INTRODUCTION

Currently many researchers studying stress in animals from different approaches (Olmos-Hernandez et al., 2008; Vahdatpour et al., 2009; Ceylan et al., 2009; Dehghan et al., 2010; Yildirim and Yurekli, 2010; Hamidi et al., 2010; Mota-Rojas et al., 2011). The handling loading transport unloading and stunning of animals for slaughter can have detrimental effects on their welfare (Peeters et al., 2005; Mota-Rojas et al., 2009, 2006; Huertas-Canen, 2010; Grandin, 2010; Dalmau et al., 2010). Several studies have confirmed the effects of short- or long-term transport to the slaughterhouse due to the signs of stress manifested by slaughter animals (Ali and Al-Qarawi, 2002; Broom, 2003a, b; Mota-Rojas et al., 2006; Becerril-Herrera et al., 2009a, b, 2010). This situation has led to intense discussions on the role of handling and transport from the point of view of animal welfare which has become a controversial topic especially when we take into account the various physiological indicators associated with stress a condition that has long been closely related to animal welfare (Bareham, 1972; Bryant, 1972; Wood-Gush et al., 1975; D’Eath et al., 2010). Earlier studies of this issue have evaluated stress levels by measuring changes in blood levels of several elements including beta endorphins (Murphy and Fordman, 1988) neutrophils/lymphocytes (Church et al., 1994) glucose and creatine kinase (Kannan et al., 2000; Mota-Rojas et al., 2009) and lactate minerals and blood gases (Gonzalez-Lozano et al., 2009; Becerril-Herrera et al., 2009a; Mota-Rojas et al., 2009). However, cortisol has been the most widely studied physiological indicator of transport-induced stress (Crookshank et al., 1979; Averos et al., 2007) because of the increased glucocorticoid secretion that occurs when animals are exposed to stressful situations (Averos et al., 2007; Becerril-Herrera et al., 2009a, b).

The negative stimuli generated by conditions that provoke stress may induce changes in homeostasis and cause adverse reactions that affect animal welfare especially when exposure is prolonged (Spraker, 1993). As a consequence of exposure to stress animals may exhibit weight loss or reduced weight gain or attain a less efficient feed-to-meat conversion index. Moreover, they may become more susceptible to disease and thus increase mortality rates (Adeola and Ball, 1992; Knights and Smith, 2007). For all these reasons assessments of the welfare of animals intended for slaughter should not be limited to just evaluating the quality of the final products obtained from them and their economic importance; i.e., the deteriorated meat quality and higher economic losses that can be caused by transport to the slaughterhouse depend on both the time required to perform the operations involved and the animals’ exposure to different stress-generating situations such as temperature changes, noise, movement and the mixing of animals from different groups (Peeters et al., 2005).

The factors that influence stress and mortality during animal transport have been reviewed previously by Warriss (1998) who concluded that the two most important factors to be considered are the ambient temperature during the journey and genetic merit. In the case of swine this latter concept refers to those animals that experience more adverse responses to stressful situations. On this topic, Averos et al. (2007) observed that transporting pigs for short periods and in winter constitute much more stressful conditions. One specific problem to contemplate in this sense is the fact that short trips do not allow animals enough time to fully recover from the initial stress caused by excessive handling during loading into the vehicle which also has a negative impact on their welfare (Broom, 2000; Averos et al., 2007).

In addition factors such as gender the use of electric prods charge densities the availability of water and bedding ventilation systems mixing animals from different groups and frequent stops during the trip have all been identified as stressors during the transport of hogs (Averos et al., 2008). In this regard no methods currently exist to accurately quantify the response of the animals nor do we have established methods that can minimize or prevent stress. Several studies have focused on the use of drugs to mitigate or avoid stress in domesticated wild or laboratory animals (Ali and Al-Qarawi, 2002) but in the specific case of animals destined for human consumption there are strict limitations on the use of such substances associated with such important factors as elimination times and the retention of drug residues or metabolites in animal tissues before and after death. Therefore, the objective of this review was to carry out an analysis of the different therapeutic methods that can be used to reduce stress in slaughter animals during transport to the slaughterhouse.

