Reasons for performing study: Trekking is a noncompetitive sport, involving maximal skeletal muscle effort. Exercise and transport may involve significant energy expenditure and give rise to substantial stress. Few studies have examined the combined effect of exercise and additional preliminary transport on adrenocortical and haematochemical responses in horses during trekking.
Objectives: To ascertain whether exercise and additional preliminary transport before trekking would affect the circulating cortisol levels and haematochemical variables of horses during a 2 day trekking event.
Materials and methods: Twenty-nine healthy horses were used. Twenty-four horses were transported over distances of 70 km the day before trekking and 5 horses were stabled at the starting place. Blood samples were taken from horses at 16.00 h the day before trekking; and at 08.30 h and 17.30 h before and after the first day of trekking; at 08.30 h and at 13.30 h before and after the second day of trekking. Serum cortisol and haematochemical variables were determined in duplicate by using commercial test kits. One-way analysis of variance for repeated measures (RM-ANOVA) was applied to determine whether trekking and transport had any effects.
Results: Trekking significantly (P<0.01) affected total protein, albumin, urea, aspartate aminotransferase (AST), alanine aminotransferase (ALT), PCV and cortisol changes in transported horses and only urea and PCV (P<0.01) changes in untransported horses. Untransported horses showed lower basal total protein (P<0.05) and albumin (P<0.01) concentrations, higher urea concentrations (P<0.001) at the second day and lower cortisol levels after the first and the second (P<0.05) day of trekking than transported horses.
Conclusion: These data show that the preliminary transport stress induced additional significant changes of cortisol and haematochemical patterns in horses after trekking.
All methods and procedures used in this study were reviewed and approved by the Messina University Institutional Board for the Care and Use of Animals.
Twenty-nine healthy horses, aged 4–15 years, habitually used for trekking events, were studied. The horses were individually fed on 4 kg of hay ration in the morning and evening and 3 kg of bran and a commercial concentrate at 12.00 h; they also had free access to water.
Twenty-four horses (Group I: 9 Thoroughbreds, 5 females, one stallion, 3 geldings, mean ± s.d. age 10.00 ± 4.25 years; 4 Appaloosas, one female, 3 geldings, age 9.33 ± 3.79 years; 11 crossbreds, 3 females, 2 stallions, 6 geldings, age 11.22 ± 4.51 years) were transported over distances of 70 km the day before trekking and 5 horses (Group II: 5 crossbreds, one female, 4 geldings, aged 9.25 ± 4.19 years) were usually stabled at the starting place. Horses were weighed the day before the trekking and at the end of the 2 day trekking event on an electronic scale.
Blood samples were taken from the jugular vein from all horses at the starting place at 16.00 h the day before trekking (within 1 h); then at the 08.30 h and 17.30 h before and after the first day of trekking (length of route: 40 km, duration of trekking: 6 h, mean speed: 7 km/h; a pause from 14.00–16.00 h for watering the horses); at 08.30 h and 13.30 h before and after the second day of trekking (length of route: 28 km, duration of trekking: 4 h, mean speed:7 km/h; without a pause for watering). Walk was the most common pace. All samples were collected by the same operator (E.G.) under quiet conditions. Blood samples were collected in evacuated tubes1 and in K3-EDTA tubes for packed cell volume (PCV) analysis and placed on ice until processing in the laboratory. Within 1 h, blood samples were centrifuged for 15 min at 1500 g and serum harvested and stored in polystyrene tubes at -20°C until used for analyses. To determine PCV, 2 capillary tubes were filled from K3-EDTA tube and centrifuged in a microcapillary centrifuge (Model Select-a-Fuge 24)2 for 5 min; PCV was the mean of the 2 capillary tubes. Serum cortisol concentrations were analysed in duplicate using a commercial test kits and a BRIO automated analyser3. Intra- and interassay coefficients of variation (CV) were 4.6 and 6.9%, respectively. Serum variables were measured on biochemistry auto analyser (SLIM)3 using commercial test kits3. The haematochemical variables analysed were: total protein, albumin, creatinine, urea, aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Total protein and albumin were measured using the biuret and bromocresol green methods, respectively. Creatinine, urea, AST and ALT were measured at 37°C using kinetic methods.
