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Dynamics of Total and Free Iodothyronines of Jumping Horses on the Responses to Competition and Transport
Journal of Equine Veterinary Science 35 (2015) 49–53
Contents lists available at ScienceDirect
Journal of Equine Veterinary Science journal homepage: www.j-evs.com
Original Research
Dynamics of Total and Free Iodothyronines of Jumping Horses on the Responses to Competition and Transport Esterina Fazio*, Pietro Medica, Cristina Cravana, Roberta Pellizzotto, Santo Fragalà, Adriana Ferlazzo Department of Veterinary Sciences, University of Messina, Polo Universitario Annunziata, Messina, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 March 2014 Received in revised form 23 September 2014 Accepted 25 November 2014 Available online 29 November 2014
The aim of the research was to study the total and free iodothyronine (T3, T4, fT3, fT4) changes of trained horses transported before and/or after competition, and performing in show jumping competition, respectively. Six horses were studied in four different sessions during four consecutive weeks. During the first week, horses were submitted to transport before competition (sessions: t0 ¼ before transport, T0 ¼ after transport). Then, during the second week, horses were submitted to transport after competition (sessions: t1 ¼ before transport, T1 ¼ after transport). During the third week, horses participated in a competitive show jumping event (sessions: t2 ¼ before competition, T2 ¼ after competition). During the fourth week, horses were submitted to transport and to a competitive show jumping event (sessions: t3 ¼ after transport and before exercise, T3 ¼ after exercise). Compared with t0 session, t1 showed higher T3 (P < .01) and lower fT4 (P < .01) concentrations. Compared with t2 session, t3 showed higher T3 (P < .01) and lower fT3 (P < .01) concentrations. A significant effect of competition on fT3 changes (F ¼ 4.227; P < .05) was recorded. The results obtained showed that transport and show jumping competition differently influenced the thyroid responses of trained sport horses to physiological and psychological stress, also according to the individual experimental sessions. Ó 2015 Elsevier Inc. All rights reserved.
Keywords: Horse Iodothyronine Show jumping Transport
1. Introduction A properly functioning thyroid gland is highly important to a horse’s good health, and it gets more difficult and complex to know when the thyroid gland is in a state of dysfunction [1,2]. Thyroid function is essential for physiological reactions and adaptations during physical exercise and transport stress; therefore, the changes in free triiodothyronine (fT3) were the most responsive to stressful conditions and may help to provide additional information for the assessment of transport stress in sport horses and about the positive effect extended by competition experience [3]. T4 and T3 are the most commonly measured * Corresponding author at: Esterina Fazio, Department of Veterinary Sciences, University of Messina, Polo Universitario Annunziata, 98168 Messina, Italy. E-mail address: [email protected] (E. Fazio). 0737-0806/$ – see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jevs.2014.11.006
thyroid hormones; all T4 circulating in the blood is released from the thyroid gland; most T3 is produced when other tissues in the body remove iodine from T4. Both T3 and T4 are very highly bound to proteins in the blood, and only the free (unbound) hormone has any active effects. The biologic activity of T3 is much higher than that of T4. Free T3 (fT3) is the most potent and active thyroid hormone in circulation, and its measurement can give a clearest picture of true contribution of extrathyroidal tissues, such as the musculoskeletal system, as a consequence of the thyroid stimulation and/or changes in capacity to bind iodothyronines [4–6]. Thyroid hormones are considered as markers of stress in horses, showing changes according to training [7], pretraining status and circadian changes [8], exercise [5,6,9], and transport and previous experience [3]. A sudden increase in plasma T3 levels 5 minutes after exercise was obtained in Thoroughbred horses [9]; in addition, endurance exercise resulted in transient
50
E. Fazio et al. / Journal of Equine Veterinary Science 35 (2015) 49–53
decreases in serum total and free iodothyronines [10]. The changes of T4 levels were found to be in direct relationship with the continuation of training cycle [11], and thyroid hormones were proposed as markers for identification of maladjustment to training programs in young Standardbreds [12]. The effect of diet composition and feeding state on the response to exercise in feed-restricted horses, with an increase of T3 concentrations in response to exercise, was described [13]. Experimental show jumping sessions induced significant increased values at 30 minutes for T3, T4, and fT4 [6]. Because limited information exists about the specific effects of show jumping competition, or transport, or competition plus transport on the thyroid hormones changes in sport horses, the aim of the research was to study the total and free iodothyronine changes of trained horses transported before and/or after competition, or performing in show jumping competition, respectively. 