Transport of Pigs Different with Respect to the ...

Transport of pigs different with respect to the halothane gene: stress assessment R. Geers, E. Bleus, T. Van Schie, H. Ville, H. Gerard, S. Janssens, G. Nackaerts, E. Decuypere and J. Jourquin J ANIM SCI 1994, 72:2552-2558.

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Transport of Pigs Different with Respect to the Halothane Gene: Stress Assessment -

R. Geers", E. Bleus", T. Van Schie", H. Villh", H. Gerard*, S. Janssens", G. Nackaerts?, E. Decuyperet, and J. Jourquin* *Laboratory of Agricultural Buildings Research and +Laboratory of Animal Physiology, Catholic University Leuven, Kard. Mercierlaan 92, B-3001 Heverlee, Belgium and SSeghers Hybrid, Buggenhout, Belgium

ABSTRACT:

Two transport experiments were carried out with 18 pigs each. These pigs originated from three genetic lines (homozygous halothane-positive and -negative and heterozygotes). Half the pigs were unfed for 12 h before transport. All pigs were transported twice for 2 h. Before and after transport pigs were anesthetized to take blood samples from the jugular vein and biopsies from the biceps femoris. At the same time equipment to measure body temperature and heart rate were attached or detached. Plasma cortisol and beta-endorphin concentrations were measured as well as the glycogen concentration in the muscle sample. Line differences were detected with

respect to body temperature ( P < .04), heart rate ( P < .05), and cortisol ( P < .01). The withholding of feed influenced ( P < .04) plasma beta-endorphin concentration. Body temperature ( P < .02), heart rate ( P< .001), cortisol ( P < . O l ) , and beta-endorphin ( P < .OO 1) were different before and after transport, whereas a training effect of the transport number was observed for heart rate ( P < .07) and plasma betaendorphin ( P < .02). No interactions between treatments were observed. The relationship between cortisol and beta-endorphin suggests a nonconcomitant release of ACTH and beta-endorphin.

Key Words: Pigs, Transport, Heart Rate, Body Temperature, Endorphins, Hydrocortisone

J. Anim. Sci. 1994. 72:2552-2558

Introduction Consumers are placing more emphasis both on meat quality and animal welfare from conception to slaughter. Meat quality may also be related to welfare during handling and transport before slaughter (MOSS, 1982). Animal welfare should be maintained as long as the animal's capacity for adaptation t o the changing environment is not jeopardized (Broom, 1991). Within that changing environment the animal-human interaction (i.e., handling) also seems to be very important (Hemsworth and Barnett, 1987; Geers, 1993). Therefore, t o evaluate animal welfare during handling and

'Transport was organized by the Logistic Service of the K. U. Leuven. K. Goossens, G. Parduyns, J. Van Bael, and W. Duchateau managed the technical service. G . Verhoeven of the Laboratory of Experimental Medicine, K. U. Leuven generously supplied the cortisol antiserum (Lambik XIII). Funding was provided by the Research Council of the K. U. Leuven (OT92/16) and the EC (ECLAIR project 106; AIR project 262). R. Geers is supported by the National Fund of Scientific Research, Belgium. Received December 20, 1993. Accepted July 1, 1994.

transport, measurements are body temperature, heart rate, cortisol, and beta-endorphin (Fordham et al., 1991; Geers et al., 1992; Vi116 et al., 1993). Those measurements are useful when it is possible to integrate appropriate sensors within injectable telemetric systems for monitoring animal welfare during handling and transport (Goedseels et al., 1990). The primary objectives of this study were 1) to evaluate the use of body temperature and heart rate as measurements of welfare during transport; 2 ) to study the relationship between cortisol and betaendorphin as reference traits for pig welfare; 3 ) to take into account in the model of behavioral homeostasis the possibility of training for transport; and 4) to test the hypothesis that 12 h of feed withholding before transport has no effect on animal welfare.