Susceptibility to stress: Before performing any therapeutic procedure designed to manage stress in transported animals we must first consider the species to be treated and its peculiarities in terms of susceptibility to stress. This will allow us to identify the specific needs of each species and determine the particular measures that can best be implemented instead of taking a generalized approach to the treatment of transport-induced stress. Thus, it is important to take into account advances in research related to certain factors that have been considered of key interest (such as gender and breed) as they relate to different animals’ susceptibility to stress.

Although it is unclear whether the gender (male/female) of the animals transported is related to susceptibility. Warriss (1996) observed that while intact males are more aggressive than females they are also more sensitive to transport-induced stress. Indeed Guardia et al. (2004) reported that male pigs exposed to stress will likely produce meat that is Pale Soft and Exudative (PSE). In contrast Van der Wal et al. (1990) and Perez et al. (2002) found that females are more sensitive to stress. Meanwhile a study by Averos et al. (2007) showed that in general the values for cortisol glucose Creatine Phospho Kinase (CPK) Lactate Dehydrogenase (LDH) albumin and total serum protein were higher in both genders after transport and unloading at the slaughterhouse findings that corroborate the effects of the journey on the hogs. Though baseline cortisol levels were higher in males compared to females it turned out that serum albumin and protein values were higher in females.

With respect to breed differences in resting muscle metabolism in hogs of different genotypes are sensitive to halothane while disorders in the regulation of Ca⁺⁺ can lead to the production of poor quality meat regardless of the degree of pre-slaughter stress. This might explain why pigs that are highly susceptible to stress produced meat with PSE characteristics even when the levels of pre-slaughter stress were relatively low (Klont et al., 1993; Cooper et al., 2004). Variations in the post-mortem metabolism of halothane among genotypes are probably caused by changes in the release of calcium, as the halothane gene encodes a release channel for this mineral in the sarcoplasmic reticulum of pigs (Harbitz et al., 1990).

An additional factor to be pondered is that transport-induced stress can increase susceptibility to infections and disease that in turn may can trigger the reactivation of viruses already present in the pigs (Broom and Kirkden, 2004; Broom, 2005). The activation of pathogens is likely associated with the presence of lymphopenia and neutropenia as Kannan et al. (2000) observed in a study of goats transported in a reduced living space for 18 h without food.

Procedures used to reduce stress in slaughter animals: Transporting animals to abattoirs is a process that entails exposing them to numerous stressful stimuli including environmental physiological and psychological factors (Knowles, 1998; Werner and Gallo, 2008; Tadich et al., 2009). Given this several studies have tested strategies that use drugs and supplements to mitigate and/or prevent stress in animals intended for human consumption during transport Table 1. Indeed the literature on this topic mentions that a common practice in the meat-producing industry consists in using medical treatments to prevent stress thus avoiding the penalties (i.e., low demand low price) that the market imposes on poor quality meat that may result directly from transport to the slaughterhouse (Cooper et al., 2004) and minimizing economic losses due to the death of animals that are more susceptible to stressful conditions. The most common cause of animal mortality in these conditions is heart failure brought on by an excessive release of catecholamines from the adrenal sympathetic nervous system (Gregory and Wilkins, 1982, 1986).

In the past, animals often received treatments to reduce stress that included tranquilizing drugs. However, due to the risk of residues in meat, the use of such sedatives has been banned in the European Union so an urgent need to find new ways of calming animals during transport has emerged. One possible solution is the use of legally-approved food additives that can be administered in the animals’ food or drinking water and whose effects on stress reduction and meat quality are well known (Peeters et al., 2005). Currently the selection and application of drugs (agonists antagonists dopaminergic agonist benzodiazepines opiates barbiturates) or dietary supplements (magnesium vitamins amino acids) as anti-stressors is done empirically and usually with no scientific justification in terms of the neurochemistry of stress. This fact becomes important when we consider that the treatments used to control stress in domesticated animals (especially those that affect the Central Nervous System [CNS]) can adversely affect the posture stability and locomotion of the animals so treated and may also have undesirable consequences from the point of view of public health if the meat from those animals is intended for human consumption (Ali and Al-Qarawi, 2002).