2. Materials and Methods 2.1. Horses and Sample Collection Six healthy trained jumping horses (five Italian Saddle and one Belgian), five geldings and 1 mare, aged 7–12 years and weighed 575 52 kg, were evaluated. All horses were housed in 4 4 stables in the Equine Research Facility, with natural lighting, allowing reciprocal visual contact. Each horse was fed a daily ration of 7 kg of alfalfa and grass hay and 4 kg of a commercially available hay cube ration, split into two feeds, at 7 AM and 5 PM, respectively; water was provide ad libitum. The horses used were daily trained for 1 hour for show jumping, approximately 4–5 d/wk with the same rider for each horse, and all had previous competition experience. All horses were studied in four different sessions during four consecutive weeks. During the first week, horses were submitted to short road transport (100 km) transport before competition (sessions: t0 ¼ before transport, T0 ¼ after transport). Then, during the second week, horses were submitted to transport (100 km) after competition (sessions: t1 ¼ before transport, T1 ¼ after transport). During the third week, horses participated in a competitive show jumping event (sessions: t2 ¼ before competition, T2 ¼ after competition). During the fourth week, horses were submitted to transport (100 km) and to a competitive show jumping event (sessions: t3 ¼ after transport and before exercise, T3 ¼ after exercise). The vehicle used was a commercial six-horse vehicle (Turbo Zeta; Iveco). The length and width of the commercial vehicle were about 9.50 and 2.5 m, respectively. The vehicle was equipped with partitions which allowed six horses to be loaded in a perpendicular configuration to the direction of travel. Stocking density was about 2 m2 per horse. Rubber padding lined the sides of the vehicles from the floor to an approximate height of 1.2 m. The competitions were carried out in an outdoor arena (33.77 m wide 70.33 m long) and performed the same circuit design over 10 fences of 110 cm, with five upright and five cross-pole fences. The show jumping competitions
were cantered at an average speed of approximately 350 m/min and were scored by time and knockdowns on the courses. The weather was sunny, with an outdoor mean temperature of approximately 15 C–16 C. At the time of blood sampling, all horses were under regular training and considered by the referring veterinarian as clinically healthy. Blood samples were collected before (8 AM) and after (10 AM) transport before competition (sessions t0-T0), before (8:00 AM) and after (10 AM) transport after competition (sessions t1-T1), before (8 AM) and after (at 5 and 30 minutes) a competitive show jumping event (sessions t2-T2), and before competition after transport (8 AM) and after (at 5 and 30 minutes) a competitive show jumping event (sessions t3-T3). All procedures, treatments and animal care were in compliance with the guidelines of the Italian Minister of health for the care and use of animals (D.L. 4/3/2014 n. 26) and UE (Directive 2010/63) and with the regulation (EC) 1/ 2005 on the protection of animals during transport and related operations. 2.2. Hormone Analyses Serum total and free iodothyronine (T3, T4, fT3, fT4) concentrations were determined in duplicate using EIA Kits that had been validated for equine serum [6]. Assays were carried out according to the manufacturer’s instructions. Testing procedure was based on enzyme-linked immunosorbent assay/competition to polyclonal biotinylated ovine iodothyronine antibodies using streptavidin technology. Limits of detection were 0.24 nmol/L for T3, 5.79 nmol/L for T4, 0.15 pmol/L for fT3, and 1.3 pmol/L for fT4. Intra- and inter-assay coefficients of variation were 7.3% and 11.4% for T3, 2.3% and 5.7% for T4, 4.2% and 11.9% for fT3, 6.6% and 9.6% for fT4, respectively. 2.3. Statistical Analyses Data are presented as mean values standard deviation, and results analyzed using a statistical analysis software PRISM package (GraphPad Software Inc, San Diego, CA). A one-way analysis of variance for repeated measures (RM-ANOVA) was applied to test for any differences in the before values of the four different experimental sessions and for any differences in values after transport and after show jumping competition. To determine the effects of competition and competition plus transport, a two-way ANOVA (RM-ANOVA) was applied. The level of significance was set at P < .05. The percentage differences (D%) of changes in thyroid hormones were also calculated by comparing the different postcompetition and posttransport values with related before values. The ratios of total (T4/T3) and free (fT4/fT4) iodothyronines, and the respective percentage of free-to-total iodothyronines (fT4/T4%; fT3/ T3%) in the different sessions were also calculated. 3. Results Data obtained are shown in Fig. 1. Compared with t0 session, t1 showed higher T3 (P < .01) and lower fT4 (P < .01)
E. Fazio et al. / Journal of Equine Veterinary Science 35 (2015) 49–53
51
Fig. 1. Total and free iodothyronine (mean standard deviation) concentrations in sport horses at different experimental sessions. Asterisk indicates significant (*P < .05) differences in average hormone concentrations versus before values. Sessions: t0 ¼ before transport, T0 ¼ after transport; t1 ¼ before transport, T1 ¼ after transport; t2 ¼ before competition, T2 ¼ after competition; t3 ¼ after transport and before exercise, T3 ¼ after exercise.
concentrations. Compared with t2 session, t3 showed higher T3 (P < .01) and lower fT3 (P < .01) concentrations. A significant effect of competition on fT3 changes (F ¼ 4.227; P < .05) was recorded. The ratios and percentages between total and free iodothyronines were shown in Table 1. T4-to-T3 and fT4-to-fT3 ratios were higher for both t0 and T0 sessions than t1 and T1 sessions. The percentages of fT4-to-T4 were higher and the percentages of fT3-to-T3 were lower for both t0 and T0 sessions than t1 and T1 sessions.
