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ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION |
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* School of Agriculture, Food Science and Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland;
and
Teagasc, Grange Beef Research Centre, Dunsany, County Meath, Ireland; and
Department of Animal Science, Michigan State University, East Lansing 48824
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: beef cattle • biomarker • disease • physiology • plasma • transportation stress
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Murata et al., 1987; Arthington et al., 2003), often resulting in bovine respiratory disease (BRD) and generating concerns of an economic as well as a welfare-related nature. Bovine respiratory disease consists of a complex interaction of genetic and environmental factors and comprises a devastating threat to young beef cattle (Lekeux, 1995). The identification of animals with BRD in the feedlot is usually performed by visual appraisal, which may be subjective. Easily measurable but accurate variables in the early diagnosis of BRD remain necessary (Duff and Galyean, 2007). In recent years, there has been a push for the discovery of novel biomarkers to aid in the early detection, diagnosis, and therapy of complex human diseases (Collins et al., 2006; Ginsburg and Haga, 2006). It is logical to hypothesize that the same approaches could be applied in livestock species for the early detection of production diseases. The objective of the current study was to characterize a profile of physiological changes in the circulation of transported beef cattle that may act as future biomarkers of transportation stress associated with decreased production and increased disease susceptibility. The hypothesis was that transportation stress would alter numerous measures of metabolism, inflammation, and steroid hormones in the circulation and that the breed of the bulls would contribute to variability in their response to the stressor. To test this hypothesis, plasma concentrations of albumin, urea, globulin, total protein, creatine kinase, β-hydroxybutyrate (BHB), haptoglobin, fibrinogen, cortisol, dehydroepiandrosterone (DHEA), calculated cortisol:DHEA ratios, testosterone, and progesterone and also circulating total leukocyte counts in transported young bulls were measured. In addition, BW and rectal temperature were recorded.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Animals and Transportation
Animals used in this study were 36 Aberdeen Angus (n = 12), Friesian (n = 12), and Belgian Blue x Friesian bulls (n = 12), 233 ± 3.0 kg in BW and 282 ± 4 d of age at the time of transportation. Bulls had been weaned at 10 d of age in the spring of 2004 and were purchased by Teagasc, Grange Beef Research Centre (County Meath, Ireland) at that time. They were housed, fed, and cared for according to accepted management practices at the Grange Beef Research Centre until use in this study in the fall of 2004. All bulls received a Bovipast RSP vaccination (Intervet UK Ltd, Milton Keynes, UK) against bovine respiratory syncytial virus, parainfluenza type 3 virus, and Mannheimia (Pasteurella) hemolytica bacteria (serotype A1) at 3 and 7 wk of age. Bulls had ad libitum access to water and grass silage (in vitro DM digestibility = 872 g/kg) supplemented with 1.5 kg of barley-soybean concentrate (CP = 104.6 g/kg of DM) per animal daily.
The transportation phase of the study lasted for 6 wk; groups of 6 bulls were transported each week. Bulls of all 3 breeds to be transported each week were penned together. They were transported at a stocking density of 0.85 m2 for 9 h under a variety of road conditions, speeds, and traffic. In accordance with European Union regulation, a 45-min rest stop was observed after 4.5 h of transportation, during which the bulls remained on the truck. Bulls were unloaded and returned to their original group pens at the end of the 9-h journey.
Blood Collection
Blood samples for the aspiration of plasma were collected at the following time points relative to commencement of transportation at 0 h: –24, 0, 4.5, 9.75, 14.25, 24, and 48 h. Blood sampling at –24, 0, 14.25, 24, and 48 h occurred in a handling chute in the cattle holding yard, whereas sampling at 4.5 and 9.75 h occurred in a handling chute on the truck. Blood (10 mL) was collected into evacuated tubes coated with the anticoagulant lithium-heparin (Greiner VACUETTE, Cruinn Diagnostics, Dublin, Ireland) for subsequent harvesting of plasma for the assay of physiological variables. An additional 10 mL of blood was collected into evacuated tubes coated with K3-EDTA (Greiner VA-CUETTE, Cruinn Diagnostics) for determination of total leukocyte numbers. Blood was stored at room temperature and was centrifuged within 1 h of blood collection.
Cell Counts
Total circulating leukocyte counts were determined using an electronic particle hematology analyzer on whole blood samples (Celltac MEK-610K, Nihon Kohden, Tokyo, Japan).