Materials and Methods Experimental Design Two experiments were carried out with 18 female pigs (approximately 20 kg) each. These pigs originated from homozygous halothane-positive ( hh) (Belgian Landrace type) and -negative (HH) (Large

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STRESS MEASUREMENTS OF PIGS DURING TRANSPORT

White type) lines and from heterozygotes (Hh)(six pigs selected at random from different litters per line). These lines were already selected during several generations based on the halothane sniff test and on backcrossing procedures within an identical group of pigs. Halothane gene carriers were also identified according to Fujii et al. (1991) (Seghers Hybrid, Belgium). Pigs were housed in climatic rooms (one per genotype) to standardize all environmental conditions. These rooms have exactly the same dimensions and layout of lying and dunging areas and feeding and drinking facilities. Effective environmental temperature was maintained within thermoneutral conditions between 18 and 22°C (Geers et al., 1990a). Commercial feed and water were provided on a continuous basis. Before measurements started, pigs were allowed to acclimatize for 2 wk. Pigs were transported twice, at BW of approximately 18 and 20 kg, to check a possible training effect. Within this range of body weights no differences of heart function were observed during resting conditions (Geers et al., 1990b). Loading density was .32 m2 per animal. Six pigs originating from the same climatic room were divided into two groups (fed and unfed) of three. Pigs were carried from their pen to the trailer and traveled with penmates to prevent stress caused by fighting. Transport took place from 1300 to 1500 along a prescribed circuit within the campus. The dimensions of the trailer were 2 x 2 x 1.2 m, with ventilation slots situated 1 m above the pigs. The air temperature within the trailer was always between 18°C and 22°C. Half the pigs were unfed during the 12 h before preparative handling started. Six measurements were performed: cortisol, betaendorphin, muscle glycogen, body temperature, electrocardiogram, and animal behavior. Blood and muscle biopsy sampling was done before and after transport when pigs were unconscious, immediately after onset of anesthesia ( 2 mgkg BW of azaperone, i.m.; 10 mgkg BW of metomidate, i.p.1. Anesthesia was completed approximately 5 min after administration and lasted for approximately 1 h, so the actual concentration of cortisol and beta-endorphin was probably not influenced by that procedure (Kallweit, 1982; Moss et al., 1990; Fordham et al., 1991; Dalin et al., 1993). During anesthesia measuring devices for body temperature (Geers et al., 1992) and electrocardiogram (Ville et al., 1993) were attached on the pig, allowing data acquisition over the whole measuring period from start of handling to end of transport ( 6 h ) . During transport, pig behavior was observed by a closed circuit video from the trailer to the jeep, but 90% of the time pigs were lying side by side. Also during anesthesia, biopsies (approximately 500 mg) were extracted from the biceps femoris muscle (Ville et al., 1992) and immediately frozen in liquid nitrogen. Further storage was at -18°C before glycogen analysis (Dahlqvist, 1961). Blood samples were

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taken from the jugular vein and immediately cooled on ice. After centrifugation plasma was stored at -18°C. Total cortisol and beta-endorphin were chosen as stress indicators (Shaw and Tume, 1990). Pigs showed no pain o r discomfort during and after these procedures. It has to be clear that results will refer to effects of handling and transport. Indeed, blood samples were taken as soon as possible after restraint, but always by handling. Before and after transport, the procedure t o restrain the pigs by hand for injection was the same and was completed within 2 min. Further procedures were performed with unconscious pigs. Transport was started 2 h after onset of awakening, a time when plasma cortisol concentrations should have returned to basal levels (Dalin et al., 1993). To check blood hormone levels 12 control pigs received exactly the same treatment but without transport. Cortisol concentrations were evaluated by RIA following dichloromethane extraction and dextrancoated charcoal separation of bound and unbound radioactivity. Liquid scintillation counting was done on the bound radioactivity that did not absorb to dextran-coated charcoal. The mean recovery after extraction was 96.5%. The intra- and interassay variabilities were 7 and 17%, respectively. Crossreactivities of the cortisol antiserum were 4.63% for ll-desoxy-cortisol, 7.1% for cortisone, .25% for corticosterone, 7.5% for ll-beta,l7-alfa-dihydroxy-4-pregnene-3,20-dione, and < .25% for other progestagens, androgens, and estrogens. The detection limit was established a t 12.5 ng/mL. Beta-endorphin was analyzed with the RIK 8843 RIA-kit of Peninsula Laboratories (Belmont, CAI.

