 450 nm of wells that received PBS alone from the absorbency of wells that received either KLH or Con-A.
Statistical Analyses
The data for all blood measures, except those sampled at 6 h, were analyzed in nine randomized blocks by a split-plot ANOVA. The main plot included effects of weaning, sex, and weaning x sex. The subplot included the effects of time, sex x time, weaning x time, and sex x weaning x time. A diurnal variation exists in plasma cortisol and leukocyte profiles. Therefore, data on all blood measures pertaining to the 6-h sampling time were analyzed separately in nine randomized blocks by a single-factor ANOVA, which described the effect of weaning. Following a significant F-test (P < 0.05), Fisher’s least significant difference test was applied to determine statistical differences between treatments (Snedecor and Cochran, 1989). Data on temperament score were analyzed using a Friedman two-way analysis to describe the effect of sex and weaning (Steel and Torrie, 1960). Linear and quadratic regressions were fitted to determine the relationship, if any, between behavioral score and plasma adrenaline, noradrenaline, and cortisol concentrations.
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Results |
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). There was no effect of time, weaning, or sex on plasma adrenaline concentration (Table 2). There was no effect of weaning on the plasma adrenaline response of bull calves at 6 h postweaning (Table 3). Plasma cortisol concentration increased after social group disruption (P < 0.001, Table 2) but not by weaning or calf sex or by weaning at 6 h (Table 3). Plasma noradrenaline concentration was influenced by weaning x sex (P < 0.05, Table 2) and time x sex (P < 0.05) interaction since the noradrenaline response of bulls only increased with weaning and time. This increase was evident (P < 0.01, Table 3) at 6 h. The response for heifers was influenced by a sharp decrease in plasma concentration at 168 h postweaning that was evident in both the control and weaned animals.
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There was no effect of weaning or sex on total white blood cell concentrations (Table 4). Social group disruption increased (P < 0.001) the total leukocyte concentration when compared with 24 and 48 h, but not 168 h, postweaning. With respect to lymphocyte proportion, male calves had a lower (P < 0.05) proportion than female calves, and weaning decreased the lymphocyte proportion at 24, 48, and 168 h postweaning (P < 0.05). This effect was also evident (P < 0.01, Table 3
) at 6 h postweaning. The neutrophil proportion was higher (P < 0.05) for male than female calves. The time x weaning (P < 0.01) interaction described an increase in the neutrophil proportion for weaned animals at 24 and 168 h postweaning. This increase was also evident for bull calves (P < 0.01) at 6 h. The N:L ratio was higher (P < 0.05) for male vs. female calves. Weaning increased (P < 0.01) the N:L ratio. The N:L ratio was higher at 24 h but not 48 or 168 h postweaning when compared with the preweaning value. Weaning increased (P < 0.05) the N:L ratio for bull calves at 6 h after weaning.
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response to the mitogen, Con-A, was reduced (P < 0.001) after social group disruption (Table 5) and did not recover by 168 h after weaning. The response to the mitogen, KLH, was also reduced (P < 0.001; Table 5) after social group disruption and did not recover by 168 h after weaning. The KLH response for weaned calves was lower (P < 0.01) than that measured for control animals, and was lower at 6 h (P < 0.05) for weaned bull calves when compared with controls.
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. For the unweaned control males, the plasma fibrinogen concentration was highest (P < 0.05) at -168 h. The fibrinogen concentration was elevated (P < 0.05) at 48 h compared with 24 h, but concentrations declined at 168 h (P < 0.05) such that they did not differ from values obtained at 24 h. For weaned males, there was no effect of social disruption on the fibrinogen concentration; however, plasma concentrations increased at 48 h (P < 0.05) when compared with concentrations recorded at 24 h, and subsequently decreased at 168 h (P < 0.05). Plasma concentrations of fibrinogen were higher for male weaned calves than control animals at 24 h, higher than all treatment groups at 48 h, and higher than the female control group at 168 h postweaning. For female controls and weaned calves, the plasma fibrinogen concentration decreased at 24 h (P < 0.05) postweaning, increased at 48 h (P < 0.05), and decreased at 168 h postweaning (P < 0.05).
