In the present study, the series of transport journeys (6 h to 24 h) by roads showed that transportation of Charolais beef bulls affected live weight, haematological and physiological measurements of metabolism and inflammation. The biological measures which were most sensitive to the stress of transport, on journeys of 6 h to 24 h duration, were total protein, urea, ßHB, glucose, NEFA, the acute phase protein (haptoglobin) and haematological variables (including WBC number, neutrophil and lymphocyte %). This was not unexpected as we reported similar responses in young bulls that were subjected to 8 h transport . However, it also flags a cautionary note when drawing conclusions based on a single cohort of animals that to increase the probability of statistically significant results when measuring other variables (for example RBC number, albumin, globulin, and haemoglobin and creatine kinase activity) it may be necessary to increase the group size per treatment.
The present experiment was designed to minimize any possible bias that would affect the study, however, we accept that it is not possible to exclude all bias since all studies are affected by some degree of bias. In the present study, the bulls were habituated to housing for 100 days pre-transport and were fed the same diet. All of the bulls were naïve to transport and each journey was carried out singly over a 6-week period. For each journey, 12 bulls were assigned to two pens on the transporter with 6 animals per pen and each journey was made by the same driver using similar road conditions. The bulls were blood sampled by the same person and the same chute was used at each experimental blood collection time point. In the statistical analysis of the data, animal was the experimental or measurement unit.
The changes in live weight post-transport were transitory and may be attributed to a loss of gut-fill over the journeys and possibly due to mild dehydration, urination and fasting during the longer journeys [15–17, 20, 21]. The loss in live weight in control animals in the present study may be attributed to the change in diet, as silage and concentrates were withdrawn and animals had access to hay and water only for 24 h corresponding to the longest duration of transport. Rectal temperature was not changed during each of the respective transport journeys indicating that there was no clinical infection induced by transport and no evidence of clinical disease. Although rectal temperature was not changed, it is a well known indicator of an inflammatory response to infection in newly arrived feedlot calves . The lack of an effect of transport on rectal body temperature may be related to the ambient temperature since animals would not have been exposed to extreme range of temperatures during the present series of journeys.
The development of electrolyte and acid-base imbalances has been reported in extended transport journeys where fasting has exceeded 2 days or more . In the present study, transportation had transient effects on metabolism as demonstrated by significant changes in the plasma concentrations of total protein, urea, βHB, NEFA and glucose. Total protein concentrations increased with journey duration in transported animals, however, by 12 h post-transport concentrations had returned to pre-transport baseline levels. Transport stress has been reported to cause dehydration and may manifest itself as a hyperproteinemia . The changes in protein concentrations reported in the present study post-transport are more likely the result of metabolic compensation for a mild metabolic acidosis secondary to water loss and feed deprivation during transport. These findings suggest that pre-transport mixing and transportation alters protein metabolism. Metabolic variables of protein, energy, and mineral metabolism in cattle as well as rumen function have been examined following transportation. Changes in circulating total protein, albumin, and urea, have been reported to increase following transportation [3, 24]. Changes in energy metabolism as evidenced by an increases in blood glucose [1, 16, 17], lactate dehydrogenase, glutamic pyruvic transaminase, and glutamic oxalacetic transaminase , decreases in βHB , increased haematocrit % and plasma corticosteroid concentration  have been reported. When the body prepares to react to a potentially stressful situation an increase in energy metabolism may be precipitated .
Increases in plasma glucose concentrations are mainly due to glycogenolysis associated with the increase in circulating catecholamines and glucocorticoids which are released during the stress of transport . Glucose levels returned to baseline in all treatments compared with baseline within 4 h of transport. Urea, NEFA and βHB concentrations were elevated in control and all transported animals and concentration remained greater than baseline for animals transported on journey durations ranging from 0 h to 24 h. Urea concentrations had returned to pre-transport baseline values by 24 h post-transport in all animals.
Physiological processes, such as the sleep-wake cycles, locomotor activity, body temperature, hormone secretion, and metabolism, are under the control of circadian clocks and are influenced by stress. Circadian control of glucose metabolism was recognized from early studies demonstrating variation in glucose tolerance and insulin action across the day [28, 29]. Increased energy metabolism is a hallmark of the stress response as the body prepares to react to a potentially stressful situation. We have previously reported increases in several of these protein metabolites in response to transportation [3, 4, 17]. These differences may be due to a number of factors including the duration of the journey and that animals did not have access to feed during transportation. Additionally, increased circulating CK is an indication of muscular activity and or/bruising and is often measured in transported cattle as a measure of bruising . Creatine and phosphocreatine are major intracellular solutes in muscle cells. Increases in plasma CK activity after different transport journeys have been described by different authors [16, 19, 30]. A direct relationship between the duration of transport and the rise in the activity of the enzyme has been reported . Fasting has also been reported to increase the activity of the enzyme, and the rise could be masked by the high values obtained after transport . In the present study, CK activities returned to pre-treatment baseline values within 12 h for all transported animals. Interestingly, control animals in the present study had elevated CK activity while the magnitude of the changes were small the return to baseline was rapid. Circulating creatine kinase activity is often measured in transported cattle as a measure of bruising , indicating that the bulls in the current study may have experienced some physical stress.