Statistical Analysis The individual pigs were chosen at random from different litters within the population of pigs available within each line. Because pigs were the experimental units and allocated at random to the fixed main effects, statistical analysis was based on a randomized layout (Snedecor and Cochran, 1971). The following fixed main effects were analyzed: genotype, feeding level, transport number, and time before and after transport. Considering all measurements together, distributions were normal. Before the final statistical model was chosen, data were analyzed with GLM procedures (SAS, 1985) to find differences between the two experiments, multiple interactions between fixed main effects, and the influence of body weight. No interactions and no relations with body weight were found, and data from both experiments could be pooled. Hence, according to the principle of parsimony data were analyzed with GLM procedures, including the interaction term, comparing fixed main effects painvise through the other main effects (SAS, 1985). Within these GLM procedures each pair of Least Squares means was tested with a t-test.


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GEERS ET AL.

Table 1. Means f SE of plasma cortisol concentration (ng/mL) before and after transport for each genotype Treatment Genotypea

Before transport

After transport

hh Hh

35.9 f 2.7b 36.5 f 2.gb 40.7 f 2.7c < .01

32.1 f 3.4b 28.6 f 3.4d 28.7 f 3.4d > .10

HH P-value

P-value > .10 < .01 < .01 -

ahh = halothane-positive; Hh = heterozygotes; HH = homozygous halothane-negative. b,c,dMeans within a row (or column only before transport) lacking a common superscript letter differ.

Results and Discussion Plasma Cortisol Content Before transport the homozygous halothane-negative pigs had a higher ( P < .01) plasma cortisol content, which disappeared after transport (Table 1 ) . For the halothane-positive pigs plasma cortisol concentration didn’t change during transport. No other differences or interactions were found. These results may reflect a different reaction to handling and a different feedback control mechanism of cortisol t o ACTH release between lines. For instance, the halothane-negative pigs showed a higher emotional response when loading before transport than halothane gene carriers.

Plasma Beta-Endorphin Concentrations Fasting resulted in an increase ( P < .04)in the concentration of plasma beta-endorphin (Table 2). This confirms data on enhanced lipolytic activity in vitro of beta-endorphin (i.e., the use of fat as energy source) (Richter et al., 1987). However, the rise is less than 40% and not sufficient to conclude impaired welfare (Rushen, 1986). A difference of the same order of magnitude ( P < .02) was observed between the first and the second transport (Table 2 ) and suggests a training effect. However, the transport effect itself was very substantial ( P < .001, Table 2). This rise of more than 40% may indicate impaired welfare because plasma concentrations of other stress hormones increased (Rushen, 1986). Beta-endorphin plasma concentrations of 12 control pigs receiving the same treatment, except transport, were 14.8 f 2.4 pg/ .1 mL, equal to values of the experimental pigs before transport (Table 2). Hence, although pigs were transported under so-called optimal conditions, they were obviously stressed and their welfare was impaired according to this criterion. Such a rise of plasma beta-endorphin concentration was not observed in transported sheep, although some training effect to handling seemed to exist (Fordham et al., 1989). No transport x genotype interactions were found. Therefore, susceptibility to transport stress is considered to be independent of the halothane gene.

The Relationship Between Cortisol and Beta-Endorphin In homozygous halothane-positive pigs, a positive linear relationship between plasma cortisol and betaendorphin concentrations was observed before transport, whereas a negative relationship was found after transport (Table 3 ). Homozygous halothane-negative pigs that were fed before transport did not show such a relationship and are considered to be less susceptible to transport stress if fed (Geers et al., 1992; Ville et al., 1993). These relationships provide evidence that the ACTH-cortisol axis is controlled by a negative feedback mechanism, but no such system seems to exist with respect to beta-endorphin (Smith and Lane, 1983). The lower plasma cortisol concentration at the end of transport (i.e., after 2 h; shown in Table 1) corresponds t o data from Dantzer and Mormede (1979). However, the same trend found in sheep was not found for halothane-positive pigs (Apple et al., 1993). The steady rise of beta-endorphin during transport supports the view (Shaw and Tume, 1990) that beta-endorphin is a good variable to evaluate a pig‘s welfare and suggests a nonconcomitant release with ACTH. Hence, further research should include the measurement of ACTH to test the hypothesis of an unequimolecular production of ACTH and beta-endorphin from pro-opiomelanocortin, including the possibility of different production sites (O’Benar et al.,

Table 2. Means k SE of plasma beta-endorphin concentration (pg/.l mL) in relation to feeding interval and transport

Treatment Feeding interval 12 h Oh Transport Before After Transport First Second


Beta-endorphin concentration

P-value

26.2 f 1.4 21.7 f 1.6