Online Issues

Journal Vol 47 (4)

Establishing an Appropriate Period of Acclimatization Following Transportation of Laboratory Animals

Jennifer A. Obernier and Ransom L. Baldwin

Jennifer A. Obernier, Ph.D., is Senior Project Director with the Institute for Laboratory Animal Research, The National Academies, Washington, DC. Ransom L. Baldwin, Ph.D., is Professor Emeritus of the University of California-Davis, Davis, CA.

Address correspondence to Dr. Obernier, Keck Center of The National Academies, 500 Fifth Street NW, Washington, DC 20001; or email ILAR@nas.edu.

Abstract

Stress associated with transportation has widespread effects on physiological systems in laboratory animals, including changes in the cardiovascular, endocrine, immune, central nervous, and reproductive systems. Although short-lived, these changes can confound research if animals are utilized before homeostasis is restored and physiological measures return to normal. Therefore, some period of acclimatization following transportation is generally suggested to restore homeostasis. The following two questions should be considered to establish an adequate period for acclimatization: (1) Will anticipated physiological changes confound the research to be conducted? (2) What is the length of time necessary for confounding physiological changes to normalize? Finding answers to those questions in the literature can be a challenge. Most literature on the physiological impact of transportation involves agricultural animals, although the limited literature in common laboratory animal species generally parallels changes documented in agricultural animals. The literature documents elevated heart rate and weight loss, as well as elevated concentrations of adrenaline, noradrenaline, glucose, cortisol, free fatty acids, and β-hydroxybutyrate. Carbohydrate, protein, and lipid metabolism (both lipolysis and lipogenesis) are altered, and plasma osmolality, albumen, protein, and pack-cell volume increase. Neutrophilia and lymphopenia are also evident. These measures generally return to baseline within 1 to 7 days of transportation, although animals that are young, severely stressed, and have stress-sensitive genotypes may show altered physiological measures for several weeks. Other measures such as circadian rhythm and reproductive performance may take several weeks to months to normalize.

Key Words: acclimatization; agricultural animals; immune system; physiological effects; rodents; stress; transportation

Introduction

Transportation unavoidably causes stress in animals. Although stress is not always an adverse experience and is a necessary and regular aspect of life, it causes changes in an animal's physiological status during transportation and for some period thereafter. Utilizing transported animals before their physiological status normalizes can have considerable and unintended effects on research results. In this article, we briefly explore some of the physiological changes that have been documented in research animals, and we consider the length of time necessary to properly acclimatize animals following transportation and before the initiation of experiments. Notwithstanding our recognition of the profound effects that transportation can have on animal behavior and subsequent behavioral studies, we focus specifically in this article on physiological changes associated with transportation and their impact on biomedical experimental data. The information herein is based partially on the recently published Institute for Laboratory Animal Research (ILAR¹) report Guidelines for the Humane Transportation of Research Animals (NRC 2006).

Transportation Stress and Physiological Changes

Stress is the biological response animals exhibit in response to stimuli (stressors) that disrupt their homeostasis (Stokes 2000). Animals often adapt successfully to a stressor and return to a normal homeostatic state, resulting in positive health effects (McEwen 2002). Stressors and stress are therefore not inherently negative events; it is generally when stress is long lasting or severe that animals are unable to adapt successfully and adverse consequences occur (NRC 1992).

Mammals respond to stress by releasing a host of primary mediators such as glucocorticoids and catecholamines. These mediators have widespread effects on tissues and cells; they bind to receptors, ion channels, and intracellular proteins to cause primary effects such as activation of signaling cascades and gene expression. Cumulatively, the primary mediators result in secondary outcomes (McEwen and Seeman 1999). For instance, acute stress causes release of the primary mediator epinephrine, which binds to β-adrenergic receptors on the heart, increasing heart rate (Reece 2004). Secondary outcomes are possible in every physiological system including the cardiovascular system, metabolism, the central nervous system, and the immune system (McEwen and Seeman 1999). However, the magnitude and duration of secondary outcomes are greatly influenced by the intensity, duration, and type of stressor. The age, health status, genotype, and previous experiences of animals also influence secondary outcomes.