Data are presented as mean ± s.d. Statistical analysis was carried out by one-way analysis of variance for repeated measures (RM-ANOVA) to determine whether trekking and transport had any effects. Significant differences between basal and post trekking values were established using the Student's paired t test. Significant differences between different groups were established using the Student's unpaired t test. The level of significance was set at P<0.05. All calculations were performed using the PRISM package4.
The horses averaged 400 ± 20 kg bwt the day before trekking and 380 ± 10 kg at the end of the 2 day trekking event. No significant differences were observed between decreases of bodyweight of Groups I and II.
As compared to basal values, statistical analyses showed significant differences, with higher cortisol levels in Group I after the first (P<0.05) and the second (P<0.001) days (Fig 1); higher total protein (P<0.001), albumin (P<0.01), creatinine (P<0.01), AST (P<0.01) and ALT (P<0.05) values in Group I after the first day of trekking (Table 1); higher urea levels in Group I after the first (P<0.01) day, and in Group I (P<0.01) and Group II (P<0.05) after the second day (Table 1); higher PCV values both in Group I after the first (P<0.001) and the second (P<0.001) days and in Group II after the first (P<0.05) and the second (P<0.01) days (Table 1).
Table 1. Haematochemical variables (mean ± s.d.) in transported (Group I) and untransported (Group II) horses during a 2 day trekking event
First day of trekking
Second day of trekking
vs. basal values: *P<0.05; **P<0.01; ***P<0.001. vs. Group I: •P<0.05; ••P<0.01.
As compared to Group I, Group II showed lower basal total protein (P<0.05) and albumin (P<0.01) concentrations and higher urea concentrations (P<0.001) at the second day of trekking (Table 1); and lower cortisol levels after the first and the second (P<0.05) days of trekking (Fig 1).
Analysis by RM-ANOVA showed that trekking significantly (P<0.01) affected total protein, albumin, urea, AST, ALT, PCV and cortisol changes in Group I and only on urea and PCV (P<0.01) changes in Group II.
The data obtained showed that basal cortisol concentrations are in accordance with the range observed in normal horses (Irvine and Alexander 1994). The increase in cortisol levels after trekking confirm previous data observed in horses after exercise (Ferlazzo and Fazio 1997; Ferlazzo et al. 2007, 2009) and particularly after trekking (Alberghina et al. 2001). Moreover, the highest cortisol concentrations observed the day before trekking in transported horses, with blood sampling performed between 16.00 and 17.00 h, when the cortisol concentrations in horses would be the lowest (Irvine and Alexander 1994), confirm that the transport performed before trekking induced an increase of cortisol levels (Ferlazzo et al. 1993; Fazio et al. 2008a). These data were confirmed by the lower cortisol concentrations observed the day before trekking in Group II than in Group I. Since cortisol is released from the adrenocortical gland in response to sources of stress, such as transport and exercise, data obtained suggest that circulating cortisol concentration might be useful in assessing the severity of preliminary transport stress associated with exercise (Fazio et al. 2008b). However, the small number of horses in Group II (n = 5) limits the strength of the conclusions that can be drawn from these identified differences, although some of them were statistically significant.
The significant increases of creatinine, AST and ALT values observed after the first day and of urea observed both after the first and second day of trekking in transported horses, confirm previous data observed in adult horses, with an increase of creatinine concentrations during different phases of training (Nogueira et al. 2002; Latimer et al. 2003), of plasma AST activity immediately after run on a soft sand track (Brady et al. 1978) and of urea concentrations after exercise (Aguilera-Tejero et al. 2000). The significant increase of these variables in transported horses could be related to the differentiated skeletal muscle effort experienced after transport and during trekking.
The highest post exercise increases of cortisol, total protein, albumin, creatinine, urea, AST, ALT and PCV values, observed in Group I, showed that the preliminary transport stress induced additional significant changes of adrenocortical and haematochemical responses in horses after trekking.
In addition, the compensatory time of 16 h after transport and before trekking was probably inadequate for complete functional recovery. This study provides important information on the effects of type and length of trekking and preliminary transport related to the endocrine and metabolic costs that may reduce the horse's homeostasis and performance.
Special thanks to veterinarians and riders who participated in this field study.
Conflicts of interest
The authors have declared no potential conflicts.
1 Terumo, Leuven, Belgium.
2 Bio-Dynamics, Indianapolis, Indiana, USA.
3 RADIM/SEAC Co., Florence, Italy.
4 GraphPad Software Inc., San Diego, California, USA.