Research Article

Edu-Ecotourism Concept of Equine Cortisol Metabolites and Tri/Tetra-Iodothyronine Ratio

Claude Mona Airin1*, Miyayu Soneta Sofyan2, Galy Hardyta3, Khrisdiana Putri4, Pudji Astuti1

1Department of Physiology, Faculty of Veterinary Medicine, Universitas Gadjah Mada Yogyakarta, Indonesia; 2Health Departement Faculty of Vocational Study Airlangga University, Surabaya, Indonesia; 3Faculty of Agriculture, Department of Animal Husbandry, Universitas Tidar Magelang, Indonesia; 4Department of Veterinary Public Health, Faculty of Veterinary Medicine, Universitas Gadjah Mada Yogyakarta, Indonesia

Received | January 08, 2024; Accepted | March 20, 2024; Published | May 07, 2024

*Correspondence | Claude Mona Airin, Department of Physiology, Faculty of Veterinary Medicine, Universitas Gadjah Mada Yogyakarta; Email: monaairin@ugm.ac.id

Citation | Airin CM, Sofyan MS, Hardyta G, Putri K, Astuti P (2024). Edu-ecotourism concept of equine cortisol metabolites and tri/tetra-iodothyronine ratio. Adv. Anim. Vet. Sci., 12(6):1174-1181.

DOI | https://dx.doi.org/10.17582/journal.aavs/2024/12.6.1174.1181

ISSN (Online) | 2307-8316

Copyright: 2024 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

The horse has natural superiority in muscle strength to support or pull weights. This superiority then functioned as a support, weight puller, as well as tourist vehicle. Back to nature concept of tourism has become highly demanding since more farmland conversion into housing. The horse has become a perfect icon to bring a rural atmosphere into tourism, because tourists are highly concerned with the appeal of a tourist attraction (Fitriani, et al., 2021). The edu-ecotourism has attracted visitors for its offer of animal direct interaction such as riding a horse. Zoo managers or keepers tend to neglect animal welfare concepts during peak season. According to Hall et al. (2018), it is very important to consider working horse welfare. Assessing the emotional component of behavior can be used as a basis for identifying fear-provoking situations which will help reduce human risk and enhance horse learning and can be used for improvements in horse management. Today animal welfare is not only highlighting the five freedoms scope, but also positive emotional experience. A crowd of visitors or inexperience and nervous rider could contribute to creating potentially dangerous situations for the visitor and the horse (Silver, 2002). Animals under physical stressors or emotions depend on complicated interactions between different biological systems (Behavior, autonomy, neuroendocrine, and/or immunity) for stress to respond (Pawluski et al., 2017). Increased heart rate and blood and saliva cortisol are strongly associated with acute stress (Koolhaas et al., 2011). Various studies have shown how horses anticipate potential stressful events such as loading transport vehicles (Schmidt et al., 2010), racing competitions compared to regular training (Bohák et al., 2018); trekking courses (Ono et al., 2017), or when horses take part in festivals (Maneu et al., 2023). Elevated cortisol levels can affect behavior (Aggressiveness, movement, and grooming) and physiological function (Depression, hypertension, immune system suppression, gastrointestinal ulceration, electrolyte imbalance, calcium loss, reduced bone mass, and growth inhibition (Birke, 2002; Breazil, 1987; Steward, 2003).

Thyroid hormones significantly influence metabolism, heart rate, blood pressure, nutritional physiology, brain development, and thermo-regulation independent of muscle activity. Thyroid hormones will also be responsive to nutrient deficits, lowering metabolism, and allowing the body to conserve energy during nutritional emergencies (Silva, 2002). Glucocorticoid hormones (GC) in circulation and feces can indicate a response to psychological and nutritional stress. According to Airin et al. (2020), when the body experiences stress and an increase in cortisol hormone, it can affect the levels of thyroid hormones, particularly T4. Inadequate energy intake caused by stress can also impact the process of gluconeogenesis, which increases by converting T4 into T3 to aid the body’s metabolic process. This suggests that the ratio of T3/T4 can serve as an indicator to identify changes in an animal’s physiological state. Thyroid hormones and GC hormones will respond when food availability or disruption impact can be used to design practical mitigation efforts to adapt (Wasser et al., 2010). The hypothalamic-pituitary-adrenocortical axis (HPA) and sympathetic adrenomedullary system (SAM) are considered the key to the stress response. They are known to have a major role in the mobilization and redistribution of oxygen and nutrients as energy for active organs and tissues. Therefore, the HPA and SAM carry important functions to prepare metabolism and cardiovascular behavior (Sapolsky et al., 2000). Stress levels are commonly measured by blood cortisol using enzyme-linked immunosorbent assay (ELISA) technique (Gholib, 2021). However, invasive sample collection can induce stress on the animal during handling and restraint resulting in a bias in the analysis (Sheriff et al., 2011; Rain et al., 2022). Recently non-invasive techniques for monitoring hormone levels using fecal samples have become a preferred tool for field researchers (Airin et al., 2020, 2022; Laura et al., 2020; Yuniarti et al., 2022; Arif et al., 2024). The excretion rate of metabolites is correlated to hormone levels in circulation. Hormone measurement from excreta reflects the hormone level of the body. A fecal sample is routinely excreted by animals thus minimizing contact and avoiding data bias. In addition, feces also can be used to determine reproductive cyclicity, time of ovulation, pregnancy, impending parturition, and adrenocortical (stress) status (Kersey and Dehnhard, 2014).