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Discussion |
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, Burton and Kehrli, 1995), with important economic implications (Gunn and Scott, 1998, Lekeux, 1995). Two main stress pathways, whose response functions are central to survival, are also central to the stress-immune axis activation that can lead to disease susceptibility (Riad et al., 2002; Eskandari and Sternberg, 2002). The first, activation of the hypothalamus-pituitary-adrenal axis, results in a significant release of the steroid hormone, cortisol, from the cortex of the adrenal glands. Systemic cortisol concentrations increase several minutes after a perceived threat and can last for a number of hours. Cortisol is a potent glucocorticoid (Riad et al., 2002), the immunosuppressive effects of which may serve as physiological down regulators of initiated immune responses during recovery from infection or tissue damage (O’Connor et al., 2000). The second major pathway mediating physiological stress responses in animals is activation of the sympatho-adrenal axis (Minton, 1994; Morberg, 1997; O’Connor et al., 2000). This results in release of the adrenergic neurotransmitters, adrenaline and noradrenaline, from the sympathetic nerves and medullae of the adrenal glands. Sympatho-adrenal axis activation occurs in seconds relative to perceived threats (Erikson et al., 1999). All leukocytes express the ß-adrenergic receptors that bind adrenaline and noradrenaline (Black, 1994; Abo and Kawamura, 2002; Nagatomi et al., 2000). However, a paucity of information exists regarding the physiological roles of these neurotransmitters in immune function. This is attributed to the extreme difficulty in sampling and measuring blood concentrations due to their sensitivity to animal handling during sampling and to their short half-life (Minton, 1994). Past studies have documented clear changes in circulating numbers of neutrophils and lymphocytes, attenuated proliferation of T and B lymphocytes, and modified expression of surface adhesion and antigen presenting molecules on leukocytes subjected to stress hormones in vivo and in vitro (Murata et al., 1985; Burton et al., 1995; Burton and Kehrli, 1995; Anderson et al., 1999; Earley and Crowe, 2002; Hickey et al., 2003). The hypothesis of this study was that the abrupt weaning of calves is a management stressor that will increase the physiological measures of stress hormones, resulting in attenuation of immune function.
Unfamiliar human-animal interaction can create a feeling of unease that is associated with a sympathetic adrenomedullary and/or the hypothalmic response by the animal (Mason, 2000). Therefore, calves were habituated to both the restraining and sampling environment, and to handling such that the hormonal response to handling would be minimized, as would the resulting impact on measures of immune function. This approach is supported by the work of Lensink et al. (2000) and Bovin et al. (2000), who indicated that animals have the ability to adapt to human interaction. The behavioral response of each animal during the sampling procedure was evaluated at three distinct periods (restraint, clamping, and venipuncture). The animal behavioral score did not change with time, which suggests that calves had habituated to the environment and handlers before the start of the study. Also, the lack of any sex effects in the light of findings from Lanier et al. (2000), who reported that heifers were found to be more excitable than steers in an open mart environment, would support habituation. Although there were significant linear and quadratic associations between behavior and stress hormones, the low experimental variation accounted for in each of these relationships was not biologically relevant. This would suggest that the physiological and immunological responses measured in this study were dominated by the imposed treatments rather than by animal handling.
Bonding behavior between dam and calf, and between calves within social groups has been established through behavioral observations. The stability of maternal (Veissier et al., 1990a) and social counterpart (Veissier and Le Neindre, 1989) relationships is important for young calves. Abrupt weaning not only disrupts the maternal bond between the calf and its dam, but also the social bond between the animal and their familiar social group. In this study, the disruption of the established social group, an inherent aspect of weaning, increased the plasma cortisol concentration for all calves up to 168 h after weaning. Though transient diurnal alterations in cortisol concentration occur, more sustained increases are associated with situations of stress. The authors are not aware of any study reporting cortisol concentrations over a 7-d duration after psychological stress. Minton and Blecha (1990) reported that although plasma cortisol concentration increased within 15 min of the restraint and isolation of sheep for 6 h (up to 40 ng/mL), plasma concentrations remained elevated (18 ng/mL) when compared with controls (<10 ng/mL) for up to 24 h after treatment, whereas Minton et al. (1992) reported that when a psychological stress was repeated over 3 d, an attenuation of the cortisol response occurred. In the current study, plasma concentrations remained elevated for up to 168 h after weaning when compared with -168 h, though noticeably declining. Plasma cortisol concentrations were not affected by weaning or calf sex in this study, which was supported by Lefcourt and Elasser (1995). Due to the periodic sampling procedures used in the current study to facilitate minimal animal handling, it cannot be ascertained if there was also a peak in plasma cortisol concentration. In other models, peak cortisol concentrations were recorded at 15 min after regrouping, relocation (Sevi et al., 2001), or branding (Lay et al., 1992) and 43 min after castration (Earley and Crowe, 2002).
Baseline concentrations of noradrenaline are consistently higher than that of adrenaline in bovine studies and a complementary increase in adrenaline and noradrenaline was reported by Lefcourt and Elasser (1995). These authors reported that the temporary separation of a 4- to 6-mo-old calf from its mother increased both the adrenaline (maximum of 0.13 ng/mL) and noradrenaline (maximum of 0.08 ng/mL) response when separated for 24 h, but not for 45 min, with a subsequent decrease on reunification. The experimental procedure involved the temporary restraint of the calf in isolation, followed by physical but not audiovisual separation for 24 h. In the present study, abrupt weaning did not elevate the plasma adrenaline concentration of calves but did increase the noradrenaline response when compared with control animals. Discrepancies between the findings of Lefcourt and Elasser (1995) and the present study may therefore be attributed to differences in age at separation, breed, or animal management in particular since the animals in this study were subjected to continued rather than intermittent separation from the dam. Koob (1999) suggests an associative interaction between cortisol-releasing factor and noradrenaline release in the locus coeruleus of the brain in response to stress, where their interaction is important in mediating behavioral responses to the stressor. In response to this activation, noradrenaline can be released to the peripheral pool from storage vesicles in the sympathetic nervous system, where its concentration is estimated to be 90% greater than that of adrenaline (Flatmark, 2000). In light of this, the findings of this study suggests that weaned bull calves experience greater difficulty in dealing with abrupt weaning than females since noradrenaline homeostasis had not re-established at the last sampling time that occurred d 7 after weaning.