The scientific literature available includes references to the fact that the application of anesthetics sedatives and β-adrenergic blocking agents to help prevent or reduce stress responses in animals during transport to the slaughterhouse generally produces satisfactory results (Cooper et al., 2004). However, many of the procedures described in those studies are difficult to incorporate into daily farming and handling practices. Moreover when evaluating the application of drugs in day-to-day practice the precise pharmacology of each substance must be judged for example xylazine is a potent alpha2-adrenergic that is classified as a sedative/analgesic with muscle relaxant properties. Though its effects are similar to those of morphine it does not cause excitement in cattle but rathe sedation and depression of the CNS. In addition it is important to consider the species to which the drug may be administered. In the case of xylazine once again the dosage required for swine is 20-to-30 times greater than that for ruminants so it is rarely used with the former (Plumb, 2002).

Among other drugs used the phenothiazines and butyrophenol sedatives are active in the central nervous system while β-adrenergic blocking agents act on the autonomic nervous system and so cannot be considered as sedatives strictly speaking although they are used to inhibit the actions of catecholamines (adrenaline and noradrenaline). According to Cooper et al. (2004), sedatives and β-adrenergic blocking agents commonly used to prevent stress during the transport of swine include the phenothiazines acepromazine propionilpromacina and chlorpromazine and the azaperone and carazolol which act as β-blockers.

Azaperone is allowed in the United Kingdom as a veterinary product in meat producing animals (StresnilTM Janssen) though studies in pigs found high concentrations of azaperol the main metabolite of azaperone, in the kidneys. In North America azaperone is listed in Annex I of the Regulatory Council (EEC, 1990) No.2377/90 which establishes a Maximum ResidueLlimit (MRL) of 100 mg kg^-1 in the kidneys and muscles of pigs.

Table 1:	Physiological responses to administration of drugs and supplements used to alleviate transport-induced stress

In Mexico MRLs have been established at 100 mg kg^-1 in the muscles of cattle and horses and at 60 mg kg^-1 in the case of pigs, according to the “Inter-Departmental Committee for the Review of Programs on Toxic Residues and Contaminants in Foods of Animal Origin” (Comité Intersecretarial para la Revisión de los Programas de Residuos Tóxicos y Contaminantes en Alimentos de Origen Animal), an agency of Mexico’s Department of Agriculture, Livestock, Rural Development Fisheries and Alimentation (Secretaria de Agricultura, Ganadería, Desarrollo Rural Pesca y Alimentación or SAGARPA). Turning to the physiological effects of azaperone studies have shown that it may impact the cardiovascular system of pigs in three direct ways first it has a marked effect as a á-adrenergic blocker second it could have a slight effect as a β-blocker and third it appears to slow the sympathetic reflexes (Gregory and Wilkins, 1986). Other studies have demonstrated that β-adrenergic blockers can reduce the expression of malignant hyperthermia in pigs that are susceptible to stress (Lister et al., 1976) by decreasing peripheral vasoconstriction and thus causing an increase in the rate of the elimination of body heat. Therefore, Azaperone could reduce the supply of the substrates required to fuel the glycolytic reactions during transport-induced stress (Gregory and Wilkins, 1986).

Acepromazine (ACP^TM, C-Vet VP) and chlorpromazine are also allowed in the UK and in veterinary products for use with pets. Chlorpromazine is found in Annex VI of Regulatory Council (EEC, 1990) No. 2377/90. Propionilpromazine is another important derivative of phenothiazin but as no Maximum Residue Limits (MRLs) have yet been established for them their use in animals intended for human consumption is strictly prohibited.

