1Fluctuations in thermal microenvironment variables inside the vehicle during a four-hour road transportation of ostriches. http://ojvr.org/index.php/ojvr/article/downloadSuppFile/388/409
2Responses of heterophils and lymphocytes of ascorbic acid-treated (n = 10) and control (n = 10) ostriches to road transportation stress. http://ojvr.org/index.php/ojvr/article/downloadSuppFile/388/410
3Relationships between the hour of the journey, thermal load, rectal temperature and aberrant behaviours in ascorbic acid-treated (n = 10) and control (n = 10) ostriches during the road transportation period. http://ojvr.org/index.php/ojvr/article/downloadSuppFile/388/411
DiscussionThe results obtained in the present study showed that the transportation of ostrich chicks during the hot-dry season is very stressful and poses a risk to the health of the birds, findings which agreed with previous reports (Foggin 1992; Minka & Ayo 2007c, 2008; Mitchell et al. 1996). The fact that the ostrich is known to withstand very high environmental temperatures without any consequential adverse effects on its homeostasis (Stewart 1994) suggests that the mean AT value of 35 °C – recorded during transportation in the present study, and which was far below the temperature (50 °C) at which ostriches begin to experience adverse effects to their homoeostasis – may not be a major cause of transportation stress in the ostriches. This may be true because, unlike mammals and other birds, ostriches have excellent ability to thermoregulate under high AT. However, during transportation, they suffer considerable stress when the AT values are either above or below 20 °C (Payne 1993; Piccione et al. 2005), which is the recommended thermoneutral zone for ostriches (Skadhauge 1981). The mean TL value of 51.0 °C ± 7.8 °C and AT value of 33.6 °C ± 1.1 °C recorded during the journey were similar to the TL and AT values of 44.0 °C and 34.0 °C, respectively, obtained during transportation of pullets, and which were reported to induce heat stress in pullets (Minka & Ayo 2010). The fact that the TL increased with the hour of the day and journey duration suggests that as the journey progressed the effect of TL on the birds also rose. In addition, panting behaviour of the birds increased the RH in the vehicle, which in turn increased TL. This apparently exacerbated the mechanism of thermoregulation by increasing evaporative heat loss and inducing hyperthermia in the ostriches. Other studies have shown that even at an AT of 18.0 °C and RH of 89% ostriches still suffer transportation stress (Payne 1993). It appears therefore that other transportation factors such as handling, loading, vehicle vibration, motion, noise and novelty of environment may be the major stress factors affecting the ostrich during transportation, rather than high AT and RH. This requires further investigation. The base-line values of RT recorded in both treated and control ostriches before transportation did not differ from each other and from the normal range RT values of 38.3 °C to 39.3 °C established for ostrich chicks (Skadhauge 1981; Stewart 1994), although the RTs were at their lower limit values. The results were similar to those obtained by Stewart (1994), who showed that ostriches, in contrast to other birds, have an RT below 40.0 °C and in the range of the eutherian mammals. The increase in the RT value recorded in AA-treated group 30 min after the administration of AA, but before loading, was within the normal range for the ostrich, but was higher than that of the control, which demonstrated that administration of AA induced RT elevation in the ostriches. This finding supports earlier results that AA exerts a biphasic effect on RT. Firstly, it increases the RT whenever the value is at a lower limit threshold as observed in AA-treated ostriches in the present study. Secondly, it reduces RT values that are at the maximum threshold (Ayo, Minka & Fayomi 2005; Ayo, Owoyele & Dzenda 2007). The increase in RT values recorded in the control group 30 min after handling and loading, but before the transportation, showed that these procedures were stressful to the ostriches. Similar observations were made during the handling, loading and transportation of birds (Minka & Ayo 2007b, 2011). The increase in RT value at this stage of transportation may not be associated with an increase in TL, but could be explained as a result of physical activity; and also by stress-induced hyperthermia, which is the regulated shift of the thermoregulatory set-point mediated by prostaglandin E and interleukin 1 and 6 (Kent & Ewbank 1983; Piccione et al. 2007). Stress-induced hyperthermia is associated with cortisol release and is known to attain peak level within 10 min of administration of a stress factor (Piccione et al. 2007). The fact that a non-significant (p < 0.05) increase in RT value was recorded post-loading in the AA-treated ostriches suggested, for the first time, that AA alleviated the effects of handling and loading stress on RT of the ostriches. The mechanism of action of AA on the handling and loading stress factors may be caused by the fact that AA is an inhibitory vitaminergic neurotransmitter in the hypothalamus, which plays an important role in thermoregulation by inhibiting cortisol, the chief hormone of stress (Balz 2003; Kannan et al. 2002). This is an important finding because handling and loading of ostriches are the most critical procedures and the major cause of poor welfare (Minka & Ayo 2007c, 2008). The progressive increase in RT value during the transportation, especially in the control birds, suggested that transportation itself, apart from the handling and loading procedures, further increased the release of cortisol, which apparently continued to alter RT. The present finding agrees with those of Knowles et al. (1996), Hartung (2003) and Minka and Ayo (2008), who established that the period of transportation of an animal is one of the most traumatic in its life. This is particularly true of ostriches, as they are very social birds and relatively minor changes in their social status may cause severe stress (Stewart 1994). The effects of stress caused by road transportation acting concomitantly with high AT and RH on the ostriches in the vehicle further aggravated the stressful conditions of the birds. The insignificant (p > 0.05) change in RT value recorded in the AA-treated ostriches, compared to those obtained in the control, further indicated that AA did not only ameliorate the stress imposed by handling and loading, but also the stresses encountered during