Research on the influence of visitors on the welfare of zoo animals has been intensified since the 1970s. Numerous studies have indicated the presence of visitors, density, activity, and position characteristics may affect zoo animal behavior and physiology. However, the variation in the results has indicated better tolerance in some zoo animals to large numbers of visitors, and some are even not impacted. Animals placed in artificial habitats are faced with a variety of potentially provocative environmental challenges, one of which is interaction with humans (Morgan and Tromborg, 2007). This research will evaluate the cortisol level and ratio T3/T4 of the horse during direct and indirect contact with zoo visitors, so that it can be used as reference data for zoo management that carries edu ecotourism concept.

MATERIALS AND METHODS

Animal ethics

All stages of sampling in this study have received approval from the Ethics Commission of the Integrated Research and Testing Laboratory of Universitas Gadjah Mada No 00049/04/LPPT/IX/2023.

Horse husbandry

Horses are housed individually in enclosed stalls with adequate ventilation. Each stall is equipped with a grassy area with short grass and trees. The stalls are bedded with clean bedding every day. Veterinarians conduct horse examinations once every two months. Horses are fed manually based on their body weight and provided with ad libitum access to drinking water. The temperature in the stalls is maintained at 25-29°C.

Sample collection

Fecal samples (n=80) were collected in the morning (07.00-08.00), afternoon (12.00-13.00), and evening (16.00-17.00) for seven days during low and high numbers of visitors. The classification of low and peak is based on the research of Rajagopal et al. (2011) modified according to the conditions of the minizoo. Low visitor density is a minizoo condition during weekdays instead of holidays, while peak sessions are on weekends (holidays). The sample was kept cold during transportation to the Physiology Laboratory, Faculty of Veterinary Medicine, Gadjah Mada University then stored at -20 oC until processed.

Visitor data

Data on the number of visitors was obtained from ticket booth records. The number of visitors riding horses was included, as well as horse trail distance.

Sample preparation

Frozen fecal samples were subjected to freeze-drying for 72 hours, this process was removing water content by lyophilization until dry. The dried feces were finely grounded and 0.5 g was transferred to a test tube for methanol extraction. The extraction was carried out by adding 5 ml of 80% methanol to the sample then vortexed for 10 minutes followed by a centrifuge (PLC series) at 3000 rpm for 10 minutes. The supernatant was collected and stored in a microtube in the freezer before analysis.

Measurement of cortisol levels

Cortisol analysis protocols were according to EIA Cortisol KIT Batch LOT 96680006100323. Standard solutions, fecal extract samples, and sample tests were loaded at 50 µl into the wells. Fifty microliters of HRP conjugate cortisol in the ratio of 1:50 were then added followed by incubation for 60 minutes. The wells were then washed three times and 100 µl of Tetramethylbenzidine (TMB) were added. After 20 minutes of incubation at RT under minimum light intensity, 50 µl of stop solution was added and read at 450 nm (Zenix-320). Cortisol levels in ng/ml units were converted to ng/gr dry feces by considering diluent volume and feces weight.

