Effect of Dietary Inclusion of Live Microbial Cultures on Faecal Glucocorticoid Metabolites in Safari Asian Elephants

Asian elephants are the largest land animals in Asia and a species of immense eco-tourism significance (Campos-Arceiz and Blake, 2011). In recent decades, the rapid decline of their population has resulted in as endangered (Choudhury et al., 2008). Conservation of wildlife species globally, the Asian elephant has been listed on the International Union for the Conservation of Nature Red List of Threatened Species and declared as endangered migratory species at UN Convention on Migratory Species (CMS COP13, 2020).
       
Despite many positive benefits of wildlife ecotourism (Kruger, 2005) increased human interactions with wildlife can trigger stress, affecting animal physiology or behaviour (Ahlering et al., 2011). Chronic stress can compromise an animal’s immune function, reduce disease resistance and provoke uncharacteristic aggression (Teixeira et al., 2007). Faecal endocrinology has essential applications for the conservation of wildlife because it facilitates the non-invasive monitoring of adrenal activity in wild animals (Möstl and Palme, 2002). The concentration of faecal glucocorticoid hormone, released in response to perceived stress (Santymire et al., 2012) and its metabolites (fGCM) is a reliable indicator circulating in livesock’s body over a time period.
       
Although only limited information is available on dietary feeding of probiotics, increased nutrient utilization was observed in the Asiatic elephants (Senthilkumar et al., 2017). Microbiota is currently an essential issue in disease and, health and many studies have revealed it to play an indispensable role in physiological homeostasis and health promotion (Alayande et al., 2020). Under stressed conditions, probiotics can be used directly to reduce the risk or severity of diseases caused by the disruption of the healthy intestinal environment (Uyeno et al., 2015). The current study was carried out to evaluate the effect of dietary inclusion of live microbial cultures on faecal glucocorticoid metabolites (fGCM) in safari asian elephants.

Study animals and design
 
Eighteen adult captive female Asian elephants of 30-62 years age, similar body weight (3495 ± 133.34 kg) and of uniform conformation were randomly selected. They were divided into three groups with six elephants in each. The elephants were stall-fed a consistent diet of green Pearl millet fodder (approx. 300-350 kg per day per animal) as basal diet throughout the research period, i.e. for a period of two months, including of preliminary 10 days for adaptability with basal diet and fifty days for the feeding trial. The experiment was conducted from months of August to October, 2019 at Elephant Village, Jaipur (India) (26°59'47"N 75°52'35"E).
 
Experimental feeding of probiotics
 
Two different probiotics, i.e. Lactobacillus acidophilus and Saccharomyces cerevisiae, were supplemented along with basal diet to all elephants of T₂ and T₃ treatment groups, respectively, during feeding trial of 50 days. The probiotics supplemented in this study was a commercially available powder product by Meteoric Biopharmaceuticals Pvt. Ltd., Ahmedabad, Gujarat (India). The products contained 1×10⁹ colony-forming units (CFU)/g concentrate of Lactobacillus acidophilus and 1×10⁹ colony-forming units (CFU)/g concentrate of Saccharomyces cerevisiae. One gram probiotic was given, orally per 50 kg body weight. Body weight was calculated as per Hile et al., (1997).
 
Sample collection and preservation
 
A total of 72 fresh faecal samples were collected from elephants of all three groups on day 0, 30^th, 40^th and 50^th of the experiment. Faecal samples were collected between 8:00 to 9:00 hrs in the morning. Freshly collected samples were oven dried the same day at 60°C-80°C, pulverized and stored in sealed containers with silica beads at 4°C. These samples were transferred to the freezer (- 20°C) within a week and stored until further analysis (Hunt and Wasser, 2003).
 