Physiological Variables
Plasma was harvested from anti-coagulated blood after centrifugation at 1,600 x g at 4°C for 15 min and stored at –80°C until the assays were performed. Albumin, urea, globulin, total protein, BHB, and creatine kinase were measured on an automatic analyzer (AU 400, Olympus, Tokyo, Japan) using the reagents supplied by Olympus. Plasma haptoglobin concentrations were measured using an assay kit (Tridelta Development Ltd., Wicklow, Ireland) on an automated analyzer (spACE, Alfa Wassermann Inc., West Caldwell, NJ) according to the procedure of the manufacturer. Plasma fibrinogen concentrations were measured according to a previously described method (Becker et al., 1984) on an automated analyzer (spACE, Alfa Wassermann Inc.).
Plasma steroids were also assayed. Cortisol was assayed using a commercially available RIA kit (Corticote, ICN Pharmaceuticals, Orangeburg, NY) adapted and validated for bovine plasma as described previously (Buckham Sporer et al., 2007). The mean intraassay CV (n = 6) was 6.5%, and the interassay CV (n = 2) was 6.5%. The steroid DHEA was assayed using a Correlate-EIA kit (Assay Designs, Ann Arbor, MI) according to the instructions of the manufacturer. All plasma samples were diluted in sterile MilliQ water using a dilution factor of 5. The sensitivity of the DHEA assay was 2.9 pg/mL. The mean intraassay CV (n = 2) was 3.4%, and the interassay CV (n = 8) was 3.3%. Ratios of cortisol:DHEA were also computed.
Plasma progesterone was assayed using time-resolved fluoroimmunoassay (Wallac AutoDELFIA, Turku, Finland) using commercially available progesterone (Sigma, Tallaght, Ireland) in the kit assay buffer as standards. The sensitivity of the assay was 0.01 ng/ mL. The mean intraassay CV (n = 6) was 10.6%, and the interassay CV (n = 3) was 10.7%. Testosterone was assayed using a direct RIA, as described previously (Ronayne et al., 1993). The sensitivity of the assay was 0.1 ng/mL. The mean intraassay CV (n = 6) was 13.6%, and the interassay CV (n = 3) was 13.3%.
BW and Rectal Temperature
Live BW and rectal temperature were measured at –24, 9.75, and 48 h relative to the initiation of transportation. Rectal temperatures were recorded using a digital thermometer (Jørgen Kruuse, Marslev, Denmark).
Statistical Analysis
Data were analyzed using PROC MIXED (SAS Inst. Inc., Cary, NC) using bull as the experimental unit, the fixed effects of time relative to the initiation of transportation and breed, the random effects of bull and bull within group, and the covariates BW and age. Any data sets that failed to meet parametric assumptions were log-transformed before statistical analyses. Data are presented in the results section as raw means ± SEM, and the associated P-values presented were derived from the statistical analysis of appropriately transformed data using the model described above. The overall effects of time relative to transportation or breed were considered significant when P 
0.05. In addition, differences between times and breeds were determined using a Tukey-Kramer adjustment for multiple comparisons and considered significant when P 0.05.
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RESULTS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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In the current study, transportation had effects on metabolism as demonstrated by changes (P < 0.05) in plasma concentrations of albumin, globulin, urea, total protein, and creatine kinase relative to pretransportation (–24 h) values (Figure 1, Table 1). Albumin concentrations were reduced by 7% at 24 h (26.52 ± 0.33 g/L) compared with –24 h (28.26 ± 0.25 g/L; Figure 1A). Plasma globulin was decreased by 4.5 h and remained depressed by approximately 13% (41.36 ± 0.66 g/L vs. 47.04 ± 0.92 g/L at –24 h) throughout the time of blood collection (Figure 1B). Urea concentrations were not affected at 4.5 h but were decreased at 48 h (3.26 ± 0.16 nmol/L), 10% of the –24-h concentration (3.60 ± 0.18 nmol/L; Figure 1C). In concurrence, total plasma protein concentrations were decreased by 11% at 24 h (67.88 ± 0.61 g/L compared with 75.30 ± 0.92 g/L at –24 h; Figure 1D). In addition, a 39% decrease in plasma creatine kinase was observed at 9.75 h (276.52 ± 31.02 U/L), followed by an increase of 221% at 24 h (1,229.72 ± 208.15 U/L compared with 554.63 ± 68.06 U/L at –24 h; Figure 1E). No changes in the concentration of plasma BHB, an energy substrate, were observed (Figure 1F).
