Ili horses are characterized with strong pull performance, beautiful appearance, both speed and strength, high milk yield, and meat production rate. Ili horse breeding has become a pillar industry of animal husbandry in Xinjiang. Nevertheless, due to the fact that there are long distances between farms and hippodromes or slaughter plants, continuous transport for 24 h is common, and some trips last for 48 h or longer (Friend, 2000). Transportation has increasingly become one of the main causes of stress for animals (Pan et al., 2018). There are several studies reporting that transportation has been identified as a stressor for horses, and has been associated with several adverse outcomes including respiratory, gastrointestinal disease, and injury (Riley et al., 2018). For example, transportation can increase heart rate and serum cortisol (COR) concentration, which could further lead to gastric ulceration (Padalino et al., 2020). Moreover, an increased risk of transport-associated respiratory diseases is identified through clinical examination and observation of behavior along the way (Padalino et al., 2018a). Injury resulting from road transport is common in horses and is a potential welfare concern as well as a source of economic loss (Padalino et al., 2018b).

According to the theory of traditional Chinese medicine, Qi and blood are the material basis to maintain the normal function of the body. Qi can drive the movement of blood, blood can carry Qi, and Qi and blood breed mutually. Qi deficiency leads to less blood, and blood deficiency signals less blood. In recent years, using herbal medicines to relieve stress has received the widespread attention of researchers all over the world for the simple reason that herbal medicine exerts no side effects and increases a wide range of biological activity (Jiang, 2017; Liu et al., 2018; Padalino and Raidal, 2020). For example, it is reported that Sini powder, Banxiahoupu decoction, Ganmaidazao decoction, and Guizhigancaolonggumuli decoction could increase the anti-stress ability by affecting the different taches of the hypothalamus-pituitary-adrenal gland axle and changing the stress behaviors, which are based on their respective functions of regulating Qi, dispersing phlegm, tonifying Qi, and warming Yang (Ji et al., 2019). Chinese herbal medicine Yin-Chen-Hao-Tang can reduce the hepatic oxidative stress in obese mice with steatosis (Lee et al., 2010).

The Chinese herbal medicine prescription, Suanzaoren decoction, consists of the following components: Ziziphi Spinosae Semen, Poria cocos Wolf, Anemarrhena, Szechuan Lovage Rhizome, Licorice, Codonopsis, Radix Aucklandiae, and Polygala. Among them, Ziziphi Spinosae Semen, due to the effects of nourishing the heart and liver, calming the heart and mind, condensing sweat, and promoting body fluid, has been widely used in treating insomnia and anxiety (Cao et al., 2010; Yang and Zhang, 2015). Poria cocos Wolf, Radix Aucklandiae, and Anemarrhena have the beneficial efficacy of calmness, anti-inflammation, and antibacterial effect and have been reported to enhance the immune response and inhibit inflammation (Wagner et al., 2015; Lee and Cha, 2018; Ji et al., 2019). Szechuan Lovage Rhizome not only promotes Qi circulation and removes collateral obstruction, but also helps to dissipate coldness and dispel stasis and regulates vital energy and soothing liver (Cui et al., 2013). Licorice has the effects of clearing away heat and detoxification, relieving cough and expectorating, invigorating the spleen and Qi and has been used to treat many ailments including lung diseases, arthritis, kidney diseases, eczema, heart diseases, low blood pressure, gastric ulcer, liver toxicity, allergies, and certain microbial infections (Mamedov and Egamberdieva, 2019). Codonopsis has the effects of nourishing the middle and nourishing Qi and invigorating the spleen and lungs. It can also enhance immunity, dilate blood vessels, lower blood pressure, improve microcirculation, and enhance hematopoietic function (Singh et al., 2004). Polygala has the functions of soothing the nerves, improving the mind, eliminating phlegm, and reducing swelling (Farina et al., 2005). However, whether Suanzaoren decoction can alleviate the stress in horse transportation is unclear.

