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Consumption of alcohol by sows in a choice test
Physiology & Behavior 88 (2006) 101 – 107
Consumption of alcohol by sows in a choice test Sylvie Cloutier a,b,⁎, Tracy L. Skaer c , Ruth C. Newberry a,b a
b
Department of Veterinary and Comparative Anatomy, Pharmacology and Physiology, Center for the Study of Animal Well-being, Washington State University, PO Box 646520, Pullman, WA 99164-6520, USA Department of Animal Sciences, Center for the Study of Animal Well-being, Washington State University, Pullman, WA 99164-6520, USA c Department of Health Policy and Administration, Pharmacoeconomics and Pharmacoepidemiology Research Unit, Washington State University, Pullman, WA 99164-6510, USA Received 18 April 2005; received in revised form 21 February 2006; accepted 9 March 2006
Abstract The domestic pig (Sus scrofa domesticus) has been proposed as an animal model for human alcoholism because pigs have been observed to consume alcohol voluntarily to a state of intoxication and to exhibit tolerance and physical dependence. However, it has not been established whether pigs can develop psychological dependence on alcohol. We hypothesised that feed-restricted, stall-housed pregnant sows fed alcohol nonvoluntarily for 5 weeks would develop a preference for alcohol and retain this preference after removal of alcohol from the diet. We fed crossbred commercial sows (n = 10) 280 ml of 95% ethanol mixed with 0.91 kg of feed and 720 ml of water twice daily for 5 weeks during the first trimester of pregnancy. Control sows (n = 7) received dextrose in their feed as a caloric control, and water was added to give the feed a consistency similar to that of the alcohol-treated feed. Immediately before and after 5 weeks of alcohol or dextrose treatment and 3 weeks later, after termination of alcohol or dextrose treatment, we evaluated sow diet preference by comparing the amount of alcohol-supplemented, dextrose-supplemented and plain feed consumed during a 5-min choice test. Contrary to our hypothesis, there was no treatment effect on sow diet preference. Both alcoholtreated and control sows ate less of the alcohol diet than the other two diets in all choice tests. They did not discriminate between the plain and dextrose diets. We conclude that 5 weeks of non-voluntary consumption of alcohol in feed did not produce a preference for alcohol in pregnant sows, either during treatment or after withdrawal, thus providing no evidence for the development of psychological dependence on alcohol under these conditions. © 2006 Elsevier Inc. All rights reserved. Keywords: Alcohol; Swine; Behaviour; Preference
1. Introduction Similarities in sociality and physiology to humans, and ready availability, make the domestic pig (Sus scrofa domesticus) potentially useful as a model for controlled studies of alcoholseeking behaviour and alcohol effects [1–3]. Domestic pigs, like humans, form long-term social bonds with group members of overlapping generations and exhibit complex social cognition [4–7]. Pigs are also similar to humans in body mass, feeding patterns, endocrine systems, heart and kidney structure ⁎ Corresponding author. Department of Veterinary and Comparative Anatomy, Pharmacology and Physiology, Center for the Study of Animal Well-being, Washington State University, PO Box 646520, Pullman, WA 99164-6520, USA. Tel.: +1 509 335 2956; fax: +1 509 335 4650. E-mail address: [email protected] (S. Cloutier). 0031-9384/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2006.03.014
and function, and the rate at which ethanol is cleared from the blood [1]. Most reports on alcohol consumption in swine have been concerned with teratogenic effects on offspring, and effects on organs, uterine function, foetal development, and reproduction [8–16]. Here, we focus on the development of alcohol preference in adult sows. Rodent models of alcohol abuse indicate that long-term voluntary consumption of alcohol can lead to the development of psychological dependence [17–19], as indicated by loss of control over alcohol consumption (i.e., reduced ability to avoid or limit alcohol consumption) and an increased risk of reinstatement following a period of abstinence [20]. Nonvoluntary (forced) consumption of alcohol typically produces tolerance and physical dependence, but not psychological dependence, in rat studies [18,21] although a period of nonvoluntary exposure to alcohol can lead to more rapid acquisition
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of alcohol preference [22,23]. However, it is unclear whether these findings in rats are generally applicable across a broad range of taxa. In previous studies using pigs, alcohol has been offered free choice [1,8,10,16] or mixed in the food and presented as a ‘slurry’ such that alcohol consumption was nonvoluntary [11–15]. It has been reported that pigs will consume alcohol voluntarily to a state of intoxication and, following prolonged consumption of alcohol, to show tolerance (i.e., a reduced effect of alcohol after repeated exposure) and physical dependence (i.e., adaptation of the body to the presence of alcohol, as indicated by withdrawal symptoms following termination of treatment) [1]. Previous studies on pigs have not addressed the question of whether non-voluntary consumption of alcohol can produce alcohol preference in swine. In commercial agricultural production, gestating sows are feed restricted and typically housed individually in gestation stalls without bedding. These conditions contribute to the spontaneous development of environmentally induced stereotyped behaviour [24–27], a form of behavioural loss of control [28–30]. There is evidence for alterations in mesolimbic dopamine function in both stereotypy [31–34] and alcohol addiction [35–37], suggesting that environmental conditions leading to the development of stereotypy in sows might also reduce the ability of sows to limit their alcohol consumption and thus increase the risk of reinstatement. Furthermore, stressors experienced by sows such as aggression [38,39] and inability to express natural behaviour [40,41] may stimulate alcohol consumption [42,43]. Additionally, it has been suggested that food restriction enhances the responsiveness of animals to alcohol [44,45], the reinforcing properties of alcohol [46], and the sensitivity of the neural substrate for drug reward [47]. For these reasons, we hypothesised that feedrestricted, stall-housed pregnant sows fed alcohol nonvoluntarily for 5 weeks would develop a preference for alcohol and retain this preference after removal of alcohol from the diet. If so, this would provide evidence for the development of psychological dependence on alcohol under these conditions in the pig model. We had the opportunity to test our hypothesis using feedrestricted, stall-housed