Previous studies have examined phenotypic alterations of blood leukocytes as potential biological indicators of physiological stress and disease susceptibility in animals. Anderson et al. (1999) reported an increase the N:L ratio when animals were challenged with dexamethasone. In this study, the glucocorticoid, rather than the catecholamine response, may be the contributing factor to the alteration of the N:L ratio since noradrenaline concentrations remained high for the bulls, whereas the N:L ratio returned to values similar to -168 h by 48 h. Church and Hudson (1999) reported an increase in the N:L ratio up to d 14 after dam removal for Cervus elaphus calves species. These calves were not habituated to handling and may have reacted to the collection and sampling procedures at each sampling interval (d 1, 7, and 14) with attenuation of the response occurring at the later period (d 28). This in turn may have extended the measured increase in the N:L ratio. In this study, fluctuations in the N:L ratio did not affect the Con-A response and were not solely responsible for attenuation of the KLH response, which remained depressed despite the normalization of N:L by 48 h.
The production of interferon- is associated with subsets within the CD4 T-lymphocyte family (Wood and Seow, 1996), where the production of the cytokine is stimulated by mitogen/antigen challenge. The KLH antigen is a nonspecific immune response modifier, which can induce both a cell-mediated and a humoral response (Harris and Markl, 1999), whereas Con-A is a specific T-cell mitogen. The interferon- response for antigen and mitogen challenge was attenuated by social group disruption and by weaning for the KLH response only. Though the cortisol concentrations recorded in the present study were within the diurnal variations in peripheral cortisol reported by Lefcourt et al. (1993; i.e., 1 to 17 ng/mL), the increase from 7.4 to 14.2 ng/mL recorded during group disruption was associated with this decrease in cell-mediated immune function. The continued elevation of the cortisol response was also associated with the continued attenuation of both the Con-A and KLH response. It is possible that an alteration in the lymphocyte concentration and, conceivably, subpopulation profiles, was associated with the glucocorticoid response at group disruption (Anderson et al., 1999), although the influence of glucocorticoids on cell-mediated immunity per se has been questioned (Fisher et al., 1997b; Anderson et al., 1999). It has been proposed that catecholamine production can influence immune function both at the tissue and cellular level through innervation and receptors, respectively (Minton, 1994; Qui et al., 1996; Ansted-Michael, 1998). Both noradrenaline plasma concentration and interferon- production in response to KLH were influenced by weaning. However, the lack of a significant sex effect on the KLH response suggests that the measured increase in plasma noradrenaline was not associated with this measure of immune attenuation. Because KLH is a nonspecific antigen, its peripheral effects may be associated with B-cell function. However, previous studies have shown no effect of chronic stress (Fisher et al., 1997a) or weaning stress (MacKenzie et al., 1997) on humoral immunity post-KLH challenge.
Bacterial infections and physical traumata can lead to a drastic change in the synthesis of acute-phase proteins in the liver. Monitoring the circulating concentration of acute-phase proteins is important in animal health evaluation, although it is recognized that there are substantial differences between species in the physiological acute-phase protein response following stimulation (Conner et al., 1988a,b; Eckersall et al., 1996). Haptoglobin is a major acute-phase protein in cattle for which plasma concentrations can change from negligible circulating concentrations in healthy animals to increases of 100-fold upon stimulation/infection (Wittum et al., 1996; Fisher et al., 1997b; Earley and Crowe, 2002). This measure, along with fibrinogen, was therefore used in this study to determine the health of each calf during the after weaning observation period. The effect of weaning was associated with alterations in plasma fibrinogen but not haptoglobin concentration. Weaned bulls had higher plasma fibrinogen concentration when compared with all other groups, but like other groups, the plasma concentrations had returned to or were less than preweaning values by 168 h. The measured range of fibrinogen was higher than that measured in animals dealing with long-term physical stress (Hickey et al., 2003) but did not differ greatly from ranges reported by Earley and Crowe (2002) in unstressed Friesian calves. Therefore, there were no subclinical signs of poor health up to d 7 postweaning in an outdoor environment.
In this study, physiological alterations in cortisol and noradrenaline concentration and attenuation of immune function indicated that abruptly weaned suckler calves are sensitive to the social stress associated with group disruption and weaning. It may be concluded from this study that the in vitro Con-A response was sensitive to social disruption and mirrored that of cortisol, whereas the in vitro KLH response was sensitive to weaning stress that also influenced the peripheral noradrenaline concentration.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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Footnotes |
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2 Correspondence—phone +353-46-26700; fax: +353-46-26154; E-mail: mchickey{at}grange.teagasc.ie.
Received for publication December 16, 2002. Accepted for publication June 17, 2003.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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interferon. Res. Vet. Sci. 49:46–49.[Medline]
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