Under the high environmental temperatures in tropical and subtropical regions, farm animals, including rabbits, are subjected to heat stress, which results in poor production traits. Provision of natural and safe dietary supplements to mitigate the negative impacts of rising temperatures has become necessary. Recently, climate change, especially the expected global rise in surface temperature, has constituted a serious hazard for livestock production ( Dangi et al. , 2016 ). In particular, high ambient temperatures are a major stress factor for rabbits due to their dense fur and few functional sweat glands, which greatly hinder heat loss ( Marai et al. , 2002 ). In tropical and subtropical regions, heat stress is aggravated by high relative humidity, which can reach 85% during hot months ( Marai et al. , 2007 ). Alterations in the rectal, skin and ear temperatures; respiration rate; and thyroid and stress hormone, albumin, globulin, total lipid, glucose, sodium, potassium, calcium, magnesium and phosphorus levels are the main physiological responses to heat load in rabbits ( Marai et al. , 2008 ). Heat load also causes increased oxygen-derived free radicals, which create a condition of oxidative stress ( Sahin & Kucuk, 2003 ).

Various genetic, managerial, nutritional, buffering, hormonal and physical mitigating strategies have been adopted to palliate the adverse effects of heat stress ( Fayez et al. , 1994 ; Marai et al. , 1999 ). Vitamins, essential oils, fats and amino acids are the main dietary supplements with marked antioxidant properties. Recently, the use of natural antioxidants of plant origin has caught the attention of both livestock producers and nutritionists, as these supplements can be added to the daily diet with ease at an affordable price and are expected to have very few side effects ( Tawfeek et al. , 2014 ). In rabbits, vitamin C (ascorbic acid) and E are the most common and widely used antioxidants to alleviate heat stress ( Yassein, 2010 ; Arafa Mervat et al. , 2012 ). Betaine (BET), which is a trimethyl form of glycine, has been found to alleviate heat stress in sheep and poultry ( Zulkifli et al. , 2004 ; DiGiacomo et al. , 2012 ).

Lemongrass essential oil (LGEO) is a volatile oil that can be extracted directly from fresh lemongrass ( Cymbopogon citratus ) using steam extraction; the grass contains 0.035% essential oil ( Malee et al. , 2000 ). LGEO is characterized by the presence of various phyto-constituents, including α-citral, β-citral, isoneral and α-myrcene. Al-Sagheer et al. (2018) found improvements in the productive performance, immunity and disease resistance of Nile tilapia fed a diet supplemented with LGEO. Extra virgin olive oil (EVOO) is obtained through mechanical pressing of the olive mesocarp. This oil has potentially beneficial effects, including antimicrobial, antioxidant and anti-inflammatory properties, due to its components, such as phenolic compounds, carotenoids and tocopherol ( Cicerale et al. , 2012 ). Gallic acid (GA) is a natural polyphenolic that possesses several pharmacological activities, including antioxidant, anti-inflammatory, anti-mutagenic, anti-carcinogenic, and anti-allergic activities ( Jo et al. , 2006 ). However, little evidence is available to date regarding potential beneficial roles of LGEO, EVOO, and GA against heat stress in rabbits.

We previously discussed the effects of EVOO, LGEO and GA supplementation on the growth, nutrient digestibility, antioxidant status, and lipid peroxidation of growing rabbits ( Al-Sagheer et al. , 2017 ). The objective of this study was to evaluate the effects of EVOO, LGEO, GA, and BET as non-traditional antioxidants on different body temperature traits and on stress hormone and glucose levels in growing rabbits under severe heat stress compared to the effects of vitamin C (VC) and vitamin E (VE) as traditional antioxidants.

Heat stress can evoke multiple biological and physiological responses that can become fatal if not appropriately controlled ( Ducray et al. , 2016 ). In the rabbit industry, heat stress is an important stressor that affects productive performance, and the use of eco-friendly dietary additives to alleviate the negative impacts of heat stress remains a vital issue ( Ayyat et al. , 2018 ). Therefore, the present in vivo study aimed to evaluate the beneficial roles of the non-traditional feed additives EVOO, LGEO, GA, and BET in the alleviation of the effects of heat stress in growing rabbits compared to the effects of VC and VE.