T4-to-T3 ratio was higher and fT4-to-fT3 ratio was lower for both t2 and T2 sessions than t3 and T3 sessions. The percentages of fT4-to-T4 were higher and the percentages of fT3-to-T3 were lower for both t2 and T2 sessions than t3 and T3 sessions. 4. Discussion Baseline total and free iodothyronine concentrations are in agreement with previous data obtained in equine by
Table 1 Total and free iodothyronine ratios and percentages in sport horses at different experimental sessions. Hormonal ratios and percentage
Sessions
Ratio of T4/T3 Ratio of fT4/fT3 Percentage fT4/T4 Percentage fT3/T3
t0
T0
t1
T1
Before
After
Before
After
27.7:1 9.13:1 0.055 0.023 0.166 0.13
22.4:1 8.05:1 0.045 0.020 0.127 0.05
25.8:1 4.17:1 0.032 0.007 0.201 0.82
26.4:1 5.26:1 0.035 0.010 0.178 0.21
Sessions
Ratio of T4/T3 Ratio of fT4/fT3 Percentage fT4/T4 Percentage fT3/T3
t2
T2
Before
After
25.5:1 2.31:1 0.037 0.05 0.412 0.52
5 min
30 min
34.4:1 3.47:1 0.039 0.004 0.389 0.44
26.7:1 2.07:1 0.037 0.028 0.037 0.30
t3
T3
Before
After 5 min
30 min
21.5:1 5.43:1 0.033 0.017 0.131 0.14
27.9:1 5.42:1 0.039 0.01 0.168 0.41
26.6:1 5.02:1 0.034 0.022 0.189 0.33
Sessions: t0 ¼ before transport, T0 ¼ after transport; t1 ¼ before transport, T1 ¼ after transport; t2 ¼ before competition, T2 ¼ after competition; t3 ¼ after transport and before exercise, T3 ¼ after exercise. T3, total triiodothyronine; T4, total thyroxine; fT3, free triiodothyronine; fT4, free thyroxine.
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E. Fazio et al. / Journal of Equine Veterinary Science 35 (2015) 49–53
Breuhaus. The results obtained showed that transport and show jumping competition differently influenced the thyroid responses of trained sport horses to physiological and psychological stress, according to the individual experimental sessions. The increase of total and free iodothyronines after transport (session T0) confirmed previous data observed in sport horses after road transport, underlining the metabolic active effects of thyroid hormones in response to stressful stimuli previously described in sport horses [3] and in Thoroughbred and Crossbred stallions [4] after road transport of 100 and 300 km, respectively. The greatest release of T3 and T4 after transport could represent the final result of preferential release of T3 from the thyroid gland and/or a probably increase of peripheral monodeiodination of T4, although a synchronous release of T4 was observed. In addition, the higher increase of T3 (56%) than that of T4 (26%) obtained after transport confirmed that T3 is a metabolically active hormone [14], and the moderate higher increase of fT3 (18%) than that of fT4 (5%) confirmed that the changes of free hormones generally followed those for total hormones changes as previously observed in Sanfratellano horses [2] and Thoroughbred foals [15]. The different patterns of total and free iodothyronines at T0 indicated that transport stress can affect thyroid response, with effective compensatory mechanisms that could restore physiological adaptation, avoiding and/or compensating the significant hormonal and metabolic changes. The lower before fT4 concentration after competition plus transport (session t1) than session t0 showed the effect of the additional physical effort, due to competition and then to transport stimuli, as well as it showed that the recovery period between competition and transport stress was probably inadequate to restore the previous homeostasis. The absence of significant changes of total and free iodothyronines at 5 and 30 minutes after competition (session t2) confirmed previous data observed in experienced jumpers [3] and in trained horses after experimental show jumping session with fences of 1.00 m of height [6]. The highest before T3 and the lowest fT3 values of horses that were submitted to transport plus competition (session t3) showed their primary involvement in maintaining functional homeostasis after competitive exercise, with the highest release of T3 and related peripheral utilization of fT3. Within physiological ranges, the increase of thyroid response, especially of T4 and fT4 concentrations at 5 and at 30 minutes after competition (sessions T2 and T3), might be associated with a higher efficiency of the mechanical work performed by exercising muscle. Peripheral metabolism of thyroid hormones can be changed significantly by a number of physiological conditions, which can alter the deiodination pathway and lead to a change in the circulating level of thyroid hormones. Hence, it is possible to suppose that the decrease of T3 and the contextual increase of T4 both at 5 and at 30 minutes after competition plus transport (session T3) could show an increase of deiodination pathway, supported by an additional stress (competition effort plus transport stress),