Carazolol is not allowed in the UK for treating food-producing animal though it is available in Spain some Latin American countries and Mexico where it is a licensed veterinary product (DIVASA-FARMAVIC). However carazolol is found in Annex I of Regulatory Council No. 2377/90. In North America MRLs have been established at 25 and 5 mg kg^-1 in pig kidneys and muscles, respectively according to Regulatory Council No. 1442/95. In Mexico the MRLs are set at 5 mg kg^-1 in the urine and muscle of cattle horses and pigs according to the aforementioned “Inter-Departmental Committee” (coordinated by the SAGARPA).

It is well known that stress stimulates an increase in the heart beat contractile force and speed of transmission due to the excessive release of catecholamines by the sympathetic-adrenal nervous system which activates β-adrenergic receptors and can lead to heart failure. In this regard, Cardenas-Moya et al. (1987) have evaluated administering carazolol (a specificβ1-blocker) in transported pigs as a means of generating a better cardiac output and reducing both the stressful conditions and deaths from heart attack. Added to that ismain carazolol blocker β1 and β2 and therefore carazolol lactoacidemia prevents stress-induced has a hypertensive action indirectly by blocking receptors on peripheral blood vessels (Gregory and Wilkins, 1986, 1982) so its use in potential use in everyday practice must be examined in future work.

Despite the benefits of the use of drugs in managing stress in transported animals their adverse effects on animal welfare cannot be left out of consideration. Those effects may include increased urinary activity decreased platelet aggregation inhibition of glycogenolysis in the cardiac and skeletal muscles and an increased eosinophil count among others (Kannan et al., 2000).

Of the different supplements and drugs used to mitigate the effects of transport-induced stress in slaughter animals some have been shown to have more beneficial effects. This is the case of seaweed extract (Kannan et al., 2007) magnesium (Apple et al., 2005; Peeters et al., 2005) tryptophan Vitamins C and E and carazolol the latter assessed earlier by Gregory and Wilkins (1982) and Cardenas-Moya et al. (1987). Table 1 shows some of the strategies that have been assayed to reduce stress and their effects on the physiological responses of farm animals.

Dietary supplements used to reduce stress in animals destined for slaughter: Currently the most widely studied supplements used to prevent stress during the transport of slaughter animals are those based on certain vitamins minerals and amino acids. Apparently the inclusion of these and other substances in animal diets could provide a practical and less expensive alternative to drugs that act on the CNS as long as they show the ability to eliminate or at least mitigate the collateral responses that result from stress. Also these biological substances may have fewer side effects than chemical-based drugs (Ali and Al-Qarawi, 2002). Thus their implementation in animal-rearing practices could have a significant impact on the welfare of animals during their mobilization to the slaughterhouse.

Minerals: It has been shown that adding Magnesium (Mg) to the diets of farm animals can decrease plasma concentrations of cortisol and catecholamines during stress thus reducing neuromuscular stimulation (Kietzmann and Jablonski, 1985) by exerting an antagonistic effect on Ca⁺⁺ (D’Souza et al., 1998).

Though explanatory mechanisms are still unclear research has shown that Mg⁺⁺ supplements in pigs’ diets reduce plasma concentrations of cortisol and catecholamines (D’Souza et al., 1999), resulting in animals that are significantly more tranqui l after long journeys (Kuhn et al., 1981). Similarly D’Souza et al. (1998) found that pigs treated with this mineral had low levels of lactate in the Longissimus thoracis muscle (LT: 3.20 vs. 3.80 mg g^-1) and the biceps femoris (BF: 2.80 vs. 3.40 mg g^-1) compared to a control group after transport. However some studies question the effect of Mg⁺⁺ as an anti-stressor: Geesink et al. (2004) for example found no differences in lactate concentrations in Mg-supplemented animals at slaughter. It is worth noting however that in recent studies blood lactate concentrations have been used as an indicator of stress in pigs transported to slaughter (Becerril-Herrera et al., 2009a). Other indicators used to determine the degree of stress to which animals are exposed include blood concentrations of cortisol glucose and calcium. In this regard several studies have found that adding Mg²⁺ to the diets of pigs decreases plasma concentrations of cortisol (Kietzmann and Jablonski, 1985) however those observations contrast with findings from Lim et al. (2004) and Apple et al. (2005) who found no effects on blood concentrations of cortisol after the inclusion of Mg⁺⁺ in the diet of pigs transported under stressful conditions, compared to a control group (10.93 vs. 13.81 mg dL^-1).