sows that were enrolled in a study investigating effects of alcohol exposure on foetuses in early pregnancy. The sows were given alcohol mixed in feed twice daily for the first trimester of pregnancy. Over the same period, control sows were given the same amount of feed supplemented with dextrose as a caloric control. We used a threealternative free-choice test to assess the effect of 5 weeks of alcohol consumption on the motivation of these sows to consume a diet containing alcohol. Free-choice tests are commonly used to assess the preference of animals in alcohol studies [48–50]. We predicted that, when given a choice of alcohol-supplemented, dextrose-supplemented and unsupplemented (plain) feed during a 5-min free-choice test, the sows on the alcohol treatment would exhibit a preference for the alcohol-supplemented feed after, but not before, 5 weeks of alcohol exposure, and that this preference would be maintained after removal of alcohol from the diet. By contrast, we predicted that control sows, fed an iso-caloric equivalent,
would not exhibit a preference for alcohol. Relative preference for the three diet choices was assessed based on the amount of each diet consumed. 2. Materials and methods 2.1. Animals Seventeen multiparous crossbred inseminated sows (predominantly Large White, Yorkshire and Landrace with lesser and varying contributions from Duroc and Hampshire breeds) were enrolled in the study. During the first trimester of pregnancy (days 1–35 of a 114-day pregnancy), the sows were housed individually in gestation stalls in the same room and limit-fed approximately 0.91 kg of a pea/barley-based gestation diet twice daily at 0630 and 1830 h ± 15 min. The sows were returned to the animal facility's standard feeding protocol for the second and third trimesters of pregnancy (approximately 1.82 kg of the same gestation diet, fed once daily at 0630 h). The diet exceeded all nutrient requirements for gestating sows, and daily intake was restricted to a level recommended for avoiding obesity and promoting sow productivity [51], which is approximately 50% of the amount of feed that pigs would choose to eat if offered feed ad libitum [41]. At this level of feeding, sows are highly motivated to eat and will rapidly consume meals supplemented with alcohol or dextrose [11]. Water was available ad libitum. Room temperature was maintained between 14 and 18 °C with an average of 16 °C. The duration of the photoperiod was 11 h. The sows were housed in facilities accredited by the Association for the Assessment and Accreditation of Laboratory Care and all procedures were approved by the Washington State University Institutional Animal Care and Use Committee. 2.2. Treatments The sows were randomly assigned to the alcohol and dextrose control treatments. The use of a dextrose control group ensured similar energy intake. A pair-fed control group was not included because alcohol consumption did not reduce feed intake in feed restricted sows in a pilot study using a higher alcohol dose. Treatment began on the first day after breeding (day 1 of gestation). At each meal, sows assigned to the alcohol treatment received a half-dose (140 ml) of 95% ethanol and 360 ml of tap water mixed in their feed for the first 2 days followed by a full dose (280 ml) of 95% ethanol (average of 1 g/kg with a range from 0.83 to 1.3 g/kg body weight) with 720 ml of water from days 3 to 36 of gestation. The alcohol was diluted with water to facilitate its distribution evenly throughout the feed and to improve palatability. This amount of alcohol is roughly equivalent to one 750-ml bottle of wine per day for an 80-kg person. Based on preliminary data, we expected this alcoholdosing regimen to produce blood alcohol concentrations in the range of 80–120 mg/dl, equivalent to the legal intoxication level (0.08–0.1%) in most states of the USA. The concentration of
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alcohol used in the present study (27%) is within the range of concentrations (10–30%) used in previous studies of the pig as an animal model for alcoholism [1,8]. Sows assigned to the control treatment received a half-dose (219 g) of powdered dextrose plus 500 ml of tap water per feeding for the first 2 days followed by a full dose (438 g) of dextrose plus 1 l of tap water from days 3 to 36 of gestation. Water was used to produce a feed consistency and moisture content similar to that of the alcohol supplemented feed. A tapering dose of alcohol (50% dose for 2 days; 25% dose for 2 days; 12.5% dose for 2 days; discontinue) was employed at the end of the first trimester to avoid possible symptoms associated with alcohol withdrawal. The dose of dextrose control was tapered off at the same rate to maintain the caloric control. 2.3. Measurements 2.3.1. Blood sampling To determine whether blood alcohol concentrations were within our targeted range of 80–120 mg/dl, and to determine whether sows fed alcohol were adequately hydrated, a blood sample was obtained from all sows in the alcohol and dextrose groups approximately every 10–15 days during the alcohol treatment period (avoiding behaviour test days). Samples were collected by jugular or ear venipuncture approximately 3 h (2.59 ± 0.66 h) following the morning feeding, when blood alcohol was at a peak [11]. The fluid hydration status of all sows was monitored based on packed cell volume (PCV) and plasma protein (PP) level. Analysis of PCV was performed using a microhematocrit centrifuge and PP was determined using a refractometer. Blood for blood alcohol determination was collected in serum separator tubes and centrifuged for 10 min before plasma was assayed. Plasma alcohol was quantified using a commercially available kit (Sigma Laboratories, St. Louis MO), according to the manufacturer's instructions. Samples were measured spectrophotometrically (Bio-Rad Laboratories, SmartSpec 3000, Hercules, CA) for absorbance at 340 nm. The alcohol content was calculated by linear regression using a standard curve for 50, 80, 100 and 300 mg/dl of ethanol. Blood samples from control sows were not sampled for alcohol concentration because previous authors [11] reported blood alcohol concentrations below detectable limits in dextrose-fed control sows kept under similar conditions. 2.3.2. Body weight The body weight of all sows was measured to the nearest kilogram using an electronic scale at the time of breeding (baseline) and at 5 weeks of pregnancy to assess the validity of our assumption that sow condition during pregnancy would not be adversely affected by the alcohol treatment. If the alcohol treatment resulted in lower body weight than the control treatment, we would not know whether any differential preference for alcohol in the alcohol-treated sows that persisted following discontinuance of alcohol treatment was due to psychological dependence or could be accounted for by