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The current study also demonstrated a decrease in the plasma concentrations of the acute phase proteins haptoglobin (P < 0.001) and fibrinogen (P < 0.001) with the onset of transportation stress. Haptoglobin reached its lowest point at 4.5 h (39.023 ± 1.32 mg/dL) at a 53% reduction from pretransportation values (82.06 ± 1.73 mg/dL) and remained depressed through 48 h (Figure 2A). Plasma fibrinogen was decreased by 44% at 14.25 h (313.11 ± 5.40 mg/dL) compared with 551.53 ± 9.81 mg/dL at –24 h, and concentrations remained depressed at all time points after –24 h (Figure 2B). The effects of time relative to transportation and breed are represented in Table 1
for these variables. In contrast with the decrease in these possible markers of inflammation, there was an increase in circulating total leukocyte counts with transportation (P = 0.002), peaking at 9.75 h by over 21% (1.52 ± 0.15 x 103 cells/µL of whole blood) the –24-h value (1.25 ± 0.22 x 103 cells/µL of whole blood; Figure 3).
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Plasma cortisol was greatly elevated with the onset of transportation in the current study when compared with –24 h (Figure 4A), a 321% increase at 4.5 h (42.54 ± 2.10 ng/mL compared with 13.22 ± 1.30 ng/mL at –24 h). Cortisol concentrations reached nadir at 14.25 h at 6.99 ± 1.34 ng/mL before returning to basal concentrations at 24 and 48 h posttransportation. The steroid hormone DHEA was decreased by 30% at 4.5 h (1.17 ± 0.124 ng/mL vs. 1.52 ± 0.15 ng/mL at –24 h; Figure 4B), when cortisol reached its peak. The cortisol:DHEA ratio followed the curve of plasma cortisol very closely, elevated by 528% at 4.5 h (74.62 ± 16.06 compared with 14.12 ± 2.39 at –24 h; Figure 4C).
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). In contrast, plasma progesterone, although present at low concentrations in bulls, was elevated at 4.5 h (0.43 ± 0.047 ng/mL), increased by 215% its –24-h concentration (0.19 ± 0.02 ng/mL; Figure 4E). Profiles of all steroidal changes are shown in Figure 4, and P-values are shown in Table 1. BW and Rectal Temperature
Body weight and rectal temperature were both monitored and found to be altered by transportation stress in the current study (Table 2). Body weight was decreased at 9.75 h (209.42 ± 2.25 kg) by 10% (230.78 ± 1.51 kg at –24 h) and remained decreased at 48 h after transportation (224.06 ± 1.60 kg). Rectal temperature was decreased by 48 h (38.55 ± 0.04°C; 38.73 ± 0.06 at –24 h) but was still well below a febrile state that would be indicative of an infection (>40°C; Duff and Galyean, 2007).
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The breed of cattle transported appeared to have an effect (P < 0.05) on plasma albumin, globulin, total protein, haptoglobin, cortisol, DHEA, cortisol:DHEA ratio, and progesterone, with tendencies toward significance (P < 0.10) on BHB, fibrinogen, and total leukocyte count. These results are summarized in Table 1. However, there did not appear to be any consistent trend as far as one breed responding differently to the stress of transportation than other breeds across all variables. Two profiles for variables by breed, total leukocyte count, and plasma cortisol are shown in Figure 5. The total leukocyte counts of all breeds responded similarly to stress (Figure 5A). In contrast, baseline plasma cortisol concentrations were lower (P < 0.001) in Aberdeen Angus bulls than in Friesian or Belgian Blue x Friesian bulls and, although acutely increased, did not peak as highly as the other 2 breeds (Figure 5B).
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DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Ginsburg and Haga, 2006). In addition, it is recognized that a profile or pattern of multiple parameters may provide the most effective marker of the risk of disease (Collins et al., 2006; Ginsburg and Haga, 2006). In the current study, multiple measures of metabolism, inflammation, and steroid hormones were investigated in the plasma of young bulls subjected to transportation stress.
An entire discipline referred to as metabolomics has emerged in recent years to describe the study of all of the cellular processes that constitute metabolism. Metabolites may represent effective biomarkers and may be useful in the prognosis and diagnosis of pathological conditions and in drug development (Ginsburg and Haga, 2006). In the current study, it is evident that transportation stress has effects on metabolism as demonstrated by significant changes in the plasma concentrations of albumin, globulin, urea, total protein, and creatine kinase. Taken together, these results indicate that transportation stress alters protein metabolism. Interestingly, others have observed increases in several of these protein metabolites in response to transportation stress (Tarrant et al., 1992; Earley and O’Riordan, 2006); differences may be due to several factors, including the duration of the journey or whether or not animals were fasted before transportation in these studies. Additionally, circulating creatine kinase is often monitored in transported cattle as a measure of bruising (Tarrant, 1990), indicating that the bulls in the current study may have experienced some muscle damage and physical stress.