The reduction of the glycemic index has been attributed to the increased energy demand caused by the handling and movement of the animals (Becker et al., 1989). Studies have shown that the decrease in glycogen is associated with the presence of the fear of threats or actual fighting so that injuries like bumps or bruises have been related to poor levels of animal welfare that result in Dark Firm Dry meat (DFD) caused by low glycogenolysis and a high pH (Broom, 2005). Apple et al. (2005) observed no differences in blood glucose levels between pigs that suffered transport-induced stress and unstressed animals both fed with 2.50% Mg⁺⁺ (95.60 vs. 95.20 mg dL^-1). That same study also determined that the levels of Ca⁺⁺ and Mg⁺⁺ in the blood revealed no differences between groups (Ca⁺⁺: 13.20 vs. 12.98 mg dL^-1 Mg 2.13 vs. 2.10 mg dL^-1). Finally those authors noted that unstressed pigs had higher plasma insulin concentrations than transported pigs (8.76 vs 7.27 mUI mL^-1).

Vitamins: Vitamin E has been the subject of several experiments that have tested its inclusion in animal diets and effects on meat quality (Eichenberger et al., 2004) factors closely related to the stress experienced by the animals prior to slaughter. Findings showed that vitamin E may reduce stress during the transport of animals to the slaughterhouse as assessed by decreased heart rates (209 to 204 bpm) and ventricular ectopic beats that suggest a sedative and anxiolytic effect. Peeters et al. (2004) observed an effect of adding Vitamin E to the diet of pigs that decreased cortisol concentrations in saliva after transport. Coupled with that result they also observed more stable concentrations of lactate (18 vs. 20 mg dL^-1) and CK in the blood (1530 vs. 3179 IUL^-1) compared to a control group. There is a relationship between the output of CK into the bloodstream and damage to the membranes in the muscle tissue. In this respect vitamin E works as a stabilizer of the cell membranes especially during oxidative stress situations where those membranes may suffer significant damage.

Amino acids and promoters of antioxidant activity: Supplementation with the amino acid precursors of neurotransmitters involved in stress responses may offer a practical way of reducing pigs’ responses to the transport process (Adeola and Ball, 1992). In this vein, studies have shown that increasing the concentration of tryptophan in the diet resulted in higher brain serotonin synthesis in rats and pigs (Leathwood, 1987; Adeola and Ball, 1992) an increase in hypothalamic serotonin concentrations that has been shown to have a sedative effect (Leathwood, 1987; Henry et al., 1992). Results showed that serotonin levels are 28% lower in pigs that exhibit a high degree of stress compared to animals that experienced minimum stress at slaughter. These findings may indicate that increased brain serotonin reduces stress responses in pigs (Henry et al., 1992).

In addition, natural additives have been used as antioxidants in the diets of goats Kannan et al. (2007) showed that including seaweed in the diet prior to transport had no influence on the level of plasma cortisol which is a physiological indicator of stress responses to transport however when there is a significant increase in antioxidant activity a decrease in lipid peroxidation was seen. This antioxidant activity prevents the formation of O₂ radicals such as superoxide and superoxide dismutase which are produced during stress (Kannan et al., 2007). The superoxide radical is a type of oxygen that can be produced by the incomplete reduction of O₂. Superoxide dismutase converts superoxide into oxygen and hydrogen peroxide by catalyzing the dismutation reaction.

When handling pigs an additional factor that can lead to a rapid increase in blood lactic acid levels is exercise (Van den Hende et al., 1970; Bertol et al., 2005). Such metabolic responses to acute stress may also compromise meat quality when they occur just prior to slaughter (Channon et al., 2000) hence any approach that helps to decrease lactate production and its effect on reducing blood pH during handling is of practical importance (Bertol et al., 2005).