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use of alcohol as a source of calories to recover body condition [52]. 2.3.3. Assessment of preference using a free-choice test Assessment of preference for alcohol-containing feed was conducted just prior to the evening meal time: (1) 1 day before treatments commenced (day 0 of gestation, Trial 1), (2) at the end of the treatment period (5 weeks of gestation, Trial 2) and (3) after withdrawal from treatments (8 weeks of gestation, Trial 3). Trial 1 was used to determine the baseline diet preference of the sows. Trial 2 was used to assess any change in diet preference as a result of the treatments, and Trial 3, which was conducted 2 weeks after tapering off the alcohol, was used to determine whether any treatment-induced change in diet preference was retained following termination of treatment. In each trial, the sows were guided, one at a time, to a familiar test arena (3.7 × 7.3 m) and presented simultaneously with three buckets (35.5 L × 30.5 W × 14 H cm), secured to the far wall of the arena at floor level and 1 m apart, containing the following diets, respectively: (1) plain: 500 ml of tap water mixed into approximately 455 g of feed; (2) dextrose: 182 g of dextrose + 500 ml of tap water mixed into approximately 455 g of feed; (3) alcohol: 140 ml of 95% alcohol + 360 ml of tap water mixed into approximately 455 g of feed. The plain diet was included in the choice test to control for the possibility that sows would exhibit a preference for the alcohol diet if dextrose was not very palatable to them. The quantity of feed in each bucket was equivalent to one quarter of the daily ration. The order of presentation of the three diets in the three buckets was randomised across treatment and trial to control for any sow location bias. The test started when the sow entered the arena, at the opposite end from the buckets. For each sow, the following variables were recorded: (1) latency to approach the buckets of feed (i.e., to place nose in one of the buckets), (2) diet approached first (nose in the bucket), and (3) amount of each diet eaten (measured by the difference in weight of each bucket before and after the test). Based on preliminary observation that sows generally spent less than 5 min investigating the food buckets, the duration of the test was set at 5 min. Two control sows were not tested in Trial 3, and a third was not tested in Trial 1 due to reluctance to leave the gestation stall. Sows that ate food during a test received no additional food that evening. 2.4. Statistical analysis We used the general linear model procedure (SAS Proc GLM) [53] to assess the effect of treatment on mean PVC, PP, body weight, and body weight gain of each sow. A matched pairs t-test was used to test whether the body weight of each sow following 5 weeks of pregnancy and alcohol or dextrose consumption differed from her body weight at the time of breeding. Because the same amount of alcohol was given to each sow regardless of body weight, a Pearson correlation analysis was used to test for any relationship between body weight and average peak blood alcohol level of the alcoholtreated sows during the 5 weeks of alcohol exposure. For each trial, a Chi-Square test was performed to determine which diet
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3. Results The peak blood alcohol levels of the alcohol-treated sows varied between 73 and 178 mg/dl, with an average of 115 ± 10.1 mg/dl. All recorded PCVs and PPs were within normal limits for both alcohol (PCV, 37.4 ± 0.55%; PP, 7.4 ± 0.09 g/dl) and control sows (PCV, 36.6 ± 1.13%; PP, 7.3 ± 0.18 g/dl), where normal ranges for PCV and PP in swine are 32–50% and 6–8 g/ dl, respectively [56]. Neither PCV nor PP were affected by treatment (p N 0.5). Sows from the alcohol and control treatments did not differ in parity (alcohol-treated sows, 5 ± 0.7; n = 10, control sows, 6 ± 1.5; n = 7), body weight at the time of breeding (alcohol-treated sows, 205 ± 9.0 kg; control sows, 200 ± 17.4 kg) or body weight at the end of the treatment period (alcohol-treated sows, 201 ± 7.6 kg; control sows, 201 ± 16.0 kg). Neither the alcohol sows nor the control sows gained weight during the first 5 weeks of pregnancy (t = − 0.84, P = 0.42 and t = 0.40, p = 0.70, for alcohol and control sows, respectively). Body weight was not correlated with blood alcohol level in alcohol-treated sows (r = − 0.08, n = 9, p = 0.84).
Control
Latency (sec)
60
Alcohol
a 40 b
C
20 0 1
2
3
Trial Fig. 1. Median (±first and third quartile) latency (s) to approach the feed buckets (i.e., place nose in one of the buckets) in a 5-min choice test by sows fed an alcohol diet (Alcohol) or an iso-caloric dextrose diet (Control) for the first trimester of pregnancy when tested in a choice situation at week 0 (Trial 1), week 5 (Trial 2) and week 8 (Trial 3, 3 weeks after withdrawal from treatment) of pregnancy. Trials with different letters are significantly different at p b 0.05 (based on analysis of ranked data).
A Amount eaten (g)
the sows approached first. We used the mixed linear model procedure (SAS Proc Mixed) [54], with compound symmetry covariance structure and trial [1(week 0), 2 (week 5) and 3 (week 8)] as a repeated measure, to assess effects of treatment and trial on the latency to approach the buckets, the amount of each diet consumed and the total amount of feed consumed. A Proc Mixed analysis with ante-dependence covariance structure and choice of diet (plain, alcohol and dextrose) as a repeated measure was also performed to assess effects of treatment, trial and diet on the amount of each diet consumed. For these analyses, because the residuals were not normally distributed, we applied the mixed linear model to both untransformed and ranked data. Similar results for the two analyses indicated reliability of the analysis of ranked data [55]. Mean comparisons were made based on differences in least squares means, using the Tukey adjustment to control for type 1 error. Data are reported as means ± S.E.M. except where otherwise noted, and significance was set at α = 0.05.
Pl ain
Dex trose
Alcohol
1400 1200 1000 800 600 400 200 0 1
2
3
Trial
B Amount eaten (g)
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Pl ain
Dex trose
Alcohol
1400 1200 1000 800 600 400 200 0 1
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Trial Fig. 2. Mean (±S.E.M.) amount of plain, dextrose and alcohol diets consumed by sows fed (A) an alcohol or (B) an iso-caloric dextrose diet during the first trimester of pregnancy when tested in a choice situation at week 0 (Trial 1), week 5 (Trial 2) and week 8 (Trial 3, 3 weeks after withdrawal from treatment) of pregnancy. The plain and dextrose diets were preferred over the alcohol diet at all times (p b 0.0001).