as consequence of the improved circulation [16]. The additional exercise plus transport stress (session T3) induced a divergent T3 and fT3 pathway, with a constant increase of fT3 and a contextual decrease of T3 at 5 and at 30 minutes after competition and transport. This evidence also suggests that, if exercise plus transport–related energy expenditure exceeds metabolic substrates consumed, a low T3 syndrome may be induced. On the other hand, the contextual increase of T4 at 5 and at 30 minutes after competition during the session T2, with a T3 decrease only after 5 minutes, showed an early deiodination pathway of T4 in T3 after exercise. Data obtained showed that show jumping competition (session T2) synchronized the T3 and fT3 pathway, with decreases of two hormones at 5 minutes and their increases at 30 minutes after competition. Jumping horses showed high T4-to-T3 and fT4-to-fT3 ratios after sessions T1, T2, and T3, with constant percentage of fT4/T4 during all four sessions, and lower percentage for fT3/T3 during sessions T0 and T3. These results confirmed that changes in fT4 concentrations generally follow those of T4. It is well known that the fT4 fraction represents the biologically active hormone for tissue; thus, the ratio of fT4 represents a primary factor for determining the fractional turnover of thyroid hormones. Moreover, the binding fraction represents the hormonal store that balances the sudden increase and decrease of hormonal release to tissue. These data showed that the biological effects of shortterm changes in the thyroid hormone concentrations are not currently completely understood but are potentially important in the body’s adjustment to stressful or catabolic states, according to thyroid economy after exercise and/or transport. Thyroid function depends on the intensity and duration of exercise and perhaps on other factors, such as transport performed before or after competition. In addition, the major involvement of total (session T0) or free (sessions T2 and T3) iodothyronines could support the hypothesis of ac stimulated activity of thyroid gland when the homeostatic response to show jumping was associated with pretransportation or posttransportation stress, inducing increasing circulating concentrations of different hormones’ aliquot ready for intracellular metabolic utilization. Hence, total iodothyronines appear to act synergically with free iodothyronines to improve postexercise and/or posttransport adaptations, including exercise plus transport stress. References [1] Breuhaus BA. Disorders of the equine thyroid gland. Vet Clin North Am Equine Pract 2011;27:115–28. [2] Medica P, Fazio E, Cravana C, Ferlazzo A. Influence of endemic goitre areas on thyroid hormones in horses. Animal 2011a;5:82–7. [3] Fazio E, Medica P, Cravana C, Ferlazzo A. Effects of competition experience and transportation on the adrenocortical and thyroid responses of horses. Vet Rec 2008;163:713–6. [4] Fazio E, Medica P, Cravana C, Giacoppo E, Ferlazzo A. Physiological variables of horses after road transport. Animal 2009;3:1313–8. [5] Cravana C, Medica P, Prestopino M, Fazio E, Ferlazzo A. Effects of competitive and noncompetitive showjumping on total and free iodothyronines, b-endorphin, ACTH and cortisol levels of horses. Equine Vet J 2010;42:179–84.
E. Fazio et al. / Journal of Equine Veterinary Science 35 (2015) 49–53 [6] Ferlazzo A, Medica P, Cravana C, Fazio E. Effects of fence height on total and free iodothyronine changes in horses after experimental show jumping sessions. Equine Vet J 2010;42: 110–5. [7] Suwannachot P, Verkleij CB, Kocsis S, Enzerink E. Prolonged food restriction and mild exercise in Shetland ponies: effects on weight gain, thyroid hormone concentrations and muscle Naþ, Kþ-ATPase. J Endocrinol 2000;167:321–9. [8] Medica P, Cravana C, Fazio E, Ferlazzo A. 24-hour endocrine profiles of Quarter Horses under resting conditions. J Equine Vet Sci 2011b; 31:35–40. [9] González O, González E, Sánchez C, Pinto J, González I, Enríquez O, Martínez R, Filgueira G, White A. Effect of exercise on erythrocyte, beta-adrenergic receptors and plasma concentrations of catecholamines and thyroid hormones in Thoroughbred horses. Equine Vet J 1998;30:72–8. [10] Graves EA, Schott HC, Marteniuk JV, Refsal KR, Nachreiner RF. Thyroid hormone responses to endurance exercise. Equine Vet J Suppl 2006;36:32–6.