the actual process of road transportation. Such stresses included vehicle motion, noise, vibration and speed, high AT and RH, long duration of journey, deprivation of food and water, novelty of environment, and confinement. The findings of the present study demonstrated that AA reduced significantly the stress on RT induced by road transportation. The results were similar to those obtained during transportation of ostriches and poultry by road (Minka & Ayo 2008, 2010). Similar ameliorating effects of AA on the RT of pullets have been documented (Minka & Ayo 2010, 2011). The behavioural responses recorded during the third hour of transportation revealed some unusual and aberrant behaviours such as pecking (directed to the ground or to objects), wing fluffing (piloerection) and panting (polypnoea), especially in the control ostriches. The manifestations of these behaviours suggest that the ostriches were stressed and the level of stress increased with the duration of the journey and TL. Although ostriches are known to have a wide range of thermoregulatory set points, it has been reported that ostrich chicks are affected severely by hot weather (Minka & Ayo 2007c; Reiner, Seitz & Dzapo 1996; Stewart 1994), as confirmed by the present study. The aberrant behaviours of polypnoea and piloerection were responses of the birds to increased demand in oxygen and heat dissipation brought about by the high AT and RH recorded in the vehicle during the transportation. Because ostriches, like other birds, have no sweat glands, the only means available for cooling is evaporation via polypnoea and piloerection by exposing the bare skin on their thorax and upper legs to enhanced convection and radiation losses. Similar observations on the behaviours of ostriches exposed to different thermal conditions have also been reported (Samson 1996). Although the vehicle cabin was well ventilated, the air entering the vehicle was very hot and did not exert any cooling effect on the birds. This is characteristic of the hot-dry season in the zone of study. The present result showed that ostriches began to pant when the TL value was 40 °C. The result obtained in AA-treated birds demonstrated that AA has reduced the effect of TL on the birds and facilitated the physiological mechanism of thermoregulation. It has been established that AA decreases heat load through reduction in heat production or increasing heat loss by enhancing thermal exchange between the body and the environment (Chang et al. 1993; Minka & Ayo 2010; Tauler et al. 2003). Similarly, AA enhanced the adaptability of poultry and animals to environmental stress (McKee & Harrison 1995; Minka & Ayo 2010). The mechanism of action of AA in reducing heat stress may also be through the detoxification of reactive oxygen species known to be in abundance in the body during stressful conditions (Minka & Ayo 2007b; Tauler et al. 2003). The intense pecking behaviour exhibited by the ostriches in the control group may be associated with frustration, hunger and metabolic stress caused by transportation procedures, which are tasking and completely novel to the birds (Foggin 1992). The fact that pecking behaviour was less manifested in AA-treated birds suggested that AA reduced the bird’s frustration. AA is known to be the first vitaminergetic neurotransmitter and it plays a significant role in inhibiting cortisol, the chief hormone of stress, and the limbic region involved in the elicitation of fear and frustration (Balz 2003; Karanth et al. 2000). The significant (p < 0.05) increase in H to L ratio recorded in the control ostriches post-transportation indicated that the immune system of the birds was compromised as a result of transportation up to the third day of the post-transportation period. The H to L ratio has been used as the most common reliable index of stress measurement from blood analysis (Kannan et al. 2002; Minka & Ayo 2007b, 2011). A similar effect of transportation stress on H to L ratio was reported in poultry, including ostriches (Minka & Ayo 2007b, 2008, 2011) and the increase in H to L ratio has been reported to reflect the effects of elevated corticosteroids in the circulation induced by stress (Kannan et al. 2002; Schalm, Jain & Carroll 1975). The insignificant change in H to L ratio obtained in AA-treated ostriches post-loading was an indication of reduced physiological stress. The result showed, for the first time, that AA alleviated the stresses induced by both the transportation proper and the post-transportation procedures on the lymphocytes and heterophils of the ostriches. AA has been reported to be a chain-breaking antioxidant, involved in the prevention and restriction of free-radical chain formation and propagation. Consequently, AA enhances the immune status of the ostriches by protecting the blood cells, including heterophils and lymphocytes, from oxidative damage induced by free radicals and cortisol (Alok et al. 2003; Elsayed, Farghaly & Abu-Taleb 2008; Tauler et al. 2003). Although there are no recommended doses for AA as an antioxidant in ostriches, it has been shown that it ameliorates heat stress in ostriches in supplementation, at a dose of 250 mg/kg of feed (Elsayed et al. 2008). Conclusion The results of the present study have demonstrated for the first time that AA administration prior to the commencement of road transportation was beneficial to ostrich chicks and may be used to improve the bird’s welfare and make their transportation more economically viable. For example, AA ameliorated the adverse effects of stress caused by road transportation on the behaviour, RT and H to L ratio of ostrich chicks. It also enhanced the homeostatic mechanisms of behavioural, thermoregulatory and cellular immune responses of ostrich chicks to the stressful conditions imposed by road transportation during the hot-dry season. AA is readily available, cheap, safe and easy to administer in ostrich chicks, making it an ideal supplement for use during transportation. Acknowledgements The authors are grateful to all laboratory staff of the College of Agriculture and Animal Science at the Ahmadu Bello University, Kaduna for their technical assistance. Competing interests The authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this paper. Authors’ contributions M.N.S. (Ahmadu Bello University, Kaduna) was the project leader. He supervised the transportation, provided logistics, collected and prepared all samples and data. He also provided an initial draft of the manuscript, whilst A.J.O. 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