During the choice tests, the sows did not approach a particular diet first (Trial 1, χ2 = 0.24, df = 2, p = 0.33, Trial 2, χ2 = 5.00, df = 2, p = 0.08, Trial 3, χ2 = 3.26, df = 2, p = 0.20). The latency to approach the feed buckets was longer in Trial 1 than Trials 2 and 3, and also in Trial 2 than Trial 3 (F = 41.70, df = 2, 18, p b 0.0001; Tukey test: p b 0.01; Fig. 1). The latency to approach the feed buckets was not affected by treatment or treatment by trial (P N 0.20). There was a main effect of diet type on the amount of food consumed from each bucket (F = 45.76, df = 2, 26, p b 0.0001; Fig. 2A and B). In all trials, the sows from both the alcohol and control treatments consumed a larger amount of the plain and dextrose diets than the alcohol diet (Tukey test: p b 0.0001). Treatment and interactions between treatment, trial and diet did not affect the amount of each diet consumed in the choice tests (p N 0.10). The total amount of food ingested during a trial (all three diets combined) was higher in Trials 2 and 3 than in Trial 1 (F = 14.90, df = 2, 19, p b 0.0001; Tukey test: p b 0.01). Treatment, and the interaction between treatment and time, did not affect the total amount of food ingested during a trial (p N 0.1). Given the sows' rapid rate of ingestion, 5 min appeared sufficient to allow consumption of the available food in all three buckets. However, after sampling the alcohol diet and consuming most or all of the plain and dextrose diets, many sows started exploring the pen towards the end of the test rather than finishing the alcohol diet.
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4. Discussion Although the average peak blood alcohol level of our sows (115 ± 10.1 mg/dl) was lower than in previous studies using pigs [1,11–15], it was in the expected range of 80–120 mg/dl and was accompanied by occasional loss of balance when walking, a behavioural sign of intoxication. Thus, our results provide confirmation that alcohol-consuming pigs of large commercial breeds can attain blood alcohol concentrations comparable to those of intoxicated humans. Although alcohol has a diuretic effect that can lead to dehydration, low blood pressure and fluid imbalance [20], our results for PCV and PP suggest that the health of the alcohol-fed sows was not compromised. Heavy alcohol consumption can result in lower weight gain or weight loss due to reduced food intake [1] and altered physiology [14], resulting in chronic malnutrition and tissue damage [20] but we found no differences in body weight as a consequence of the alcohol treatment, suggesting that the alcohol dose given in this study did not cause nutritional deficiencies. We detected no significant change in sow body weight during the first 5 weeks of pregnancy, probably because each foetus weighs only about 3–7 g at 5 weeks of gestation [11,57]. Neither the alcohol-fed sows nor the control sows showed any bias towards a particular diet when approaching the buckets from the other end of the test arena, suggesting that, despite having a keen sense of smell, the sows were not using an odour cue to select or avoid a particular diet from a distance. Nor did their latency to approach the buckets differ, suggesting that the alcohol-fed sows were not more motivated to seek food in general, or alcohol specifically, than the control sows. The shorter latency to cross the arena and reach the buckets in Trials 2 and 3 compared with Trial 1, as well as the increased amount of feed eaten during Trials 2 and 3, may have been associated with increased hunger after 5–8 weeks of feed restriction. Restricted feeding is recommended practice for breeding swine [51] to avoid obesity [58] and, although the amount of feed offered to the gestating sows was sufficient to maintain good health, it was well below the digestive capacity and appetite of pigs this size [11]. Because the sows approached the buckets faster in the later trials (2 and 3), they had more time to feed. The amount of each diet consumed by the alcohol-treated sows in the choice tests did not differ significantly from the amount consumed by control sows in any of the trials, indicating that neither 5 weeks of consumption of the initially novel alcohol diet nor subsequent discontinuance of alcohol treatment differentially increased the willingness of sows in the alcohol treatment group to consume alcohol. We found no evidence of neophobia to the alcohol and dextrose diets, given that there was no treatment difference in the amount of each diet consumed in the different trials. Many of the sows started exploring the pen towards the end of the test, after having visited the three feed buckets at least once, rather than finishing the feed in the alcohol bucket. Pigs are known to be highly exploratory [59,60]. It appears that, although the sows were
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familiar with the test arena, the opportunity to explore the arena was more rewarding to both the alcohol-treated and control sows than consumption of the alcohol diet. This exploratory behaviour can be explained as environmental monitoring (patrolling) or rebound following confinement [61]. Feed restriction and confinement in gestation crates are known risk factors for the development of oral stereotypies in sows [24–27]. Based on evidence for stress-induced deficits in mesolimbic dopamine function in both stereotypy and alcohol addiction [31–37,62], we hypothesized that environmental conditions leading to the development of stereotypy in sows might also predispose sows to psychological dependence on alcohol. Nevertheless, despite the performance of oral stereotypies by the sows in our study (unpublished data), our findings suggest that the sows retained flexibility and control over their food choices. They did not lose their ability to avoid or limit their alcohol consumption and did not show reinstatement following 5 weeks of alcohol consumption. They ingested the alcohol-soaked food when it was their only source of food, but they avoided it when given the choice. Given that alcohol preference tends to be stronger at lower concentrations [44,63], it is possible that the sows were not attracted to the alcohol because of the relatively high concentration used (27%). The sows may have experienced positive effects from consuming alcohol but been unable to associate these with the food if it was perceived as being unpleasant. Although the sows did not exhibit a preference between the plain and dextrose diets, they were highly motivated to eat when tested shortly before their evening meal and were easily able to consume all of the plain and dextrose feed during the 5-min test. It is possible that, if more feed had been placed in each bucket, a difference in intake of the plain and dextrose diets may have been detected. However, in that case, sows may have become temporarily satiated after consuming their most preferred diet and we would not have been able to detect that the alcohol diet was their least preferred choice. In conclusion, 5 weeks of non-voluntary consumption of alcohol in feed, at the dose and in the form given in this study, did not result in the development of a preference for alcohol in stall-housed, feed-restricted pregnant sows, either during treatment or following withdrawal. Acknowledgments The authors are grateful to the animal care staff of the Washington State University Swine Center and to D. Barone, P. Cain, T.M. Donaldson, J. Sosa and J. Tilton for technical assistance and Dr. B. Slinker for statistical advice. This research was supported by a grant from the Washington State University Alcohol and Drug Abuse Program. References [1] Dexter JD, Tumbleson ME, Hutcheson DP, Middleton CC. Sinclair (S-1) miniature swine as a model for the study of human alcoholism. Ann NY Acad Sci 1976;273:188–93. [2] Falk JL. Drug dependence myth or motive. Pharmacol Biochem Behav 1983;19:385–92.