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rzev G. Dynamics of the [11] Georgiev P, Petkov PI, Georgiev KH, Bu functional, biochemical and hormonal indices of racehorses. Vet Med Nauki 1983;20:51–8. [12] Leleu C, Haentjens F. Morphological, haemato-biochemical and endocrine changes in young Standardbreds with “maladaptation” to early training. Equine Vet J Suppl 2010;38:171–8. [13] Powell D, Lawrence LM, Brewster-Barnes T, Fitzgerald B, Warren LK, Rokuroda S, Parker A, Crum A. The effect of diet composition and feeding state on the response to exercise in feed-restricted horses. Equine Vet J Suppl 1999;30:514–8. [14] Larsen PR, Silva JE, Kaplan MM. Relationships between circulating and intracellular hormones: physiological and clinical implications. Endocr Rev 1981;2:87–102. [15] Fazio E, Medica P, Cravana C, Messineo C, Ferlazzo A. Total and free iodothyronine levels of growing Thoroughbred foals: effects of weaning and gender. Livest Sci 2007;110:207–13. [16] Art T, Amory H, Desmecht D, Lekeux P. Effect of show jumping on heart rate, blood lactate and other biochemical values. Equine Vet J Suppl 1990;9:78–82.
Contents lists available at ScienceDirect
Journal of Equine Veterinary Science journal homepage: www.j-evs.com
Original Research
Dynamics of Total and Free Iodothyronines of Jumping Horses on the Responses to Competition and Transport Esterina Fazio*, Pietro Medica, Cristina Cravana, Roberta Pellizzotto, Santo Fragalà, Adriana Ferlazzo Department of Veterinary Sciences, University of Messina, Polo Universitario Annunziata, Messina, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 March 2014 Received in revised form 23 September 2014 Accepted 25 November 2014 Available online 29 November 2014
The aim of the research was to study the total and free iodothyronine (T3, T4, fT3, fT4) changes of trained horses transported before and/or after competition, and performing in show jumping competition, respectively. Six horses were studied in four different sessions during four consecutive weeks. During the first week, horses were submitted to transport before competition (sessions: t0 ¼ before transport, T0 ¼ after transport). Then, during the second week, horses were submitted to transport after competition (sessions: t1 ¼ before transport, T1 ¼ after transport). During the third week, horses participated in a competitive show jumping event (sessions: t2 ¼ before competition, T2 ¼ after competition). During the fourth week, horses were submitted to transport and to a competitive show jumping event (sessions: t3 ¼ after transport and before exercise, T3 ¼ after exercise). Compared with t0 session, t1 showed higher T3 (P < .01) and lower fT4 (P < .01) concentrations. Compared with t2 session, t3 showed higher T3 (P < .01) and lower fT3 (P < .01) concentrations. A significant effect of competition on fT3 changes (F ¼ 4.227; P < .05) was recorded. The results obtained showed that transport and show jumping competition differently influenced the thyroid responses of trained sport horses to physiological and psychological stress, also according to the individual experimental sessions. Ó 2015 Elsevier Inc. All rights reserved.
Keywords: Horse Iodothyronine Show jumping Transport
1. Introduction A properly functioning thyroid gland is highly important to a horse’s good health, and it gets more difficult and complex to know when the thyroid gland is in a state of dysfunction [1,2]. Thyroid function is essential for physiological reactions and adaptations during physical exercise and transport stress; therefore, the changes in free triiodothyronine (fT3) were the most responsive to stressful conditions and may help to provide additional information for the assessment of transport stress in sport horses and about the positive effect extended by competition experience [3]. T4 and T3 are the most commonly measured * Corresponding author at: Esterina Fazio, Department of Veterinary Sciences, University of Messina, Polo Universitario Annunziata, 98168 Messina, Italy. E-mail address: [email protected] (E. Fazio). 0737-0806/$ – see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jevs.2014.11.006
thyroid hormones; all T4 circulating in the blood is released from the thyroid gland; most T3 is produced when other tissues in the body remove iodine from T4. Both T3 and T4 are very highly bound to proteins in the blood, and only the free (unbound) hormone has any active effects. The biologic activity of T3 is much higher than that of T4. Free T3 (fT3) is the most potent and active thyroid hormone in circulation, and its measurement can give a clearest picture of true contribution of extrathyroidal tissues, such as the musculoskeletal system, as a consequence of the thyroid stimulation and/or changes in capacity to bind iodothyronines [4–6]. Thyroid hormones are considered as markers of stress in horses, showing changes according to training [7], pretraining status and circadian changes [8], exercise [5,6,9], and transport and previous experience [3]. A sudden increase in plasma T3 levels 5 minutes after exercise was obtained in Thoroughbred horses [9]; in addition, endurance exercise resulted in transient
50
E. Fazio et al. / Journal of Equine Veterinary Science 35 (2015) 49–53