Although literature on effects of acute and chronic stressors continues to grow, it is difficult to predict physiological changes that will occur in response to transportation from available literature. Transportation involves physical (e.g., temperature changes), physiological (e.g., limited access to food), and psychological stressors (e.g., exposure to novel environments) (NRC 2006). Predicting secondary physiological outcomes resulting from transportation is a challenge because multiple stressors occur, often in combinations unique to a particular transportation scenario or species. For example, swine and cattle are often shipped in large groups on truck beds without food or water (FASS 1999). In this situation, heat/cold stress, social stress, food/water deprivation stress, noise stress, and restraint stress may all occur. Nonhuman primates, however, are often shipped by plane in environmentally controlled compartments with food and water (NRC 2006). In such cases, noise stress, vibration stress, and stress from a novel cage environment are unavoidable and should be expected.

The majority of animals utilized in research are rodents (Trull and Rich 1999) generally shipped in environmentally controlled ground vehicles by commercial vendors (NRC 2006). With the physical environment tightly controlled, the main stressors are psychological. Laboratory animals, particularly rodents, live in uniform environments. Cages, bedding, food, conspecifics, sounds, and odors animals are exposed to in laboratories and breeding facilities comprise their familiar and constant environment. During and after transportation, almost every aspect of the animal's environment changes, with no way for the animal to return to the previously familiar physical and social environments.

Due to the complexity of the stressors associated with transportation, research efforts have been, appropriately, evaluations of physiological changes resulting from actual transportation events. The majority of this literature involves agricultural animals because transport of livestock is an integral part of the production cycle in the United States, and there are social and economic pressures for animals to arrive in good condition. This literature is reviewed briefly in the following section to provide a broad overview of the major physiological changes that have been documented. For an in-depth review of transportation in agricultural animals, we advise readers to consult the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching (FASS 1999).

After review of physiological changes associated with transportation of agricultural animals, we review the limited research available on physiological changes in rodents following transportation. We refer researchers seeking additional information about the effects of transportation on research animals to the recently published ILAR report Guidelines for the Humane Transportation of Research Animals (NRC 2006).

Physiological Changes in Agricultural Animals Following Transportation

When an animal is transported, psychological, physiological, and physical stress result in the release of catecholamines such as adrenaline (epinephrine) (Dalin et al. 1993) and noradrenaline (norepinephrine) (Dalin et al. 1993; Parrott et al. 1998). These releases correspond to elevated heart rates (Ingram et al. 2002; Parrott et al. 1998) and blood glucose concentrations (Kannan et al. 2000; Stull and Rodiek 2000).

Transportation also activates the hypothalamic-pituitary-adrenocortical (HPA¹) axis, which is generally considered to be a response to psychological stress (Knowles and Warriss 2000). Activation of the HPA axis leads to the release of glucocorticoids, of which cortisol is the most commonly measured (Lay et al. 1996; McGlone et al. 1993). Elevated levels of cortisol and other glucocorticoids have been shown to have a significant impact on the immune system (McEwen et al. 1997). Therefore many immunological changes are associated with transportation (see Table 1). These immune changes can lead to delayed morbidity and mortality, particularly in young animals (Swanson and Morrow-Tesch 2001). Glucocorticoids also increase blood glucose levels (Kannan et al. 2000; Kent and Ewbank 1983) through effects on carbohydrate, protein, and lipid metabolism including lipolysis, lipogenesis and gluconeogenesis (Sapolsky et al. 2000).