Throughout the experimental period, thermoregu­latory parameters, including rectal, ear and skin temperatures, were estimated as indicators for the initial response of rabbits to air temperature fluctuations, as adopted by previous investigators ( Marai et al. , 2007 , 2008 ). In the control group, exposure to severe heat stress (a THI of 84.67) resulted in a significant increase in all body temperature traits compared to those of the other additive-supplemented groups. Failure of the physiological mechanisms of animals to balance the excessive heat loads caused by exposure to high ambient temperatures could be responsible for the increase in both the rectal and ear temperatures of the heat-stressed rabbits ( Habeeb et al. , 1998 ). Moreover, the high skin temperatures in hot climates may be linked to the insulating effect of the hair coat ( Marai et al. , 2008 ).

Among the treated groups, EVOO and BET showed significant improvements in different body temperature traits with potencies similar to those of VE, followed by GA and LGEO. Similarly, in slow-growing chicks, dietary supplementation with BET at 1 g/kg and VC at 250 mg/kg was equally potent for partial amelioration of the effects of heat stress on performance ( Qota et al. , 2008 ). As an osmolyte and methyl group donor, betaine may maintain the thermo-neutral state of animals by reversing the heat-induced inhibition of the osmotic equilibrium and maintaining the tertiary structures of macromolecules in the kidney and other tissues ( Lever et al. , 2004 ; Huang et al. , 2007 ).

High temperatures in mammals are known to cause physiological stress in organisms with enhanced generation of reactive oxygen species (ROS), leading to oxidative damage ( Jena et al. , 2013 ). Hence, in the current study, the significant palliative effects of EVOO on various body temperature traits of growing rabbits may have been associated with the phenolic antioxidant components of EVOO, including hydroxytyrosol and oleuropein, and its monounsaturated fatty acids (MUFAs, especially oleic acid). In our earlier study using GC-MS analysis, EVOO was shown to contain 1-octadecene, octacosanol, delta-3-carene, docosane, 17-pentatriacontane, and n-hexadecanoic acid, whereas the major identified fractions of LGEO were citral, a-myrcene, and cis-geraniol ( Al-Sagheer et al. , 2017 ). The components of both EVOO and LGEO were reported to have antioxidant activity ( Kumar et al. , 2010 ; Guimarães et al. , 2011 ; Gurnani et al. , 2016 ). Consequently, their antioxidant activity may be the key for their roles in improving rectal, ear and skin temperatures in heat-stressed growing rabbits ( Nhu-Trang et al. , 2006 ). GA has also been reported to have potent free radical scavenging and antioxidant activities ( Priscilla & Prince, 2009 ).

In the current experiment, hormonal and glucose assays were performed to further characterize the effects of the tested feed additives on the thermoregulation of heat-stressed growing rabbits. Primarily, a sharp rise in the cortisol levels was found in the control rabbits under heat stress. This outcome could be attributed to activation of the hypothalamic-pituitary-adrenal axis during heat stress conditions that subsequently caused an increase in the serum glucocorticoid concentrations (mainly cortisol). In line with these findings, Kowalska (2011) reported that the blood cortisol level was increased in NZW rabbits exposed to thermal stress. Nevertheless, all tested feed additives had a lowering effect on the rising cortisol levels compared with the control group levels during the overall trial period. This reduction could presumably be linked to their antioxidant activities, because the glucocorticoid response was shown to be associated with changes in redox physiology ( Costantini et al. , 2011 ). In our previous study, dietary supplementation with EVOO, GA, or LGEO in heat-stressed growing rabbits enhanced catalase activity and reduced glutathione content, whereas EVOO-treated rabbits had the highest catalase activity and reduced glutathione content. Malondialdehyde activity was also reduced in response to all tested additives ( Al-Sagheer et al. , 2017 ). The cortisol-reducing effect of BET may have been due to its positive effect on nitric oxide production, which in turn may inhibit the biosynthesis of glucocorticoids ( Monau et al. , 2010 ; Messadek, 2012 ). GA has also been shown to inhibit the production of both prostaglandins and the enzymes involved in glucocorticoid production ( Hsu et al. , 2007 ; Seo et al. , 2016 ).