decreases in serum total and free iodothyronines [10]. The changes of T4 levels were found to be in direct relationship with the continuation of training cycle [11], and thyroid hormones were proposed as markers for identification of maladjustment to training programs in young Standardbreds [12]. The effect of diet composition and feeding state on the response to exercise in feed-restricted horses, with an increase of T3 concentrations in response to exercise, was described [13]. Experimental show jumping sessions induced significant increased values at 30 minutes for T3, T4, and fT4 [6]. Because limited information exists about the specific effects of show jumping competition, or transport, or competition plus transport on the thyroid hormones changes in sport horses, the aim of the research was to study the total and free iodothyronine changes of trained horses transported before and/or after competition, or performing in show jumping competition, respectively. 2. Materials and Methods 2.1. Horses and Sample Collection Six healthy trained jumping horses (five Italian Saddle and one Belgian), five geldings and 1 mare, aged 7–12 years and weighed 575 52 kg, were evaluated. All horses were housed in 4 4 stables in the Equine Research Facility, with natural lighting, allowing reciprocal visual contact. Each horse was fed a daily ration of 7 kg of alfalfa and grass hay and 4 kg of a commercially available hay cube ration, split into two feeds, at 7 AM and 5 PM, respectively; water was provide ad libitum. The horses used were daily trained for 1 hour for show jumping, approximately 4–5 d/wk with the same rider for each horse, and all had previous competition experience. All horses were studied in four different sessions during four consecutive weeks. During the first week, horses were submitted to short road transport (100 km) transport before competition (sessions: t0 ¼ before transport, T0 ¼ after transport). Then, during the second week, horses were submitted to transport (100 km) after competition (sessions: t1 ¼ before transport, T1 ¼ after transport). During the third week, horses participated in a competitive show jumping event (sessions: t2 ¼ before competition, T2 ¼ after competition). During the fourth week, horses were submitted to transport (100 km) and to a competitive show jumping event (sessions: t3 ¼ after transport and before exercise, T3 ¼ after exercise). The vehicle used was a commercial six-horse vehicle (Turbo Zeta; Iveco). The length and width of the commercial vehicle were about 9.50 and 2.5 m, respectively. The vehicle was equipped with partitions which allowed six horses to be loaded in a perpendicular configuration to the direction of travel. Stocking density was about 2 m2 per horse. Rubber padding lined the sides of the vehicles from the floor to an approximate height of 1.2 m. The competitions were carried out in an outdoor arena (33.77 m wide 70.33 m long) and performed the same circuit design over 10 fences of 110 cm, with five upright and five cross-pole fences. The show jumping competitions
were cantered at an average speed of approximately 350 m/min and were scored by time and knockdowns on the courses. The weather was sunny, with an outdoor mean temperature of approximately 15 C–16 C. At the time of blood sampling, all horses were under regular training and considered by the referring veterinarian as clinically healthy. Blood samples were collected before (8 AM) and after (10 AM) transport before competition (sessions t0-T0), before (8:00 AM) and after (10 AM) transport after competition (sessions t1-T1), before (8 AM) and after (at 5 and 30 minutes) a competitive show jumping event (sessions t2-T2), and before competition after transport (8 AM) and after (at 5 and 30 minutes) a competitive show jumping event (sessions t3-T3). All procedures, treatments and animal care were in compliance with the guidelines of the Italian Minister of health for the care and use of animals (D.L. 4/3/2014 n. 26) and UE (Directive 2010/63) and with the regulation (EC) 1/ 2005 on the protection of animals during transport and related operations. 2.2. Hormone Analyses Serum total and free iodothyronine (T3, T4, fT3, fT4) concentrations were determined in duplicate using EIA Kits that had been validated for equine serum [6]. Assays were carried out according to the manufacturer’s instructions. Testing procedure was based on enzyme-linked immunosorbent assay/competition to polyclonal biotinylated ovine iodothyronine antibodies using streptavidin technology. Limits of detection were 0.24 nmol/L for T3, 5.79 nmol/L for T4, 0.15 pmol/L for fT3, and 1.3 pmol/L for fT4. Intra- and inter-assay coefficients of variation were 7.3% and 11.4% for T3, 2.3% and 5.7% for T4, 4.2% and 11.9% for fT3, 6.6% and 9.6% for fT4, respectively. 2.3. Statistical Analyses Data are presented as mean values standard deviation, and results analyzed using a statistical analysis software PRISM package (GraphPad Software Inc, San Diego, CA). A one-way analysis of variance for repeated measures (RM-ANOVA) was applied to test for any differences in the before values of the four different experimental sessions and for any differences in values after transport and after show jumping competition. To determine the effects of competition and competition plus transport, a two-way ANOVA (RM-ANOVA) was applied. The level of significance was set at P < .05. The percentage differences (D%) of changes in thyroid hormones were also calculated by comparing the different postcompetition and posttransport values with related before values. The ratios of total (T4/T3) and free (fT4/fT4) iodothyronines, and the respective percentage of free-to-total iodothyronines (fT4/T4%; fT3/ T3%) in the different sessions were also calculated. 3. Results Data obtained are shown in Fig. 1. Compared with t0 session, t1 showed higher T3 (P < .01) and lower fT4 (P < .01)
E. Fazio et al. / Journal of Equine Veterinary Science 35 (2015) 49–53
51
Fig. 1. Total and free iodothyronine (mean standard deviation) concentrations in sport horses at different experimental sessions. Asterisk indicates significant (*P < .05) differences in average hormone concentrations versus before values. Sessions: t0 ¼ before transport, T0 ¼ after transport; t1 ¼ before transport, T1 ¼ after transport; t2 ¼ before competition, T2 ¼ after competition; t3 ¼ after transport and before exercise, T3 ¼ after exercise.