Many animals respond to the transportation experience by reducing their intakes of food and water (Friend et al. 1998; Knowles et al. 1999). In other cases, it may be necessary for personnel to withhold food or water during transport for safety reasons. Normally, animals lose weight during transport; however, this weight loss is greater than that lost during the same length of time without food and water (Knowles and Warriss 2000). When food intake decreases because food is withheld or due to travel-associated anorexia, the animal's body mobilizes energy stores. Triglycerides mobilized from adipose tissue and are released as nonesterified fatty acids (NEFAs¹). NEFAs are converted, in part, by the liver to ketones, including β-hydroxybutyrate (β-OHB¹). Both NEFA and β-OHB are utilized as energy sources in peripheral tissues (Knowles and Warriss 2000). Elevated levels of both NEFA and β-OHB have been found in the blood of transported animals (Broom 2003). A decrease in water intake also has the direct effect of decreasing intracellular and extracellular fluid volumes (Knowles and Warriss 2000). As a result, plasma osmolality, total plasma protein and albumen concentrations, and packed-cell volume increase (Broom 2003).

Physiological Changes in Rodents Resulting from Transportation

It is reasonable to suggest that rodents likely exhibit similar physiological responses during and after transport. In fact, the limited literature on transportation of rodents (Table 2) indicates that basic measures of stress and immune function are affected in a manner similar to that documented in agricultural animals. In general, it has been reported that glucocorticoid concentrations increased for 1 to 2 days, body weight decreased, and measures of the immune system indicated a suppressed immune response up to 48 hr after transport. These changes have also been documented in dogs (Bergeron et al. 2002) and rabbits (Toth and January 1990).

Acclimatization Following Transportation

Because even the overseas transport of animals takes only a few days, chronic stress resulting in long-term health effects (Irwin 1994; Kiecolt-Glaser et al. 2002) is unlikely to occur during a routine transportation event. The available, albeit limited, literature supports this supposition, documenting that the physiological changes are short lived. Nevertheless, even transient physiological changes can affect biomedical research results substantially if experiments are performed before the animal can acclimate to its new environment and before its physiological status has normalized.

To minimize effects of transportation-induced physiological changes on subsequent biomedical research, it is advisable to consider two factors. The first factor is whether the anticipated physiological changes could confound the research to be conducted. In general, studies suggest that the physiological responses of animals to transportation include changes in the cardiovascular, endocrine, immune, and reproductive systems as a result of stress (Knowles and Warriss 2000). Researchers should carefully consider whether the focus of their research utilizing transported animals involves physiological functions that can be affected by changes in these several systems. For example, it is reasonable to suggest that metabolic studies could be confounded by transportation-induced elevations in glucocorticoids. Researchers should also consider whether a physiological measure that is used as a humane endpoint could be altered by transportation. For example, body weight is a physiological measure commonly used as a humane endpoint in some types of research (Toth 2000). Most animals show decreases in body weight following transportation (see Tables 1 and 2) therefore use of body weight in this situation may not accurately indicate an animal's condition.

The second factor that should be considered is how long it takes for potentially confounding physiological changes to normalize. Generally, primary mediators of a stress response (i.e., catecholamines and glucocorticoids) return to normal concentrations within 24 hr of transportation. Secondary physiological outcomes may take longer to normalize. Although literature on effects of transportation on rodents is limited, it generally includes information on the length of time necessary for different physiological parameters to normalize. As shown in Table 2, most physiological changes in the immune and endocrine systems take 1 to 7 days to normalize. Similar acclimatization periods were documented in rabbits from studies in which hyperglycemia, neutrophilia, lymphopenia, and elevated plasma cortisol levels required 48 hr to normalize (Toth and January 1990). The only physiological parameters in common laboratory species that took longer than 1 wk to normalize were measures of reproductive performance, which required 1 mo for normalization (Hayssen 1998).

We advise researchers to consult the literature for guidance on effects of stress that may confound their study and the required duration for acclimatization. However, before a final determination can be made, it is necessary to consider several other factors. These factors include the intensity and duration of stress; the age, genotype, health status, and previous experience of the animal; and allometric differences. All of these factors can have an impact on the magnitude and duration of physiological changes.

Particularly lengthy or traumatic transportation events might be expected to increase the magnitude of physiological changes and the length of time for these measures to return to normal. Generally, investigators who have compared length of transport with loss of body weight have reported that the longer the animals are transported, the more body weight an animal loses and the longer it takes for animals to regain their initial condition (e.g., Brown et al. 1999). Other studies have documented that physiological measures were affected by transport duration in a linear fashion, including serum glucose, urea nitrogen, and urea nitrogen:creatinine ratio (Cole et al. 1988).