Measurement of thyroid levels

Thyroid hormone analysis is performed using the KIT EIA T3 and T4 manual with LOT numbers t3g6560 and T4s6559. A standard solution of 25 μl is put into each designated well in two different microplates. A sample of fecal extract was taken using a 25 μl micropipette and inserted into every other well available in each. A working solution containing thyroxine-enzyme conjugates and 50 μl triiodothyronine-enzyme conjugates was added to each microplate well. On the triiodothyronine plate, Biotin was added as much as 50 μl. The plate is placed on the machine mixer for 20-30 seconds and incubated for 60 minutes at room temperature. Wells are washed three times using 300 μl deionized water for each sump, then add 100 μl 3, 3ʹ, 5, 5ʹ-Tetramethylbenzidine (TMB) to each well and incubate under closed conditions for 30 minutes at room temperature. The stop solution is inserted as much as 50 μl, then read using an EIA machine reader with a wavelength of 450 nm. The OD value obtained is converted into a grade using a formula generated by the standard standard curve. The levels obtained in units of ng/ml are then converted into ng/gr dry feces, while the results of the ratio of T3/ T4 are obtained by dividing the levels of hormone T3 with T4.

Data analysis

The study analyzed data on the number of tourists, horsemen, cortisol metabolite levels, and T3/T4 ratio using the SPSS software. The T-test was used to determine the effect of the number of visitors on cortisol metabolite levels and the T3/T4 ratio. The correlation test was conducted to determine the relationship between the number of visitors, horsemen, cortisol levels, and the T3/T4 ratio. A significant effect is observed if P<0.05, while a strong correlation is indicated when R is close to 1 or -1.

RESULTS and DISCUSSION

Average cortisol levels and the number of daily visitors (morning, afternoon, evening) during peak sessions are higher than low sessions (Table 1). The number of horsemen at peak session is more than during low session (Table 1). Horse cortisol levels in the morning during the peak session were higher than during the low session, but in the afternoon, cortisol levels in both conditions decreased (Table 2 and Figure 1).

 

Table 1: The average cortisol levels, number of visitors, and horse riders during low season and peak season.

	Cortisol level (ng/gr dry feces)	Number of visitors (people)	Number of visitor riding a horse (people)
Low season	392,41 ± 34,9a*	21 ± 19*	0 ± 0
Peak season	413,56 ± 35,8b*	759 ± 202*	12 ± 6

 

Note: a,b different letters in the same column indicate significant differences; *indicates a positive correlation

 

Table 2: The average morning, afternoon, and evening cortisol levels of the riding horse during low dan peak season.

Horse ID	Cortisol level in low season (ng/gr dry feces)			No. of rider	Cortisol level in peak season (ng/gr dry feces)			No. of rider
	Morning	Afternoon	Evening		Morning	Afternoon	Evening
1	257,37± 75,19	259,68±114,83	256,18±100,05	0	485,38± 239,30	234,61±85,43	320,56±84,51	16 ± 4
2	309,65± 36,91	517,51±170,19	500,13±172,75	0	416,17± 125,30	468,25±136,11	351,09±214,20	14 ± 4
3	385,64± 74,31	490,16±108,2	465,49±240,52	0	539,38± 159,12	527,46±91,17	492,66±199,72	7 ± 2
Ave	344,41± 108,27a	425,7±144,1a	407,27±131,98a	0	462,26± 67,21b	401,64±169,56a	376,98±103,15a	12 ± 6

 

Note: a,b different letters in the same column and row indicate significant differences.

 

 

Measured cortisol reflected its level condition in the body. According to (Pawluski, et al., 2017) there is a positive correlation between plasma cortisol levels and cortisol metabolites. The cortisol level in horses at JE during the low and peak season of visitors showed a positive correlation (r= 0.5237, r2= 0.2838) but was not significant (Table 1). This positive, insignificant correlation indicated the increase in cortisol levels also occurred in the absence of visitor direct interaction with the horses. Various studies have shown that the presence of visitors affects wild animals in zoos and results in behavioral changes such as increased intra-group aggression or stereotyping and decreased exploration (Birke, 2002; Davis et al., 2005; Hosey, 2005; Sekar et al., 2008; Skyner et al., 2004). However, other researchers also reported no effect of zoo visitors on animals (Synder, 1975). Furthermore, zoo visitors even could enrich some zoo animals (Cook and Hosey, 1995).

The number of tourists riding horses was also positively correlated with cortisol levels, although not significant. Evening cortisol levels tend to be lower than during the day in peak and low seasons even though the numbers of riders were varied. This shows that the distance trail and the riding load were within reasonable limits since the cortisol was decreasing in the afternoon following the circadian rhythm. The horse trail distance for a riding horse is 200 m/tourist. If the average number of tourists is 12±6 on a range of 6-20 tourists during peak sessions meaning every day the horses cover a maximum distance of 4000 m or 4 km. Based on Kentucky Equine Research (2006), stress and muscle fatigue associated with transportation are comparable to exercise. Trips of up to 90 km (50 miles) usually do not significantly affect blood profile. However, a trip of up to 900 km (540 miles) or more should ideally be followed by several days of rest before the horse is assigned for vigorous exercise.