concentrations. Compared with t2 session, t3 showed higher T3 (P < .01) and lower fT3 (P < .01) concentrations. A significant effect of competition on fT3 changes (F ¼ 4.227; P < .05) was recorded. The ratios and percentages between total and free iodothyronines were shown in Table 1. T4-to-T3 and fT4-to-fT3 ratios were higher for both t0 and T0 sessions than t1 and T1 sessions. The percentages of fT4-to-T4 were higher and the percentages of fT3-to-T3 were lower for both t0 and T0 sessions than t1 and T1 sessions.
T4-to-T3 ratio was higher and fT4-to-fT3 ratio was lower for both t2 and T2 sessions than t3 and T3 sessions. The percentages of fT4-to-T4 were higher and the percentages of fT3-to-T3 were lower for both t2 and T2 sessions than t3 and T3 sessions. 4. Discussion Baseline total and free iodothyronine concentrations are in agreement with previous data obtained in equine by
Table 1 Total and free iodothyronine ratios and percentages in sport horses at different experimental sessions. Hormonal ratios and percentage
Sessions
Ratio of T4/T3 Ratio of fT4/fT3 Percentage fT4/T4 Percentage fT3/T3
t0
T0
t1
T1
Before
After
Before
After
27.7:1 9.13:1 0.055 0.023 0.166 0.13
22.4:1 8.05:1 0.045 0.020 0.127 0.05
25.8:1 4.17:1 0.032 0.007 0.201 0.82
26.4:1 5.26:1 0.035 0.010 0.178 0.21
Sessions
Ratio of T4/T3 Ratio of fT4/fT3 Percentage fT4/T4 Percentage fT3/T3
t2
T2
Before
After
25.5:1 2.31:1 0.037 0.05 0.412 0.52
5 min
30 min
34.4:1 3.47:1 0.039 0.004 0.389 0.44
26.7:1 2.07:1 0.037 0.028 0.037 0.30
t3
T3
Before
After 5 min
30 min
21.5:1 5.43:1 0.033 0.017 0.131 0.14
27.9:1 5.42:1 0.039 0.01 0.168 0.41
26.6:1 5.02:1 0.034 0.022 0.189 0.33
Sessions: t0 ¼ before transport, T0 ¼ after transport; t1 ¼ before transport, T1 ¼ after transport; t2 ¼ before competition, T2 ¼ after competition; t3 ¼ after transport and before exercise, T3 ¼ after exercise. T3, total triiodothyronine; T4, total thyroxine; fT3, free triiodothyronine; fT4, free thyroxine.