In this study, thyroid hormone activities were significantly depressed in the non-supplemented heat-stressed growing rabbits. These results are in a harmony with those reported by Habeeb et al. (1993) in growing male rabbits, in which the thyroxine hormone decreased significantly by 17.86% when the rabbits were exposed to 35°C for six hours daily. Gad (1996) also estimated declines in the T ₃ levels of 21.7 and 20.7% in NZW and Californian rabbits, respectively. Thyroid hormones are the key hormones in the regulation of metabolism and adaptation of animals to stress ( Brecchia et al. , 2010 ). At high ambient temperatures, the activity of thyroid hormones has been suggested to decline due to the decrease in endogenous heat production by the animals and thus depress its growth performance ( Leung et al. , 1984 ). In contrast, both BET and EVOO supplementation elevated the serum T ₃ and T ₄ levels, which could be directly linked to their antioxidant activities; indeed, hypothyroidism has been shown to be related to oxidative stress and cellular damage ( Cano-Europa et al. , 2012 ). Notably, VC and VE produced the highest increased in T ₄ . Similarly, serum concentrations of T ₄ increased to a greater extent by increasing dietary VC or VE levels of Japanese quails reared under heat stress ( Sahin et al. , 2002 ). Mechanism for increasing the levels of T ₄ by VC or VE is not well known ( Omidi et al. , 2015 ). However, it may occur in part because of their antioxidant properties ( Al-Sagheer et al. , 2017 ).

The blood glucose levels showed significant elevation in the control heat-stressed growing rabbits in the present work. In the same line, Rashidi et al. (2010) reported that heat stress condition increased blood glucose levels. The increase in the serum glucose levels under hot climatic conditions could be akin to the decrease in glucose utilization, depression of both catabolic and anabolic enzyme secretions and subsequent reduction in metabolic rates and the preservation of energy ( Hassan et al. , 2016 ). Also, the increase in glucose concentration is directly responsive to an increase in glucocorticoids resulting from heat stress ( Borges et al. , 2007 ). In contrast, a significant decline in the blood glucose concentration was recorded in all dietary supplement groups. Similarly, in Japanese quails reared under heat stress, blood glucose level was observed to move down constantly with increasing dietary levels of VC or VE ( Sahin et al. , 2002 ). Also, Kutlu & Forbes (1993) reported that VC supplementation in heat-stressed broilers significantly decreased blood glucose concentration. Because oxidative stress in the cell blocks normal glucose metabolism, the antioxidant properties of the tested additives could be the key to their beneficial roles in the manipulation of glucose levels ( Shah et al. , 2007 ). Additionally, the lowering of glucose level could be related to the reduced secretion of glucocorticoids following antioxidants dietary supplementation ( Sarica et al. , 2017 ). Also, the decline in the glucose levels could be correlated to lesser extent with the effects of the insulin hormone, because most of the dietary groups showed a trend toward increase in insulin levels which was significant in VC treated rabbits compared with the control group. The observed increase in the insulin level is linked to the decrease in glucose due to an improvement of non-oxidative glucose metabolism ( Paolisso et al. , 1994 ).

These results suggest that in the warm, subtropical environmental conditions of Egypt, the adverse impacts of the exposure of growing rabbits to severe heat stress can be alleviated using natural feed additives of plant origin, including extra virgin olive oil, lemongrass essential oil, gallic acid and betaine. The former outcome was obvious in terms of thermoregulatory parameters and glucose and stress hormone levels. Notably, extra virgin olive oil and betaine showed high potency in heat-stress amelioration that was similar to that of the traditional antioxidants vitamin C and vitamin E.