Herein, we investigated the efficacy of Suanzaoren decoction in relieving stress of horses during transportation. The efficacy and safety of Chinese herbal medicine Suanzaoren decoction was analyzed. Our findings may provide evidence for the use of Suanzaoren decoction as an intervention to alleviate stress of horses caused by transportation.

The transportation stress of livestock and poultry is referred to as an adaptive and defensive response of livestock under the stimulation of stressors such as fasting and water prohibition, environmental changes (temperature and humidity), and turbulence during transportation, which will affect the production performance, endocrine metabolism, and immunity of the animals (Padalino et al., 2018a). Transportation is considered stressful because horses are confined in a small space, and have higher arousal and the difficulty of maintaining balance during transport (Padalino and Raidal, 2020).

Ziziphi Spinosae Semen, a prototypical hypnotic-sedative herb in oriental medicine, exhibits anxiolytic effects by improving amygdaloid CRF/CRF1 receptor signalling (Li et al., 2019). In addition, the Ziziphi Spinosae Semen can be effectively used as a healthy functional food for preventing or alleviating inflammatory diseases or a therapeutic agent for inflammatory diseases (Kim et al., 2018). Poria extract has the effect of nourishing the liver through regulation of lipid metabolism, inhibiting ER stress, and activating autophagy via AMPK activation (Kim et al., 2019). Anemarrhena and Szechuan lovage rhizome extracts have certain antiplatelet and antithrombotic activities (Lu et al., 2011; Wang and Lu, 2011). Horses in transportation may be inflicted by long-term confining in a limited space, causing prolonged hypoxic exposures and resulting in procoagulant state. Codonopsis extract has the effect of improving the procoagulant state induced by hypoxia (Das et al., 2019). Licorice has the effects of clearing away heat and detoxification (Mohseni et al., 2014).

Biochemical and physiological blood indexes can reflect the physiological changes and are usually used to assess animal welfare and health (Galapero et al., 2015). Among them, WBC, GRAN%, and LYM% are generally used as indicators of immune function. In addition, elevation in serum COR concentration in response to stress decreases LYM count (Morán et al., 2017). Similar to previous studies, horses during transport showed increase in neutrophils (GRAN%), COR, and WBC, but showed a decrease in LYM% in our study. However, Suanzaoren decoction intervention could alleviate these abnormalities in inflammation-related indicators caused by transportation stress.

Serum CK activity is considered an important indicator of muscle damage and muscle exhaustion. When animals are subjected to transport stress, CK from muscles is released into the blood (Alcalde et al., 2017). Similarly, we found that Suanzaoren decoction supplementation decreased serum CK activity, suggesting a lower level of tissue damage or physical fatigue. This is possible due to the antioxidant activity of chlorogenic acids in Poria and Licorice. Previous study has reported that stress can inhibit membrane ATPases activities and damage the integrity and permeability of cell membrane. However, antioxidants can restore the ATPases activities, and dietary antioxidants supplementation can decrease serum CK activity in horses subjected to transport and heat stress (Liu et al., 2016). Elevation in serum COR concentrations can increase serum glucose concentrations by increasing glycogenolysis. In the present study, horses during transport showed increase in serum COR concentrations, and GLU had a significant increase. The rapid increase in GLU in horses after transport may be partially due to the gluconeogenic activity of COR as well as hemoconcentration. Our research showed that Suanzaoren decoction relieved GLU and serum COR reduction caused by transportation stress. This may be achieved by reducing the amount of red blood cells and hematocrit in the blood and via reducing the dehydration of animals. There are some limitations for this study due to factors including geographical location, climate, sample size, and temperament of horses.

We intended to investigate the efficacy of Suanzaoren decoction in transport stress of horse, and believe that conscientiously performed studies and positive outcomes can benefit animals during transportation. This study demonstrates that dietary Suanzaoren decoction supplementation relieves horse transportation stress, and can contribute as a useful clinical therapy for relieving transportation stress of horses.