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Consumption of alcohol by sows in a choice test Sylvie Cloutier a,b,⁎, Tracy L. Skaer c , Ruth C. Newberry a,b a
b
Department of Veterinary and Comparative Anatomy, Pharmacology and Physiology, Center for the Study of Animal Well-being, Washington State University, PO Box 646520, Pullman, WA 99164-6520, USA Department of Animal Sciences, Center for the Study of Animal Well-being, Washington State University, Pullman, WA 99164-6520, USA c Department of Health Policy and Administration, Pharmacoeconomics and Pharmacoepidemiology Research Unit, Washington State University, Pullman, WA 99164-6510, USA Received 18 April 2005; received in revised form 21 February 2006; accepted 9 March 2006
Abstract The domestic pig (Sus scrofa domesticus) has been proposed as an animal model for human alcoholism because pigs have been observed to consume alcohol voluntarily to a state of intoxication and to exhibit tolerance and physical dependence. However, it has not been established whether pigs can develop psychological dependence on alcohol. We hypothesised that feed-restricted, stall-housed pregnant sows fed alcohol nonvoluntarily for 5 weeks would develop a preference for alcohol and retain this preference after removal of alcohol from the diet. We fed crossbred commercial sows (n = 10) 280 ml of 95% ethanol mixed with 0.91 kg of feed and 720 ml of water twice daily for 5 weeks during the first trimester of pregnancy. Control sows (n = 7) received dextrose in their feed as a caloric control, and water was added to give the feed a consistency similar to that of the alcohol-treated feed. Immediately before and after 5 weeks of alcohol or dextrose treatment and 3 weeks later, after termination of alcohol or dextrose treatment, we evaluated sow diet preference by comparing the amount of alcohol-supplemented, dextrose-supplemented and plain feed consumed during a 5-min choice test. Contrary to our hypothesis, there was no treatment effect on sow diet preference. Both alcoholtreated and control sows ate less of the alcohol diet than the other two diets in all choice tests. They did not discriminate between the plain and dextrose diets. We conclude that 5 weeks of non-voluntary consumption of alcohol in feed did not produce a preference for alcohol in pregnant sows, either during treatment or after withdrawal, thus providing no evidence for the development of psychological dependence on alcohol under these conditions. © 2006 Elsevier Inc. All rights reserved. Keywords: Alcohol; Swine; Behaviour; Preference
1. Introduction Similarities in sociality and physiology to humans, and ready availability, make the domestic pig (Sus scrofa domesticus) potentially useful as a model for controlled studies of alcoholseeking behaviour and alcohol effects [1–3]. Domestic pigs, like humans, form long-term social bonds with group members of overlapping generations and exhibit complex social cognition [4–7]. Pigs are also similar to humans in body mass, feeding patterns, endocrine systems, heart and kidney structure ⁎ Corresponding author. Department of Veterinary and Comparative Anatomy, Pharmacology and Physiology, Center for the Study of Animal Well-being, Washington State University, PO Box 646520, Pullman, WA 99164-6520, USA. Tel.: +1 509 335 2956; fax: +1 509 335 4650. E-mail address: [email protected] (S. Cloutier). 0031-9384/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2006.03.014
and function, and the rate at which ethanol is cleared from the blood [1]. Most reports on alcohol consumption in swine have been concerned with teratogenic effects on offspring, and effects on organs, uterine function, foetal development, and reproduction [8–16]. Here, we focus on the development of alcohol preference in adult sows. Rodent models of alcohol abuse indicate that long-term voluntary consumption of alcohol can lead to the development of psychological dependence [17–19], as indicated by loss of control over alcohol consumption (i.e., reduced ability to avoid or limit alcohol consumption) and an increased risk of reinstatement following a period of abstinence [20]. Nonvoluntary (forced) consumption of alcohol typically produces tolerance and physical dependence, but not psychological dependence, in rat studies [18,21] although a period of nonvoluntary exposure to alcohol can lead to more rapid acquisition
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of alcohol preference [22,23]. However, it is unclear whether these findings in rats are generally applicable across a broad range of taxa. In previous studies using pigs, alcohol has been offered free choice [1,8,10,16] or mixed in the food and presented as a ‘slurry’ such that alcohol consumption was nonvoluntary [11–15]. It has been reported that pigs will consume alcohol voluntarily to a state of intoxication and, following prolonged consumption of alcohol, to show tolerance (i.e., a reduced effect of alcohol after repeated exposure) and physical dependence (i.e., adaptation of the body to the presence of alcohol, as indicated by withdrawal symptoms following termination of treatment) [1]. Previous studies on pigs have not addressed the question of whether non-voluntary consumption of alcohol can produce alcohol preference in swine. In commercial agricultural production, gestating sows are feed restricted and typically housed individually in gestation stalls without bedding. These conditions contribute to the spontaneous development of environmentally induced stereotyped behaviour [24–27], a form of behavioural loss of control [28–30]. There is evidence for alterations in mesolimbic dopamine function in both stereotypy [31–34] and alcohol addiction [35–37], suggesting that environmental conditions leading to the development of stereotypy in sows might also reduce the ability of sows to limit their alcohol consumption and thus increase the risk of reinstatement. Furthermore, stressors experienced by sows such as aggression [38,39] and inability to express natural behaviour [40,41] may stimulate alcohol consumption [42,43]. Additionally, it has been suggested that food restriction enhances the responsiveness of animals to alcohol [44,45], the reinforcing properties of alcohol [46], and the sensitivity of the neural substrate for drug reward [47]. For these reasons, we hypothesised that feedrestricted, stall-housed pregnant sows fed alcohol nonvoluntarily for 5 weeks would develop a preference for alcohol and retain this preference after removal of alcohol from the diet. If so, this would provide evidence for the development of psychological dependence on alcohol under these conditions in the pig model. We had the opportunity to test our hypothesis using feedrestricted, stall-housed sows that were enrolled in a study investigating effects of alcohol exposure on foetuses in early pregnancy. The sows were given alcohol mixed in feed twice daily for the first trimester of pregnancy. Over the same period, control sows were given the same amount of feed supplemented with dextrose as a caloric control. We used a threealternative free-choice test to assess the effect of 5 weeks of alcohol consumption on the motivation of these sows to consume a diet containing alcohol. Free-choice tests are commonly used to assess the preference of animals in alcohol studies [48–50]. We predicted that, when given a choice of alcohol-supplemented, dextrose-supplemented and unsupplemented (plain) feed during a 5-min free-choice test, the sows on the alcohol treatment would exhibit a preference for the alcohol-supplemented feed after, but not before, 5 weeks of alcohol exposure, and that this preference would be maintained after removal of alcohol from the diet. By contrast, we predicted that control sows, fed an iso-caloric equivalent,