Researchers should also consider the age, genotype, health status, and previous experience of the animal being transported. Young calves, for example, do not exhibit the large changes in heart rate, cortisol, and glucose levels that mature cattle show (Knowles et al. 1997). However, after being transported, young pigs and calves have been found to have unstable metabolic rates, which require ≥1 wk to stabilize (del Barrio et al. 1993; Heetkamp et al. 2002; Schrama et al. 1992).

Genotype and previous experience can also increase or decrease physiological changes. For example, stress susceptibility in swine, characterized by hyperthermia and sudden death following stress, is inherited by a single recessive gene (Halⁿ). When subjected to transport, the three Hal genotypes (Hal^N/N, Hal^N/n, Hal^n/n) had different cortisol concentrations immediately after transport as well as 2 wk after transport (Nyberg et al. 1988).

The allometric differences between small animals (e.g., rodents and young animals) and large animals (e.g., agricultural or adult animals) in energy expenditures should also be considered. Small animals require more calories per unit of body mass and become dehydrated more quickly than larger animals. For this reason, small animals may become stressed more quickly and more severely by food or water deprivation than larger animals, leading to larger and longer-lasting stress responses (NRC 2006).

Conclusion

Prudent investigators would be well advised to ensure that transported animals used in their investigations have had adequate time to acclimatize prior to the initiation of experiments because transportation causes physiological changes that can confound subsequent research. To avoid this consequence, researchers must consider both the physiological changes caused by transportation stress and the durations of these changes. Investigating pertinent literature (reviewed briefly in this article) and considering mitigating factors such as age, genotype, health, and previous experience will help guide decisions regarding the length of time necessary to properly acclimatize animals.

Researchers must be aware that the physiological changes caused by transport may not be limited to those identified in this article because comprehensive studies to identify all the physiological changes associated with transportation have not been published to date. In addition, generalities discussed in this article may not hold true for every species. For example, cortisol levels have not been found to correlate with stress in swine during transport (Brown et al. 1999; Hicks et al. 1998). Based on this and other likely differences between species, we urge researchers to thoroughly examine the literature for relevant information. We hope that the information in this article will guide and facilitate their search.

Abbreviations used in this article: β-OHB, β-hydroxybutyrate; HPA, hypothalamic-pituitary-adrenocortical; ILAR, Institute for Laboratory Animal Research; NEFA, nonesterified fatty acid.

References

Aguila HN, Pakes SP, Lai WC, Lu YS. 1988. The effect of transportation stress on splenic natural killer cell activity in C57bl/6j mice. Lab Anim Sci 38:148-151.

Bergeron R, Scott SL, Edmond JP, Mercier F, Cook NJ, Schaefer AL. 2002. Physiology and behavior of dogs during air transport. Can J Vet Res 66:211-216.

Broom DM. 2003. Transport stress in cattle and sheep with details of physiological, ethological and other indicators. Deutsche Tierärztliche Wochenschrift 110:81-89.

Brown SN, Knowles TG, Edwards JE, Warriss PD. 1999. Behavioural and physiological responses of pigs to being transported for up to 24 hr followed by 6 hr recovery in lairage. Vet Rec 145:421-426.

Cole N, Camp TH, Rowe LD, Steven DG, Hutcheson DO. 1988. Effect of transport on feeder calves. Am J Vet Res 49:178-183.

Dalin AM, Magnusson U, Haggendal J, Nyberg L. 1993. The effect of transport stress on plasma levels of catecholamines, cortisol, corticosteroid-binding globulin, blood cell count, and lymphocyte proliferation in pigs. Acta Vet Scand 34:59-68.

del Barrio AS, Schrama JW, van der Hel W, Beltman HM, Verstegen MWA. 1993. Energy metabolism of growing pigs after transportation, regrouping, and exposure to new housing conditions as affected by feeding level. J Anim Sci 71:1754-1760.