Physical activities of elephants
 
Elephants were taken out for safari from elephant village to Amer fort and vice versa from 8:00 AM to 12:00 PM, which covers approximately 10 kilometres distance both sides. Amer fort is located high up on the Aravali hill. Elephants carried two passengers per safari maximum five times a day and each safari took approximately 30 minutes, which covers about 500 meters. Then, elephants were allowed for safari tours from 1:00 PM to 6:00 PM within elephant village, average 2-5 safari tours of approximately 30 minutes/tour. Elephants were given feed, water and rest from 12:00 PM to 1:00 PM and other than safari tours. Elephants had a weekly off from safari. Apart from this, elephants were taken out for rehearsal and procession of Elephant Festival; Hindu festivals like Teej and Gangaur, etc. Elephants were also used for other commercial activities like marriage and resorts. Usually an elephant spends 90% of its time to feed, but working elephants spend less time for feeding and this could be stressful, resulting in elevated glucocorticoid (Kumar et al., 2014).
 
Extraction of steroid metabolites
 
The faecal samples were extracted according to the previously described procedure (Ganswindt et al., 2005; Kumar et al., 2014). The pulverized faecal sample was sieved and 0.2 g of faecal powder was transferred to a 15 mL falcon tube, 5 mL of 80% methyl alcohol was added and it was vortexed vigorously at least for 20 min. Then, the sample was centrifuged at 3300´g for 20 min; the supernatant was transferred into 5 mL of a cryogenic vial and stored at - 30°C until further assay. The extraction efficiency was calculated by adding a known amount of ³H labelled cortisol to faecal samples before extraction (Umapathy et al., 2013; Kumar et al., 2014). Extraction efficiency was found to be 85.6% ± 6.3 for cortisol.
 
Glucocorticoid immunoassay and procedure
 
The faecal glucocorticoid metabolites were measured using a single antibody competitive enzyme immunoassay incorporating a polyclonal cortisol antibody (R4866), as described earlier (Munro and Lasley, 1988; Kumar et al., 2014). The cortisol antibody was diluted to 1:9000, HRP (Horseradish peroxidase)-conjugated cortisol 1:250,000 (Dr C. Munro, University of California, Davis, CA, USA) and cortisol standards used as 1000-1.9 pg/well. The cortisol assay was successfully validated in elephants for reliable quantification of adrenal activity (Kumar et al., 2014).
 
Statistical analysis
 
The experiment design was a Complete Randomized Design. All statistical analysis of data was performed using SPSS 16 version for windows. The difference among groups was determined by one way ANOVA analysis. The significant effects of different means were compared by Duncan’s Multiple Range Test. Significance was defined at P<0.05. All values represent mean ± standard errors of the mean.

The mean faecal glucocorticoid metabolite (fGCM) concentration per elephant varied from 1.69 to 2.81 ng/g (mean ± SE=2.24 ± 0.20 ng/g), 1.36 to 2.13 ng/g (mean ± SE=1.89 ± 0.12ng/g), 1.01 to 3.36 ng/g (mean ± SE=2.11 ± 0.33ng/g) dry weight of faeces in the animals of T₁, T₂, T₃ groups, respectively on 0 day, 1.59 to 2.27 ng/g (mean ± SE=1.85 ± 0.12 ng/g), 1.27 to 2.66 ng/g (mean ± SE=2.19 ± 0.22ng/g), 1.71 to 2.27 ng/g (mean ± SE=1.92 ± 0.08 ng/g) dry weight of faeces in the animals of T₁, T₂, T₃ groups, respectively on 30^th day, 1.05 to 2.46 ng/g (mean ± SE=1.80 ± 0.19ng/g), 1.55 to 2.25 ng/g (mean ± SE=1.87 ± 0.10 ng/g), 1.32 to 2.17 ng/g (mean ± SE=1.82 ± 0.12 ng/g) dry weight of faeces in the animals of T₁, T₂, T₃ groups, respectively on 40^th day, 2.13 to 2.80 ng/g (mean ± SE=2.48 ± 0.10ng/g), 2.07 to 3.06 ng/g (mean ± SE=2.47 ± 0.15ng/g), 1.66 to 3.12 ng/g (mean ± SE=2.25 ± 0.21ng/g) dry weight of faeces in the animals of T₁, T₂, T₃ groups, respectively on 50^th day.
                     