would not exhibit a preference for alcohol. Relative preference for the three diet choices was assessed based on the amount of each diet consumed. 2. Materials and methods 2.1. Animals Seventeen multiparous crossbred inseminated sows (predominantly Large White, Yorkshire and Landrace with lesser and varying contributions from Duroc and Hampshire breeds) were enrolled in the study. During the first trimester of pregnancy (days 1–35 of a 114-day pregnancy), the sows were housed individually in gestation stalls in the same room and limit-fed approximately 0.91 kg of a pea/barley-based gestation diet twice daily at 0630 and 1830 h ± 15 min. The sows were returned to the animal facility's standard feeding protocol for the second and third trimesters of pregnancy (approximately 1.82 kg of the same gestation diet, fed once daily at 0630 h). The diet exceeded all nutrient requirements for gestating sows, and daily intake was restricted to a level recommended for avoiding obesity and promoting sow productivity [51], which is approximately 50% of the amount of feed that pigs would choose to eat if offered feed ad libitum [41]. At this level of feeding, sows are highly motivated to eat and will rapidly consume meals supplemented with alcohol or dextrose [11]. Water was available ad libitum. Room temperature was maintained between 14 and 18 °C with an average of 16 °C. The duration of the photoperiod was 11 h. The sows were housed in facilities accredited by the Association for the Assessment and Accreditation of Laboratory Care and all procedures were approved by the Washington State University Institutional Animal Care and Use Committee. 2.2. Treatments The sows were randomly assigned to the alcohol and dextrose control treatments. The use of a dextrose control group ensured similar energy intake. A pair-fed control group was not included because alcohol consumption did not reduce feed intake in feed restricted sows in a pilot study using a higher alcohol dose. Treatment began on the first day after breeding (day 1 of gestation). At each meal, sows assigned to the alcohol treatment received a half-dose (140 ml) of 95% ethanol and 360 ml of tap water mixed in their feed for the first 2 days followed by a full dose (280 ml) of 95% ethanol (average of 1 g/kg with a range from 0.83 to 1.3 g/kg body weight) with 720 ml of water from days 3 to 36 of gestation. The alcohol was diluted with water to facilitate its distribution evenly throughout the feed and to improve palatability. This amount of alcohol is roughly equivalent to one 750-ml bottle of wine per day for an 80-kg person. Based on preliminary data, we expected this alcoholdosing regimen to produce blood alcohol concentrations in the range of 80–120 mg/dl, equivalent to the legal intoxication level (0.08–0.1%) in most states of the USA. The concentration of
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alcohol used in the present study (27%) is within the range of concentrations (10–30%) used in previous studies of the pig as an animal model for alcoholism [1,8]. Sows assigned to the control treatment received a half-dose (219 g) of powdered dextrose plus 500 ml of tap water per feeding for the first 2 days followed by a full dose (438 g) of dextrose plus 1 l of tap water from days 3 to 36 of gestation. Water was used to produce a feed consistency and moisture content similar to that of the alcohol supplemented feed. A tapering dose of alcohol (50% dose for 2 days; 25% dose for 2 days; 12.5% dose for 2 days; discontinue) was employed at the end of the first trimester to avoid possible symptoms associated with alcohol withdrawal. The dose of dextrose control was tapered off at the same rate to maintain the caloric control. 2.3. Measurements 2.3.1. Blood sampling To determine whether blood alcohol concentrations were within our targeted range of 80–120 mg/dl, and to determine whether sows fed alcohol were adequately hydrated, a blood sample was obtained from all sows in the alcohol and dextrose groups approximately every 10–15 days during the alcohol treatment period (avoiding behaviour test days). Samples were collected by jugular or ear venipuncture approximately 3 h (2.59 ± 0.66 h) following the morning feeding, when blood alcohol was at a peak [11]. The fluid hydration status of all sows was monitored based on packed cell volume (PCV) and plasma protein (PP) level. Analysis of PCV was performed using a microhematocrit centrifuge and PP was determined using a refractometer. Blood for blood alcohol determination was collected in serum separator tubes and centrifuged for 10 min before plasma was assayed. Plasma alcohol was quantified using a commercially available kit (Sigma Laboratories, St. Louis MO), according to the manufacturer's instructions. Samples were measured spectrophotometrically (Bio-Rad Laboratories, SmartSpec 3000, Hercules, CA) for absorbance at 340 nm. The alcohol content was calculated by linear regression using a standard curve for 50, 80, 100 and 300 mg/dl of ethanol. Blood samples from control sows were not sampled for alcohol concentration because previous authors [11] reported blood alcohol concentrations below detectable limits in dextrose-fed control sows kept under similar conditions. 2.3.2. Body weight The body weight of all sows was measured to the nearest kilogram using an electronic scale at the time of breeding (baseline) and at 5 weeks of pregnancy to assess the validity of our assumption that sow condition during pregnancy would not be adversely affected by the alcohol treatment. If the alcohol treatment resulted in lower body weight than the control treatment, we would not know whether any differential preference for alcohol in the alcohol-treated sows that persisted following discontinuance of alcohol treatment was due to psychological dependence or could be accounted for by