Dymsza HA, Miller SA, Maloney JF, Foster HL. 1963. Equilibration of the laboratory rat following exposure to shipping stresses. Lab Anim Care 13:60-65.

FASS [Federation of Animal Science Societies]. 1999. Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching. 1st revised ed. Savoy IL: FASS.

Friend TH, Martin MT, Householder DD, Bushong DM. 1998. Stress responses of horses during a long period of transport in a commercial truck. J Am Vet Med Assoc 212:838-844.

Hayssen V. 1998. Effect of transatlantic transport on reproduction of agouti and nonagouti deer mice, Peromyscus maniculatus. Lab Anim 32:55-64.

Heetkamp MJ, Schrama JW, Schouten WG, Swinkels JW. 2002. Energy metabolism in young pigs as affected by establishment of new groups prior to transport. J Anim Physiol Anim Nutr-Zeitschrift Fur Tierphysiologie Tierernahrung Und Futtermittelkunde 86:144-152.

Hicks TA, McGlone JJ, Whisnant CS, Kattesh HG, Norman RL. 1998. Behavioral, endocrine, immune, and performance measures for pigs exposed to acute stress. J Anim Sci 76:474-483.

Ingram JR, Cook CJ, Harris PJ. 2002. The effect of transport on core and peripheral body temperatures and heart rate of sheep. Anim Welfare 11:103-112.

Irwin M. 1994. Stress-induced immune suppression: Role of brain corticotrophin releasing hormone and autonomic nervous system mechanisms. Adv Neuroimmunol 4:29-47.

Kannan G, Terrill TH, Kouakou B, Gazal OS, Gelaye S, Amoah EA, Samake S. 2000. Transportation of goats: Effects on physiological stress responses and live weight loss. J Anim Sci 78:1450-1457.

Kegley EB, Spears JW, Brown TT Jr. 1997. Effect of shipping and chromium supplementation on performance, immune response, and disease resistance of steers. J Anim Sci 75:1956-1964.

Kent JE, Ewbank R. 1983. The effect of road transportation on the blood constituents and behavior of calves. I. Six months old. Br Vet J 139:228-235.

Kiecolt-Glaser JK, McGuire L, Robles TF, Glaser R. 2002. Emotions, morbidity, and mortality: New perspectives from psychoneuroimmunology. Annu Rev Psychol 53:83-107.

Knowles TG, Warriss PD. 2000. Stress physiology of animals during transport. In: Grandin T, ed. Livestock Handling and Transport. 2nd ed. Cambridge, MA: CABI Publishing. p 385-407.

Knowles TG, Warriss PD, Brown SN, Edwards JE. 1999. Effects on cattle of transportation by road for up to 31 hours. Vet Rec 145:575-582.

Knowles TG, Warriss SN, Brown SN, Edwards JE, Watkins PE, Phillips AJ. 1997. Effects on calves less than one month old of feeding or not feeding them during road transport of up to 24 hours. Vet Rec 140:116-124.

Landi MS, Kreider JW, Lang CM, Bullock LP. 1982. Effects of shipping on the immune function in mice. Am J Vet Res 43:1654-1657.

Lay DC Jr, Friend TH, Randel RD, Jenkins OC, Neuendorff DA, Kapp GM, Bushong DM. 1996. Adrenocorticotropic hormone dose response and some physiological effects of transportation on pregnant Brahman cattle. J Anim Sci 74:1806-1811.

McEwen BS. 2002. The End of Stress as We Know It. Washington DC: Dana Press.

McEwen BS, Seeman T. 1999. Protective and damaging effects of mediators of stress: Elaborating and testing the concepts of allostasis and allostatic load. Ann N Y Acad Sci 896:30-47.

McEwen BS, Biron CA, Brunson KW, Bulloch K. Chambers WH, Bhabhar FS, Goldfarb, RH, Kitson RP, Miller AH, Spencer RL, Weiss JM. 1997. The role of adrenocorticoids as modulators of immune function in health and disease: Neural, endocrine and immune interactions. Brain Res Rev 23:79-133.