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Breuhaus. The results obtained showed that transport and show jumping competition differently influenced the thyroid responses of trained sport horses to physiological and psychological stress, according to the individual experimental sessions. The increase of total and free iodothyronines after transport (session T0) confirmed previous data observed in sport horses after road transport, underlining the metabolic active effects of thyroid hormones in response to stressful stimuli previously described in sport horses [3] and in Thoroughbred and Crossbred stallions [4] after road transport of 100 and 300 km, respectively. The greatest release of T3 and T4 after transport could represent the final result of preferential release of T3 from the thyroid gland and/or a probably increase of peripheral monodeiodination of T4, although a synchronous release of T4 was observed. In addition, the higher increase of T3 (56%) than that of T4 (26%) obtained after transport confirmed that T3 is a metabolically active hormone [14], and the moderate higher increase of fT3 (18%) than that of fT4 (5%) confirmed that the changes of free hormones generally followed those for total hormones changes as previously observed in Sanfratellano horses [2] and Thoroughbred foals [15]. The different patterns of total and free iodothyronines at T0 indicated that transport stress can affect thyroid response, with effective compensatory mechanisms that could restore physiological adaptation, avoiding and/or compensating the significant hormonal and metabolic changes. The lower before fT4 concentration after competition plus transport (session t1) than session t0 showed the effect of the additional physical effort, due to competition and then to transport stimuli, as well as it showed that the recovery period between competition and transport stress was probably inadequate to restore the previous homeostasis. The absence of significant changes of total and free iodothyronines at 5 and 30 minutes after competition (session t2) confirmed previous data observed in experienced jumpers [3] and in trained horses after experimental show jumping session with fences of 1.00 m of height [6]. The highest before T3 and the lowest fT3 values of horses that were submitted to transport plus competition (session t3) showed their primary involvement in maintaining functional homeostasis after competitive exercise, with the highest release of T3 and related peripheral utilization of fT3. Within physiological ranges, the increase of thyroid response, especially of T4 and fT4 concentrations at 5 and at 30 minutes after competition (sessions T2 and T3), might be associated with a higher efficiency of the mechanical work performed by exercising muscle. Peripheral metabolism of thyroid hormones can be changed significantly by a number of physiological conditions, which can alter the deiodination pathway and lead to a change in the circulating level of thyroid hormones. Hence, it is possible to suppose that the decrease of T3 and the contextual increase of T4 both at 5 and at 30 minutes after competition plus transport (session T3) could show an increase of deiodination pathway, supported by an additional stress (competition effort plus transport stress),
as consequence of the improved circulation [16]. The additional exercise plus transport stress (session T3) induced a divergent T3 and fT3 pathway, with a constant increase of fT3 and a contextual decrease of T3 at 5 and at 30 minutes after competition and transport. This evidence also suggests that, if exercise plus transport–related energy expenditure exceeds metabolic substrates consumed, a low T3 syndrome may be induced. On the other hand, the contextual increase of T4 at 5 and at 30 minutes after competition during the session T2, with a T3 decrease only after 5 minutes, showed an early deiodination pathway of T4 in T3 after exercise. Data obtained showed that show jumping competition (session T2) synchronized the T3 and fT3 pathway, with decreases of two hormones at 5 minutes and their increases at 30 minutes after competition. Jumping horses showed high T4-to-T3 and fT4-to-fT3 ratios after sessions T1, T2, and T3, with constant percentage of fT4/T4 during all four sessions, and lower percentage for fT3/T3 during sessions T0 and T3. These results confirmed that changes in fT4 concentrations generally follow those of T4. It is well known that the fT4 fraction represents the biologically active hormone for tissue; thus, the ratio of fT4 represents a primary factor for determining the fractional turnover of thyroid hormones. Moreover, the binding fraction represents the hormonal store that balances the sudden increase and decrease of hormonal release to tissue. These data showed that the biological effects of shortterm changes in the thyroid hormone concentrations are not currently completely understood but are potentially important in the body’s adjustment to stressful or catabolic states, according to thyroid economy after exercise and/or transport. Thyroid function depends on the intensity and duration of exercise and perhaps on other factors, such as transport performed before or after competition. In addition, the major involvement of total (session T0) or free (sessions T2 and T3) iodothyronines could support the hypothesis of ac stimulated activity of thyroid gland when the homeostatic response to show jumping was associated with pretransportation or posttransportation stress, inducing increasing circulating concentrations of different hormones’ aliquot ready for intracellular metabolic utilization. Hence, total iodothyronines appear to act synergically with free iodothyronines to improve postexercise and/or posttransport adaptations, including exercise plus transport stress. References [1] Breuhaus BA. Disorders of the equine thyroid gland. Vet Clin North Am Equine Pract 2011;27:115–28. [2] Medica P, Fazio E, Cravana C, Ferlazzo A. Influence of endemic goitre areas on thyroid hormones in horses. Animal 2011a;5:82–7. [3] Fazio E, Medica P, Cravana C, Ferlazzo A. Effects of competition experience and transportation on the adrenocortical and thyroid responses of horses. Vet Rec 2008;163:713–6. [4] Fazio E, Medica P, Cravana C, Giacoppo E, Ferlazzo A. Physiological variables of horses after road transport. Animal 2009;3:1313–8. [5] Cravana C, Medica P, Prestopino M, Fazio E, Ferlazzo A. Effects of competitive and noncompetitive showjumping on total and free iodothyronines, b-endorphin, ACTH and cortisol levels of horses. Equine Vet J 2010;42:179–84.
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