Increasing the load on the horse can increase its metabolic rate (Wickler et al., 2001) and the biomechanical, physiological, biochemical, and behavioral parameters of the horse during work. According to Christensen et al. (2020), increasing rider weight by 15 and 25% does not have a significant effect on heart rate, salivary cortisol, behavior, and gait symmetry. Some research has shown variations in the permitted maximum load of a horse. There are variations in the load that can be given to horses, a native Japanese horse-drawn carriage (body weight 339.9±37.5 kg) is allowed to load less than 100 kg (Matsuura, 2013a, b); the maximum permitted load for a Japanese land racehorse to race is less than 70 kg (33% of body weight) (Matsuura et al., 2016); the permitted load carriage for Taishuh horse (Japanese small breed horse) is less than 100 kg (43 % of BW) with running speed 3.00 ms−1 (Matsuura et al., 2013a). The load received by each horse must be adjusted to its capacity and body weight. This follows Matsuura et al. (2013a) that to improve welfare, it is very important to know the loading capacity for each horse breed that varies in average body weight; the maximum horse to rider weight ratio is 12-23% (Christesen et al., 2020); or 29% of an adult horse’s body weight (Matsuura et al., 2013a, b). In this study, the average horseman in a day during peak session was 12±6 people. The load received by each horse was different, at the moment the arrangement was based only on the length of time the horse had been used as a riding horse and adjusted to the ability of the horse by the keeper. This is supported by the results of research by Dobec et al. (1994) that individual variations in muscle fiber proportions can influence metabolic responses and genetics can have an important role in a horse’s weight-carrying capacity (Stefánsdóttir et al., 2017). The highest cortisol levels in the study were seen in the morning samples during the peak session compared to the low session. Cortisol levels during the day were stable in both low and peak seasons (Table 2 and Figure 1).

The average morning cortisol levels during peak season are higher compared to afternoon and evening following Pawluski et al. (2017). Plasma cortisol concentrations were significantly lower in the evening, after a full day of work, and after one day of rest, rather than in the morning. According to Lestari et al. (2023), horses are more comfortable in evening on environmental temperature conditions. Horses have a circadian cortisol rhythm that reaches the maximum in the morning, and minimum in the evening (Bohak et al., 2013). This is because the horse is not exposed to any disturbances that could create a false diurnal rhythm. The mean cortisol levels of horses during the afternoon and evening during both low and peak sessions were not differentially significant.

The mean cortisol levels increased during the day in the low season when the number of visitors and horse riders was lower than during the peak season. This is following Henshall et al. (2022), the salivary cortisol concentration of trained horses decreased during learning while the concentration of other groups (inactive group) increased. Exercise is a physical activity that leads to increased oxygen consumption, metabolic load, and as a consequence, increased cardiac output as well as various other physiological processes to support body compensation (Caspersen et al., 1985). Exercise has been showing positive effects on horses and other species. Sports activities have been proven to benefit the brain for learning, memory, and cognitive function (Basso and Suzuki, 2017). Domesticated horses routinely perform a warm-up session before training or competition (Chatel and Walliams, 2021), although the intensity of the warm-up was found to be negatively correlated with subsequent performance in a steeplechase competition (Stachurska, 2018).

The analysis results showed morning cortisol levels were significantly different (p<.05) during low and peak season. This is strongly correlated to the number of visitors in the zoo and who made direct interactions with the horses. Venugopal and Sha (1993) have reported that behavioral problems in animals could be triggered when visitors tease the animals. Social integration decreased in captivity groups and increased behavioral abnormalities during the presence of visitors have been reported (Mitchell et al., 1991). A different opinion is expressed by Carlstead and Brown (2005) that chronic exposure of captive animals to zoo visitors can reduce stress in some species. Animals are more resilient to disturbance from tourists when they are in larger groups (Newsome et al., 2001) since individuals require less alert time within collective alertness in large groups (Blumstein and Daniel, 2003). Horse-human interactions have influenced the subjective emotional experience and behavioral expression of horses. This might be due to the intensive management, handling, and interactions (Hall, 2018). This study showed the highest average cortisol levels in the morning at peak season were 462.26±67.21 with the number of visitors at 759±202 and 12±6 horse riders.