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use of alcohol as a source of calories to recover body condition [52]. 2.3.3. Assessment of preference using a free-choice test Assessment of preference for alcohol-containing feed was conducted just prior to the evening meal time: (1) 1 day before treatments commenced (day 0 of gestation, Trial 1), (2) at the end of the treatment period (5 weeks of gestation, Trial 2) and (3) after withdrawal from treatments (8 weeks of gestation, Trial 3). Trial 1 was used to determine the baseline diet preference of the sows. Trial 2 was used to assess any change in diet preference as a result of the treatments, and Trial 3, which was conducted 2 weeks after tapering off the alcohol, was used to determine whether any treatment-induced change in diet preference was retained following termination of treatment. In each trial, the sows were guided, one at a time, to a familiar test arena (3.7 × 7.3 m) and presented simultaneously with three buckets (35.5 L × 30.5 W × 14 H cm), secured to the far wall of the arena at floor level and 1 m apart, containing the following diets, respectively: (1) plain: 500 ml of tap water mixed into approximately 455 g of feed; (2) dextrose: 182 g of dextrose + 500 ml of tap water mixed into approximately 455 g of feed; (3) alcohol: 140 ml of 95% alcohol + 360 ml of tap water mixed into approximately 455 g of feed. The plain diet was included in the choice test to control for the possibility that sows would exhibit a preference for the alcohol diet if dextrose was not very palatable to them. The quantity of feed in each bucket was equivalent to one quarter of the daily ration. The order of presentation of the three diets in the three buckets was randomised across treatment and trial to control for any sow location bias. The test started when the sow entered the arena, at the opposite end from the buckets. For each sow, the following variables were recorded: (1) latency to approach the buckets of feed (i.e., to place nose in one of the buckets), (2) diet approached first (nose in the bucket), and (3) amount of each diet eaten (measured by the difference in weight of each bucket before and after the test). Based on preliminary observation that sows generally spent less than 5 min investigating the food buckets, the duration of the test was set at 5 min. Two control sows were not tested in Trial 3, and a third was not tested in Trial 1 due to reluctance to leave the gestation stall. Sows that ate food during a test received no additional food that evening. 2.4. Statistical analysis We used the general linear model procedure (SAS Proc GLM) [53] to assess the effect of treatment on mean PVC, PP, body weight, and body weight gain of each sow. A matched pairs t-test was used to test whether the body weight of each sow following 5 weeks of pregnancy and alcohol or dextrose consumption differed from her body weight at the time of breeding. Because the same amount of alcohol was given to each sow regardless of body weight, a Pearson correlation analysis was used to test for any relationship between body weight and average peak blood alcohol level of the alcoholtreated sows during the 5 weeks of alcohol exposure. For each trial, a Chi-Square test was performed to determine which diet
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3. Results The peak blood alcohol levels of the alcohol-treated sows varied between 73 and 178 mg/dl, with an average of 115 ± 10.1 mg/dl. All recorded PCVs and PPs were within normal limits for both alcohol (PCV, 37.4 ± 0.55%; PP, 7.4 ± 0.09 g/dl) and control sows (PCV, 36.6 ± 1.13%; PP, 7.3 ± 0.18 g/dl), where normal ranges for PCV and PP in swine are 32–50% and 6–8 g/ dl, respectively [56]. Neither PCV nor PP were affected by treatment (p N 0.5). Sows from the alcohol and control treatments did not differ in parity (alcohol-treated sows, 5 ± 0.7; n = 10, control sows, 6 ± 1.5; n = 7), body weight at the time of breeding (alcohol-treated sows, 205 ± 9.0 kg; control sows, 200 ± 17.4 kg) or body weight at the end of the treatment period (alcohol-treated sows, 201 ± 7.6 kg; control sows, 201 ± 16.0 kg). Neither the alcohol sows nor the control sows gained weight during the first 5 weeks of pregnancy (t = − 0.84, P = 0.42 and t = 0.40, p = 0.70, for alcohol and control sows, respectively). Body weight was not correlated with blood alcohol level in alcohol-treated sows (r = − 0.08, n = 9, p = 0.84).
Control
Latency (sec)
60
Alcohol
a 40 b
C
20 0 1
2
3
Trial Fig. 1. Median (±first and third quartile) latency (s) to approach the feed buckets (i.e., place nose in one of the buckets) in a 5-min choice test by sows fed an alcohol diet (Alcohol) or an iso-caloric dextrose diet (Control) for the first trimester of pregnancy when tested in a choice situation at week 0 (Trial 1), week 5 (Trial 2) and week 8 (Trial 3, 3 weeks after withdrawal from treatment) of pregnancy. Trials with different letters are significantly different at p b 0.05 (based on analysis of ranked data).
A Amount eaten (g)
the sows approached first. We used the mixed linear model procedure (SAS Proc Mixed) [54], with compound symmetry covariance structure and trial [1(week 0), 2 (week 5) and 3 (week 8)] as a repeated measure, to assess effects of treatment and trial on the latency to approach the buckets, the amount of each diet consumed and the total amount of feed consumed. A Proc Mixed analysis with ante-dependence covariance structure and choice of diet (plain, alcohol and dextrose) as a repeated measure was also performed to assess effects of treatment, trial and diet on the amount of each diet consumed. For these analyses, because the residuals were not normally distributed, we applied the mixed linear model to both untransformed and ranked data. Similar results for the two analyses indicated reliability of the analysis of ranked data [55]. Mean comparisons were made based on differences in least squares means, using the Tukey adjustment to control for type 1 error. Data are reported as means ± S.E.M. except where otherwise noted, and significance was set at α = 0.05.
Pl ain
Dex trose
Alcohol
1400 1200 1000 800 600 400 200 0 1
2
3
Trial
B Amount eaten (g)
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Pl ain
Dex trose
Alcohol
1400 1200 1000 800 600 400 200 0 1
2
3
Trial Fig. 2. Mean (±S.E.M.) amount of plain, dextrose and alcohol diets consumed by sows fed (A) an alcohol or (B) an iso-caloric dextrose diet during the first trimester of pregnancy when tested in a choice situation at week 0 (Trial 1), week 5 (Trial 2) and week 8 (Trial 3, 3 weeks after withdrawal from treatment) of pregnancy. The plain and dextrose diets were preferred over the alcohol diet at all times (p b 0.0001).