McGlone JJ, Salak JL, Lumpkin EA, Nicholson RI, Gibson M, Norman RL. 1993. Shipping stress and social status effects on pig performance, plasma cortisol, natural killer cell activity, and leukocyte numbers. J Anim Sci 71:888-896.

Murata H, Takahashi H, Matsumoto H. 1985. Influence of truck transportation of calves on their cellular immune function. Nippon Juigaku Zasshi 47:823-827.

NRC [National Research Council]. 1992. Recognition and Alleviation of Pain and Distress in Laboratory Animals. Washington DC: National Academy Press.

NRC [National Research Council]. 2006. Guidelines for the Humane Transportation of Research Animals. Washington DC: National Academies Press.

Nyberg L, Lundstrom K, Edfors-Lilja I, Rundgren M. 1988. Effects of transport stress on concentrations of cortisol, corticosteroid-binding globulin and glucocorticoid receptors in pigs with different halothane genotypes. J Anim Sci 66:1201-1211.

Parrott RF, Hall SJG, Lloyd DM, Goode JA, Broom DM. 1998. Effects of a maximum permissible journey time (31 h) on physiological responses of fleeced and shorn sheep to transport, with observations on behaviour during a short (1 h) rest-stop. Anim Sci 55:197-207.

Phillips WA, Juniewicz PE, Zavy MT, von Tungeln DL. 1989. The effect of the stress of weaning and transport on white blood cell patterns and fibrinogen concentration of beef calves of different genotypes. Can J Anim Sci 69:333-340.

Reece WO, ed. 2004. Dukes' Physiology of Domestic Animals. 12th ed. Ithaca: Cornell University Press.

Rowland RT, Cleveland JC, Upadhya P, Harken AH, Brown JM. 2000. Transportation or noise is associated with tolerance to myocardial ischemia and reperfusion injury. J Surg Res 89:7-12.

Sapolsky RM, Romero LM, Munck AU. 2000. How do glucocorticoids influence stress responses? Integrating permissive, suppressive, stimulatory, and preparative actions. Endocrine Rev 21:55-89.

Schaefer AL, Jones SD, Stanley RW. 1997. The use of electrolyte solutions for reducing transport stress. J Anim Sci 75:258-265.

Schrama JW, van der Hel W, Arieli A, Verstegen MW. 1992. Alteration of energy metabolism of calves fed below maintenance during 6 to 14 days of age. J Anim Sci 70:2527-2532.

Stokes WS. 2000. Reducing unrelieved pain and distress in laboratory animals using humane endpoints. ILAR J 41:59-61.

Stull CL, Rodiek AV. 2000. Physiological responses of horses to 24 h of road transportation using a commercial van during summer conditions. J Anim Sci 78:1458-1466.

Swanson JC, Morrow-Tesch J. 2001. Cattle transport: Historical, research, and future perspectives. J Anim Sci 79(Suppl E):E102-E109.

Tarrant PV, Kenny FJ, Harrington D, Murphy M. 1992. Long distance transportation of steers to slaughter: Effect of stocking density on physiology, behavior and carcass quality. Livestock Prod Sci 30:223-238.

Toth LA. 2000. Defining the moribund condition as an experimental endpoint for animal research. ILAR J 41:72-79.

Toth LA, January B. 1990. Physiological stabilization of rabbits after shipping. Lab Anim Sci 40:384-387.

Trull FL, Rich BA. 1999. More regulation of rodents. Science 284(5419):1463.

van Ruiven R, Meijer GW, Wiersman A, Baumans V, van Zutphen LF, Ritskes-Hoitinga J. 1998. The influence of transportation stress on selected nutritional parameters to establish the necessary minimum period for adaptation in rat feeding studies. Lab Anim 32:446-456.

Wallace ME. 1976. Effects of stress due to deprivation and transport in different genotypes of house mouse. Lab Anim 10:335-347.

Weisbroth SH, Paganelli RG, Salvia M. 1977. Evaluation of a disposable water system during shipment of laboratory rats and mice. Lab Anim Sci 27:186-194.