Stress experienced by animals can be known through changes in levels of the hormone T3 (triiodothyronine) and hormone T4 (thyroxine) in addition to the hormone cortisol. Both hormones are hormones produced by the thyroid gland. Generally, T3 and T4 regulate basal metabolic rate, oxygen use, cellular metabolism, average tissue growth, and differentiation (Cochran, 2011). In this study, the ratio of T3/ T4 in the morning during the peak session was lower than during the intense session, while during the day, the ratio in the peak session was higher than in the joint session. This shows that when cortisol is high, it will impact a low T3/T4 ratio; conversely, when cortisol decreases, there will be an increase in the T3/T4 ratio again. A high T3/T4 ratio can be caused by the absence of conversion of T4 into a more active T3 hormone, so there will be an increase in the value of the T3/T4 ratio. According to Ferlazzo et al. (2018), increased levels of the hormone T3 are positively correlated with the hormone cortisol. The sudden increase in the T3 hormone is the body’s physiological response in carrying out metabolic adaptations in dealing with changes that occur (Fazio et al., 2022). In horses that exercise, there will be a sudden increase in T3; this phenomenon is most likely a way for horses to adjust to the exercise given (Gonzales et al., 2010). Based on Figure 2 and Table 3 the high ratio of T3/T4 during the day during the peak session is the body’s response after an increase in morning cortisol. The body’s ability to convert T4 into T3 is an indicator of the body’s recovery response, as happened in this study; the value of the T3/T4 ratio changes in the afternoon shows that horses used as riding horses can adjust metabolism in the recovery process from exercise carried out in a day.

 

Table 3: The average morning, afternoon, and evening T3/T4 ratio of the riding horse during low dan peak season.

	Ratio T3/T4
	Morning	Afternoon	Evening
Low session	0.57 ± 0.068	0.55 ± 0.047	0.52 ± 0.014
Peak session	0.50 ± 0.061	0.58 ± 0.056	0.55 ± 0.19

 

The occurrence of fluctuations in fecal cortisol metabolites indicates the functioning of HPA axis regulation of cortisol, and chronic stress does not occur that changes the regulation (Starr et al., 2019). According to Ferlazzo et al. (2018), there is an interaction between the HPA and HPT axes; their research stated that horse exercise can cause an endocrine response and the role of iodothyronine in overcoming the stimulation. Spiga et al. (2014) state that the hypothalamic-pituitary-adrenal (HPA) axis regulates circulating glucocorticoid hormone levels. The central neuroendocrine system in mammals provides a quick response and defense against stress. According to Ewen (2007), glucocorticoids (cortisol in humans and corticosterone in rodents) are vital hormones that regulate many physiological functions, including glucose, fat, and protein metabolism. In basal conditions without pressure, glucocorticoids are released with a pronounced circadian rhythm, characterized by peak levels of glucocorticoids during the active phase (Spiga et al., 2014).

The reduction of average cortisol levels in the afternoon at both seasons shows the normal circadian rhythm of the riding horse in the JE mini zoo. The hypothalamic-pituitary-adrenal (HPA) axis regulates circulating glucocorticoid hormone levels and is the primary neuroendocrine system in mammals that provides rapid response and defense against stress; when unpressurized basal conditions, glucocorticoids are released with circadian rhythms (Spiga et al., 2014). Cortisol production has an independent daily circadian rhythm with the lowest concentration at night (Ljubijanke et al., 2008). According to Pawluski et al. (2017) cortisol levels can be used as an indicator of animal welfare. Horses kept in a welfare-supporting environment will have low plasma and fecal (FCM) cortisol levels.

Conclusions and Recommendations

The cortisol will reach its highest level in the morning and then decrease in the afternoon and evening during both low and peak sessions. The number of tourists and the number of horse riders are positively correlated with cortisol levels elevation but not significantly different. The T3/T4 ratio is highest during the day at peak session but will return to basal levels in the afternoon. This shows that the concept of edu ecotourism in minizoos has considered animal welfare.

ACKNOWLEDGMENTS

We are thankful to the research team, Wagimin, DVM is the leader of the Jogja Exotarium Mini Zoo and staff. This research was funded by the Ministry of Research and Technology DIKTI with Contract No 3137/UN1/DITLIT/Dit-Lit/PT.01.03/2023.

NOVELTY STATEMENT

The findings of this study provide evidence that incorporating riding horses in tourism, especially in Indonesia, with the concept of Edu-ecotourism, can be done while maintaining animal welfare. This can serve as a useful reference for tour managers who share the same concept.

AUTHOR’S CONTRIBUTION

All authors participate from the beginning of this study until the publications are released.

Conflict of interest

The authors declare have no conflict of interest. All co-authors have seen and agree with the contents of the manuscript and there is no financial interest to report. We certify that the submission is original work and is not under review at any other publication.

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