L-carnitine can also be used as an adjunct to the metabolism of fatty acids (Ags) because it acts as a carrier of long chain Ags from the cytosol into the mitochondrial matrix and plays a key role in the use of Ags as an energy substrate in tissues (Bertol et al., 2005).

Though including additives in animal diets may modify responses to stress more experiments are needed with large numbers of animals and different species before we can arrive at any firm conclusions as to the efficacy and safety of such agents. Economic considerations for their inclusion are certainly an important factor in considering their use as anti-stressors however, improved transport conditions for the animals and reducing or preventing exposure to stressor stimuli could be ethically and financially more effective than using drugs or supplements to alleviate or reduce the effects of stress (Ali and Al-Qarawi, 2002).

Management strategies to control stress in animals during transport: Another alternative for resolving the problem of stress in transported animals is to ensure good management at this stage of the meat chain. As is well known, transport time often ranges from 1-to-12 h though it can go as high as 60 h (Gallo, 2008). In the case of cattle and sheep the duration of transport commonly increases due to intermediary distributors or delays caused by inclement weather (heat rain storms) or poor road conditions. Results from Gallo (2008) indicate that non-stop journeys that carry cattle for 24-to-36 h are detrimental to animal welfare as reflected in the measurement of blood variables known to be related to stress (e.g., cortisol glucose package cell lactate CPK).

It has also been suggested that assuring the proper handling of animals before during and after transport can diminish stress levels (Gonsalvez et al., 2006). Results from Averos et al. (2008) indicate that changes in animal management procedures designed to counteract the high ambient temperatures during the summer months can help maintain animal welfare and meat quality in swine because long trips adversely affect their wellbeing and therefore the quality of the meat they produce (Perez et al., 2002). Broom (2005) argues that all animals can be better prepared for transport through appropriate pretreatment regimes.

There is no question that transport modifies animal physiology (Broom, 2000; Averos et al., 2007) and that pre-slaughter rest aids in the recovery from previously experienced stress (Brown et al., 1999). According to Warriss et al. (1992), an average of 6 h of pre-slaughter rest is sufficient to assure a full recovery from transport-induced stress. Averos et al. (2007) observed a decrease of CPK and LDH during pre-slaughter rest suggesting a probable recovery from the earlier stressful condition. The handling of animals without the use of electric prods or sticks also results in improved welfare and a reduced risk of poor quality meat. Moreover sound knowledge of animal behavior and the quality of the facilities involved in their handling loading transport etc. are all keys to maintaining an adequate state of welfare during those processes (Broom, 2005). Other common problems include measures that are taken in attempts to reduce overhead and transportation costs and malpractice during loading and unloading; conditions often made worse by the poor design and maintenance of handling equipment and transport vehicles. Gallo (2008) and Klont et al. (1993) for example, mention that the stress caused by premedication procedures can provoke high lactate levels in seconds due to increased blood levels of lactic acid and calcium. However, when stress conditions were very low no differences were observed in muscle metabolism at slaughter (Klont et al., 1993).

Implications for public health: Some of the drugs used to reduce the adverse effects of stress during animal transport have relatively long elimination periods that may compromise the safety of the final product. For example the accumulation of waste or its metabolites can affect human health if consumed causing a public health problem. This is the case of sedatives such as the phenothiazines and butyrophenols. Mexico however does not enforce any legislation to regulate the application of drugs or supplements designed to decrease stress in animals transported to abattoirs though efforts have been made to ensure that animal products do meet international requirements of safety and quality, in order to guarantee the absence of very high-risk substances for the health of human populations and the environment. The institution responsible for these controls has been the SAGARPA’s “Interdepartmental Committee” (mentioned above) but more research is needed to support local laws on the transport and slaughter of animals destined for human consumption. It is also important to ensure that animal handlers and carriers all along the meat-producing chain are adequately trained and sufficiently well-informed to assure that significant improvements are achieved in animal welfare and meat quality whenever possible (Gallo, 2008). Finally another key aspect is the creation or improvement of laws that govern the application of drugs used to reduce stress in transported animals intended for human consumption and their possible effects on public health.