Acute phase proteins, such as haptoglobin and fibrinogen, are released by hepatocytes and mediate the inflammatory response to injury, trauma, or infection (Baumann and Gauldie, 1994). Their presence in the circulation may be an excellent biomarker of inflammation, because they are readily measurable in serum or plasma and may even discriminate between acute and chronic inflammation in cattle (Horadagoda et al., 1999). Serum haptoglobin concentrations may also be indicators of BRD (Godson et al., 1996). Although circulating concentrations of the acute phase proteins haptoglobin and fibrinogen were reduced posttransportation in the current study, the literature concerning the effects of transportation on acute phase protein concentrations in cattle is variable (Murata and Miyamoto, 1993; Arthington et al., 2003; Earley and O’Riordan, 2006). In contrast, the profound leukocytosis shown in the current study has been observed in other transportation studies and is usually marked by an increase in circulating neutrophils (Blecha et al., 1984; Earley et al., 2006; Buckham Sporer et al., 2007). Murata et al. (1987) have shown that this neutrophilia consists of an increase of mature neutrophils coupled with immature neutrophils just released from the bone marrow in response to the stress of transportation. More investigation into acute phase proteins as biomarkers is necessary, because transportation has been shown to both stimulate and suppress circulating concentrations (Arthington et al., 2003; Earley and O’Riordan, 2006); however, cellular variables would suggest a proinflammatory state during transportation stress.
Elevated circulating concentrations of the glucocorticoid cortisol are a hallmark of stress in livestock species (Grandin, 1997; von Borell, 2001; Mstl and Palme, 2002), especially transportation stress (Buckham Sporer et al., 2007; Gupta et al., 2007). However, DHEA is a steroid exerting antiglucocorticoid effects that has rarely been monitored in stressed animals. In recent research, the cortisol:DHEA ratio has been of importance as a marker of inflammation in elderly humans (Butcher et al., 2005) and potentially in dairy cows with lameness (Almeida et al., 2008). Antiinflammatory and immunoprotective properties of DHEA oppose the often immunosuppressive actions of glucocorticoids (Kalimi et al., 1994; Saccò et al., 2002). In addition, although glucocorticoids have historically been used as antiinflammatory therapeutics, there is ample evidence to suggest that they may also be proinflammatory in some cases (Sorrells and Sapolsky, 2007). The cortisol:DHEA ratio was also highly correlated with the expression of several inflammatory neutrophil genes in a related study (Buckham Sporer et al., 2008), indicating that these steroid hormones may support a proinflammatory state after transportation of cattle. Elevations in glucocorticoids may drive an attenuation in other circulating steroid hormones (Thibier and Rolland, 1976; Welsh et al., 1979), which is supported in the current study by changes in testosterone and progesterone concentrations. Bulls have been found to have lesser serum cortisol concentrations during transportation than steers (Tennessen et al., 1984), thus, it may be interesting to monitor these other gonadal steroid hormones in steers or in female cattle to determine if similar results are found, and these steroid hormones may be additional biomarkers of stress and disease susceptibility.
Body weight and rectal temperature are routinely monitored upon arrival at the feedlot as easily obtained measures of well-being and possible infection. The loss of BW observed in this study corroborates the results of others and is attributed to a loss of gutfill over short distances and possibly dehydration and fasting during longer transports (Knowles, 1999; Arthington et al., 2003). Although rectal temperature was not increased, it is a well known indicator of an inflammatory response to infection in newly arrived feedlot calves (Galyean et al., 1995).
Differences in the physiological changes observed here between breeds were significant but variable. Some breed differences have been observed between calves of Bos taurus breeding as opposed to those of Bos indicus breeding, which have greater baseline cortisol concentrations (Zavy et al., 1992). From the current study and other transportation studies (Phillips et al., 1987; Zavy et al., 1992), clear conclusions concerning the genetic effect of breed on response to transportation stress cannot be declared. However, this may be of interest in future research for genetic selection toward cattle that may be less susceptible to the effects of stress.
In conclusion, we have determined that transportation stress in young beef bulls alters concentrations of physiological variables of metabolism and inflammation as well as steroid hormones in the circulation that, taken together, may be effective biomarkers of stress and disease susceptibility. Effects of breed on several physiological variables were also found to be significant but with no clear trend. Early detection of susceptible animals may aid in treatment and separation from other animals before disease increases in severity and incidence. Further validation of these potential bio-markers is required before they may be utilized in accurate diagnosis, though the current study presents a profile of possibilities.
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Footnotes |
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2 Corresponding author: Bernadette.Earley{at}teagasc.ie.
Received for publication November 28, 2007. Accepted for publication February 28, 2008.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
