*2.5. Data Analysis*

For descriptive analyses, variables are described by mean and standard error (SE). To assess the variation in the physiological variables measured in calves, after transport and fasting times, linear mixed model (LMM) analyses were performed. The model used was:

$$
\gamma i \dot{\jmath} = \mu + \beta \dot{\imath} + \varepsilon \dot{\jmath},
$$

where *γ* corresponds to a dependent variable measured; *μ* is time (three evaluations for each calf), considered as fixed effect; *β* represents the calf, included as random effect; and *ε* is the error not explained by the model. Data were analyzed using the lme4 statistical package and multiple comparisons were explored using a Tukey's adjustment included in the lsmeans function in R software version 3.2.2 [26].

#### **3. Results and Discussion**

A significant increase of 0.55 ◦C in TT was found between the temperatures before loading and after unloading (Table 2). However, a 1.1 ◦C daily variation can occur even under moderate thermal conditions in beef cattle [27]. The body temperature represents an integrated response to both internal and external factors such as climatic conditions, heat production, and the heat losses that an animal experiences [25]. Moreover, core body temperature can also be dramatically increased with muscular activity, and by nervous and hormonal factors (such as sympathetic nervous activity), catecholamines, and thyroid hormones [28]. The relationship between body temperature and the level of stress, either physical and/or psychological, at several stages of animal handling has also been shown [29,30].

**Table 2.** Mean (±SE) of all the variables measured before loading, after unloading, and after the 24-h fasting period.


Different letters (a, b, c) represent statistical differences (*p* < 0.05) between sampling times. \* Reference values: for cortisol according to DCPAH, Michigan State University [31]; other blood variables according to Wittwer [32].

MET also increased significantly after transport and then returned to initial values after the 24 h fasting period. The rise in temperature after transport was 1.6 ◦C (Table 2). An increase in MET has previously been reported following castration in calves [33], after jumping competitions in horses [21], and during the veterinary clinic examination phase, compared with both pre- and post-examination phases, in dogs [23]. Nevertheless, some authors described a rapid drop in eye temperature during the minutes following a stressful and/or painful procedure, for example, calves disbudded without local anesthetic [18] and heifers exposed to fear related handling procedures such as being hit with a plastic tube, sudden flag movements, shouting, and being shocked with an electric prod [19]. The rapid drop in eye temperature in cattle during the first seconds after the stimuli has been explained as a sympathetic response, which is part of the autonomic nervous system reaction [17,18]. If the stressor persists for a longer time, the HPA axis produces a cortisol response that could be maintained from minutes to hours. In addition, the nature of the stimulus, or the level of fear and/or pain, that the animals experience may affect the duration of the drop in eye temperature [19]. The results of the present study sugges<sup>t</sup> that transportation was a strong enough stimulus to cause a later increase in eye temperature in calves, as described by Stewart et al. [17,18]. However, no relationship between the increase in eye temperature and HPA axis activation has been shown [33,34]. The increases in MET and TT could reflect a body temperature increase due to stress and physical exercise during transport. It has been proposed that MET, measured with infrared thermography, may be associated with body core temperature [13,35]. In addition, although thermography is described as a non-invasive measurement, in the case of the MET measurements the animals had to be immobilized in a chute, which was undoubtedly a stressful procedure by itself. Otherwise, if there had been a drop in the eye temperature of the calves due to the handling procedures, it would probably have happened during loading, after the current measurement was made. MET in the calves decreased after unloading, returning to initial levels after the fasting period (Table 2). Cattle are a homeothermic species that need to maintain a high metabolic rate to generate heat, and so they require a high level of feed intake [36].

Regarding the blood variables; cortisol and haptoglobin did not change significantly after 3 h of transport or after 24 h of fasting (Table 2). When animals are transported, the effects of this process

can be assessed by monitoring glucocorticoid concentrations, but the limitations described for this stress indicator, such as the diurnal fluctuation in plasma cortisol concentration and the effects of the sampling procedure itself, may prevent a correct evaluation [10,37]. However, all mean levels were higher than reference values for cortisol after unloading and after the 24 h fasting period, probably due to HPA axis stimulation during sampling. Animal scientists have hypothesized that glucocorticoids may have an impact on the production of acute phase proteins such as haptoglobin [9]. Literature suggests that haptoglobin is less sensitive and slower reacting than other acute phase proteins, like serum amyloid A (SAA) [38,39]. Werner et al. [8] showed a significant increase in haptoglobin levels 24 h after unloading beef calves transported for a duration of 63 h.

Creatine kinase increased significantly after transport and returned to initial values during fasting in pens (Table 2). CK appears in the circulating plasma because of tissue damage (e.g., bruising), and also when there is vigorous exercise, and is relatively organ specific (i.e., muscle) [37]. In this study, calves were transported at a low stocking density which probably meant that an additional physical effort from calves was required in order to maintain balance during transport. Additionally, this same factor could have influenced bruising when the truck was in motion. The significant decrease in CK after the 24 h fasting period is likely associated with the elimination of the physical stressor, and a rise in excretion, as observed previously in steers after transport for slaughter [40]. CK values were higher than reference values (Table 2) at all sampling times, possibly because of previous handling, such as immobilization for sampling.

After strenuous exercise, ketone oxidation by muscles is reduced and this leads to an increase in plasma levels of betahydroxibutyrate [9]. Additionally, the high weight loss observed was consistent with the significantly higher concentration of β-HB found after the 24 h fasting period (with transport included). This indicated that, even with the short time off feed, the calves had to make use of their body reserves, even though mean β-HB values were within normal ranges for the species for all sample times (Table 2).

Blood glucose levels reflect the nutritional status of an animal, and during food deprivation the glucose blood levels decrease [41]. The results here show that transport may have had a greater impact on plasma glucose than food deprivation (Table 2). There were significant differences in glucose values between sampling times (Table 2). Glycemia increased over the reference values after transport and then returned to basal levels, but remained higher than before loading. The maintenance of glycemia may have been associated with the use of liver glycogen, as practically all glucose available to ruminants comes from the glycogenolysis process [42,43]. Increases in glucose levels after transportation and fasting have been previously reported in calves after 8 h of fasting and 8 h of transport [44]. The explanation for this increase is related to the primary response to stress [9], so glucose levels can be used as an indirect indicator of stress [41].

The calves lost 10 kg live weight (LW). Fifty percent of the total LW loss occurred between sampling before loading and sampling after unloading (within approximately 5 h), whereas the other 50% occurred while the calves were fasting in the pen (Table 2). Generally, weight loss during transport is accelerated compared with deprivation of food and water without transport [9]. The calves lost 6.8% of LW in 24 h, which is consistent with Knowles et al. [9] who described an initial loss of LW in ruminants, predominantly due to loss of gu<sup>t</sup> fill, of approximately 7% during the first 18 to 24 h without food and water. Metabolic changes due to stress are greatest if increased heat production and decreased feed intake occur concomitantly. In this situation, the required calories come from the catabolism of tissues [45]. Body weight loss in animals is probably the most significant economic effect of transport [8].