During the choice tests, the sows did not approach a particular diet first (Trial 1, χ2 = 0.24, df = 2, p = 0.33, Trial 2, χ2 = 5.00, df = 2, p = 0.08, Trial 3, χ2 = 3.26, df = 2, p = 0.20). The latency to approach the feed buckets was longer in Trial 1 than Trials 2 and 3, and also in Trial 2 than Trial 3 (F = 41.70, df = 2, 18, p b 0.0001; Tukey test: p b 0.01; Fig. 1). The latency to approach the feed buckets was not affected by treatment or treatment by trial (P N 0.20). There was a main effect of diet type on the amount of food consumed from each bucket (F = 45.76, df = 2, 26, p b 0.0001; Fig. 2A and B). In all trials, the sows from both the alcohol and control treatments consumed a larger amount of the plain and dextrose diets than the alcohol diet (Tukey test: p b 0.0001). Treatment and interactions between treatment, trial and diet did not affect the amount of each diet consumed in the choice tests (p N 0.10). The total amount of food ingested during a trial (all three diets combined) was higher in Trials 2 and 3 than in Trial 1 (F = 14.90, df = 2, 19, p b 0.0001; Tukey test: p b 0.01). Treatment, and the interaction between treatment and time, did not affect the total amount of food ingested during a trial (p N 0.1). Given the sows' rapid rate of ingestion, 5 min appeared sufficient to allow consumption of the available food in all three buckets. However, after sampling the alcohol diet and consuming most or all of the plain and dextrose diets, many sows started exploring the pen towards the end of the test rather than finishing the alcohol diet.
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4. Discussion Although the average peak blood alcohol level of our sows (115 ± 10.1 mg/dl) was lower than in previous studies using pigs [1,11–15], it was in the expected range of 80–120 mg/dl and was accompanied by occasional loss of balance when walking, a behavioural sign of intoxication. Thus, our results provide confirmation that alcohol-consuming pigs of large commercial breeds can attain blood alcohol concentrations comparable to those of intoxicated humans. Although alcohol has a diuretic effect that can lead to dehydration, low blood pressure and fluid imbalance [20], our results for PCV and PP suggest that the health of the alcohol-fed sows was not compromised. Heavy alcohol consumption can result in lower weight gain or weight loss due to reduced food intake [1] and altered physiology [14], resulting in chronic malnutrition and tissue damage [20] but we found no differences in body weight as a consequence of the alcohol treatment, suggesting that the alcohol dose given in this study did not cause nutritional deficiencies. We detected no significant change in sow body weight during the first 5 weeks of pregnancy, probably because each foetus weighs only about 3–7 g at 5 weeks of gestation [11,57]. Neither the alcohol-fed sows nor the control sows showed any bias towards a particular diet when approaching the buckets from the other end of the test arena, suggesting that, despite having a keen sense of smell, the sows were not using an odour cue to select or avoid a particular diet from a distance. Nor did their latency to approach the buckets differ, suggesting that the alcohol-fed sows were not more motivated to seek food in general, or alcohol specifically, than the control sows. The shorter latency to cross the arena and reach the buckets in Trials 2 and 3 compared with Trial 1, as well as the increased amount of feed eaten during Trials 2 and 3, may have been associated with increased hunger after 5–8 weeks of feed restriction. Restricted feeding is recommended practice for breeding swine [51] to avoid obesity [58] and, although the amount of feed offered to the gestating sows was sufficient to maintain good health, it was well below the digestive capacity and appetite of pigs this size [11]. Because the sows approached the buckets faster in the later trials (2 and 3), they had more time to feed. The amount of each diet consumed by the alcohol-treated sows in the choice tests did not differ significantly from the amount consumed by control sows in any of the trials, indicating that neither 5 weeks of consumption of the initially novel alcohol diet nor subsequent discontinuance of alcohol treatment differentially increased the willingness of sows in the alcohol treatment group to consume alcohol. We found no evidence of neophobia to the alcohol and dextrose diets, given that there was no treatment difference in the amount of each diet consumed in the different trials. Many of the sows started exploring the pen towards the end of the test, after having visited the three feed buckets at least once, rather than finishing the feed in the alcohol bucket. Pigs are known to be highly exploratory [59,60]. It appears that, although the sows were
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familiar with the test arena, the opportunity to explore the arena was more rewarding to both the alcohol-treated and control sows than consumption of the alcohol diet. This exploratory behaviour can be explained as environmental monitoring (patrolling) or rebound following confinement [61]. Feed restriction and confinement in gestation crates are known risk factors for the development of oral stereotypies in sows [24–27]. Based on evidence for stress-induced deficits in mesolimbic dopamine function in both stereotypy and alcohol addiction [31–37,62], we hypothesized that environmental conditions leading to the development of stereotypy in sows might also predispose sows to psychological dependence on alcohol. Nevertheless, despite the performance of oral stereotypies by the sows in our study (unpublished data), our findings suggest that the sows retained flexibility and control over their food choices. They did not lose their ability to avoid or limit their alcohol consumption and did not show reinstatement following 5 weeks of alcohol consumption. They ingested the alcohol-soaked food when it was their only source of food, but they avoided it when given the choice. Given that alcohol preference tends to be stronger at lower concentrations [44,63], it is possible that the sows were not attracted to the alcohol because of the relatively high concentration used (27%). The sows may have experienced positive effects from consuming alcohol but been unable to associate these with the food if it was perceived as being unpleasant. Although the sows did not exhibit a preference between the plain and dextrose diets, they were highly motivated to eat when tested shortly before their evening meal and were easily able to consume all of the plain and dextrose feed during the 5-min test. It is possible that, if more feed had been placed in each bucket, a difference in intake of the plain and dextrose diets may have been detected. However, in that case, sows may have become temporarily satiated after consuming their most preferred diet and we would not have been able to detect that the alcohol diet was their least preferred choice. In conclusion, 5 weeks of non-voluntary consumption of alcohol in feed, at the dose and in the form given in this study, did not result in the development of a preference for alcohol in stall-housed, feed-restricted pregnant sows, either during treatment or following withdrawal. Acknowledgments The authors are grateful to the animal care staff of the Washington State University Swine Center and to D. Barone, P. Cain, T.M. Donaldson, J. Sosa and J. Tilton for technical assistance and Dr. B. Slinker for statistical advice. This research was supported by a grant from the Washington State University Alcohol and Drug Abuse Program. References [1] Dexter JD, Tumbleson ME, Hutcheson DP, Middleton CC. Sinclair (S-1) miniature swine as a model for the study of human alcoholism. Ann NY Acad Sci 1976;273:188–93. [2] Falk JL. Drug dependence myth or motive. Pharmacol Biochem Behav 1983;19:385–92.
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