Changes in acute phase protein concentrations during transportation have been reported but the results are variable. Haptoglobin, an acute phase protein, is released by hepatocytes and mediate the inflammatory response to injury, trauma, or infection . The presence of acute phase proteins in the circulation may be an excellent biomarker of inflammation as they are readily measurable in serum or plasma and may even discriminate between acute and chronic inflammation in cattle . Acute phase proteins are present in very low concentrations in plasma and increase in concentration following tissue injury and inflammation [35, 36]. In the present study haptoglobin concentrations were increased relative to the baseline in control and transported animals up to 24 h post-transport, with the exception of the animals transported for 6 h. Serum haptoglobin was reported to be elevated in calves transported for 2 days and levels were negatively correlated with lymphocyte function . In a separate study transporting bulls at different stocking densities, plasma haptoglobin concentrations were unchanged, while plasma fibrinogen levels were reduced [3, 4]. Fibrinogen, ceruloplasmin, serum amyloid-A, and α-acid glycoprotein were assayed in the plasma of transported and commingled calves and found to be increased post-transportation; however, haptoglobin concentrations were greater in non-transported versus transported calves .
Alterations in immunity are of great importance following transportation stress as these alterations are thought to be associated with increased incidence and severity of respiratory diseases. Many measures of immunological changes relate to immune cell numbers in the blood. Similar to the findings of the present study, most studies observe a leukocytosis that is marked by neutrophilia, which may occur in conjunction with a decrease in the number of other cells (lymphopaenia, eosinopaenia) [9, 10, 38]. Changes in the haematological responses of cattle to transport have been reported with increases in RBC number, haematocrit percentage and haemoglobin concentration following transportation of steers [17, 39]. In the present study, all transported animals had greater neutrophil percentage and lower lymphocyte percentage post-transport. Haemoglobin concentrations and RBC numbers were within normal blood referenced ranges [40–43]. The neutrophilia observed in control animals is most likely due to the effect of stress related to the mixing and the handling procedures. Blood lymphocytes contain concentrations of glucocorticoid and adrenergic receptors , which are down-regulated in response to stress  and suggests that alterations in the blood cell composition of leukocytes may have an important role in the responsiveness of the immune system when stress challenged. There was no major change in haematocrit % compared with baseline in animals transported for 6 h to 24 h. Animals in the present study had ad libitum access to water on the transporter and they received the last feeding immediately before loading and these factors may have prevented the animals from showing signs of dehydration. Elevated haematocrit % has been reported following transport in association with greater erythrocyte counts in the circulation [17, 25, 46, 47] and a significant increase in haematocrit values indicates mainly dehydration.
Measures of immunological changes relate to immune cell numbers in the blood and immune cell function. A number of studies have reported leukocytosis that is marked by neutrophilia, and which may be present with a decrease in the number of other cells (lymphopaenia, eosinopaenia) [8, 10, 17]. Bovine alveolar macrophages, isolated from bronchoalveolar lavage (BAL) fluid, have a reduced respiratory burst function after 4 h of transportation . The respiratory burst function is necessary to produce reactive oxygen species that are toxic to phagocytosed pathogens, and these results may represent impaired lung defence. In contrast, enhanced respiratory burst activity has been found in neutrophils of transported calves . Apoptosis of neutrophils in combination with increased migratory capacity in dairy cows have been reported after 4 h of transportation . The normal referenced ranges for differential counts, neutrophils are in the range 15-45 [40, 50]. Within the range of transport times analysed, there were no significant changes in MCV and MCHC values. In the present study, the changes in the composition of the blood cell variables reflect the physiological response of the bulls to the stress of mixing, fasting and/or transportation.
In the current study, several measures of physiological, metabolic and haematological variables were investigated in the plasma of bulls subjected to transport journeys ranging from 6 to 24 h durations. It is evident that transportation of bulls has effects on biomarkers of metabolism as demonstrated by significant changes in the plasma concentrations of protein, glucose and NEFA. Additionally, circulating creatine kinase activity is a useful measure which is often monitored in transported cattle as a measure of bruising. Changes in CK activity indicate that the bulls in the current study may have experienced some muscle damage and physical stress, particularly after the longer duration journeys. The acute phase protein, haptoglobin, is a useful biomarker of inflammation and together with changes in haematological cellular variables would suggest a pro-inflammatory state during transportation stress. The pronounced neutrophilia and lymphopenia following transportation observed in this study are in agreement with previously reported findings following a variety of stressors, including transport stress [8, 10, 17]. Taken together, these results indicate that transportation stress alters physiological measures of metabolism and haematology. Thus, a profile or pattern of multiple physiological, metabolic and haematological variables may provide the most effective marker of altered homeostasis to allow an assessment of an animal's response to transport.