The mean fGCM concentration, demonstrated by the elephants of the control group i.e.T₁ was not significantly (P>0.05) different from the elephants of T₂ and T₃ groups (Table 1). However, mean fGCM concentration were found to be higher in T₁ group and lowest in T₃ group at 0 day and 50^th day of experiment whereas, fGCM concentration were found to be higher in T₂ group and lowest in T₁ group at 30^th day and 40^th day of the experiment. The fGCM concentration level gradually decreased up to 40^th day then increased on 50^th day in T₁ control group and T₃ group, whereas unstable variation was seen in T₂ group (Fig 1).
 

 

       
The overall mean fGCM levels were found to be higher in the study elephants of T₂ group and lowest in T₃ group (Table 2). Therefore, none of the probiotics had any significant difference of faecal glucocorticoid concentration of study elephants with elephants of the control group and which demonstrated that fGCM concentration values remained unaffected on dietary inclusion of live microbial cultures.
 

       
The present study investigated monitoring of non-invasive stress using 72 faecal samples from 18 captive Asian elephants at Elephant Village, Jaipur and suggests that supplementation of live microbial cultures to working elephants were not significantly related to fGCM concentration. The health status of elephants was found to be normal as they were regularly monitored as part of management; however, the availability of resources and physical activities vary widely across the facilities.
       
Similar to this study, previous researcher (Klasing, 2005) revealed that metabolites of stress hormones that are analyzed in faeces are the products of extensive alteration by bacteria in the gut. Alteration by bacteria has also been made liable for changes in metabolite concentration of hormones during the storage of faecal samples (Hunt and Wasser, 2003). Dietary changes may affect plasma glucocorticoids levels to some extent but more seriously fGCM concentration (Goymann, 2005). Diet and microbial composition can potentially alter the metabolism of glucocorticoids. It can also affect faecal bulk and metabolic rate; both can affect fGCM levels. Dietary effects may be more pronounced in avian species, because their diet can change dramatically over a year and because they excrete faeces and urine together in the form of droppings (Goymann, 2005; Klasing, 2005). However, there was no effect of diet (P = 0.385) or probiotic (P = 0.388) on plasma cortisol concentration. No difference between treatment groups was found for change in plasma cortisol in response to exogenous ACTH (Swanson, 2002).
 
The noticed difference in glucocorticoid elevation in Asian elephants and a shortage of it in African elephants might be due to the differences in management practices and may depend on individual livestock’s physiology. The average glucocorticoid metabolite level is shown to be lower by captive female Asian elephants exhibited by female elephants, which live in restricted environmental and space conditions (Kumar et al., 2014).
       
Nishanth (2013) revealed that easy availability of forage materials and the absence of other stress-causing factors might have contributed to the less faecal cortisol levels in elephants of Mudumalai sanctuary than in Anamalai sanctuary.
       
In the present study, mean fGCM value of the groups was, however, lower than that of normal values of elephant, i.e. about 65 ng/g, as observed by (Wasser et al., 2000) during transportation and values of 90 ng/g, should be considered high and indicate a stressed elephant.
       
Lama (2017) quoted that the increase in average fGCM concentration was relatively small (4.0 ng/g) on days with the ride and mixed ride/walk activities versus days when these activities did not occur.
       
The wild adult male African elephants (Ahlering et al., 2011) and captive male Asian elephants (Kumar et al., 2014) exhibited relatively higher fGCM concentration than did females. The literature on African and Asian elephants suggests that musth is not necessarily associated with elevated glucocorticoid levels (Ganswindt et al., 2003; Ahlering et al., 2011).
       
The overall mean basal concentration of fGCM was observed as 2.40 ± 0.33 ng/g to 9.4 ± 0.72ng/g (Kumar et al., 2014), 17.35 ± 6.23 ng/g (Wong et al., 2016), 0.45 to 18 ng/g (mean ± SE=3.09 ± 0.14 ng/g) (Vijayakrishnan et al., 2018) and 7.36 to 21.55 ng/g. (Kumar et al., 2019) in Asian elephants.

In general, the elevations in faecal glucocorticoids might be regarded as indicative of physiological stress response in Asian elephants. Diet and microbial composition can possibly affect the metabolism of glucocorticoids. The current study concluded that supplementation of live microbial cultures to working elephants had no significant effect to fGCM concentration. This could probably be attributed to the easy availability of green forage and devoid of other stress-causing factors which might have contributed to the less faecal cortisol levels in Asian elephants.