J. Anim. Sci. 2006. 84:1015-1021
© 2006 American Society of Animal Science
Effects of halothane sensitivity on mobility status and blood metabolites of HAL-1843-normal pigs after rigorous handling1,2
C. P. Allison*,
A. L. Marr
,
N. L. Berry*,
D. B. Anderson,
D. J. Ivers,
L. F. Richardson,
K. Keffaber,
R. C. Johnson
and
M. E. Doumit*,
,3
* Departments of Animal Science and
and
Food Science and Human Nutrition, Michigan State University, East Lansing 48824;
and
Farmland Foods, Director of Pork Quality, Denison, IA 51442; and
and
Elanco Animal Health, Greenfield, IN 46140
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Abstract
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The objective of this study was to determine if HAL-1843-normal pigs that respond abnormally to halothane anesthesia were more likely to become nonambulatory (NA) when subjected to rigorous handling than pigs that exhibit a normal response to halothane. After a 1,100-km transport, pigs exhibiting low (HS-L; n = 33), intermediate (HS-I; n = 10), and high (HS-H; n = 47) sensitivity to halothane were moved through a 36.6-m long aisle that was 2.1 m wide at each end and 0.6 m wide in the middle 18.3 m. Ten groups of 8 pigs were briskly moved down the aisle and back 4 times, receiving a minimum of 1 electrical prod per pass (8 prods/pig). Before testing, rectal temperature was measured, open-mouth breathing and skin discoloration were visually evaluated, and a blood sample was collected from each pig. After the test, the pigs were returned to their pens, and the same measurements were taken immediately posttest and 1 h posttest (no blood at 1 h posttest). Pigs that were HS-H were more prone to becoming NA compared with HS-L pigs (P < 0.02). Regardless of halothane status, a greater number of pigs exhibited open-mouth breathing and skin discolorations immediately posttest than at the pretest or 1 h posttest times (P < 0.05). No differences were observed in blood metabolites between the different halothane sensitivity categories. However, pigs that became NA had elevated blood levels of creatine phosphokinase, lactate, glycerol, nonesterified fatty acids, ammonia, and urea nitrogen before testing (P < 0.05). Collectively, these data suggest HS-H pigs are more susceptible to becoming NA than HS-L. The elevated pretest blood metabolites of NA pigs suggest that they were in a hypermetabolic state that predisposed them to becoming NA.
Key Words: halothane • nonambulatory • swine
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INTRODUCTION
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Nonambulatory (NA) pigs are those that either arrive at slaughtering facilities unable to move on their own or lose this ability before slaughter. Because of the low frequency of occurrence in the industry (Ellis et al., 2003
; Ritter et al., 2004), causative biological factors associated with pigs becoming NA have not been identified. In a preliminary report, Benjamin et al. (2001) described a handling test that provides an experimental approach to examine the problem of fatigued NA pigs. This approach increased the frequency of fatigued pigs relative to that typically observed in the industry due to more rigorous handling of an entire treatment group rather than just evaluating slow-moving pigs.
Fujii et al. (1991) identified a single nucleotide polymorphism (C1843T) in the skeletal muscle calcium release channel gene, RYR1, of swine that results in an arginine to cysteine change at amino acid residue 615. This polymorphism was associated with stress susceptibility and halothane sensitivity of pigs and is considered a primary cause of classical porcine stress syndrome. However, Rempel et al. (1993) demonstrated that 30% of the pigs considered to be free of the detrimental polymorphism (termed HAL-1843-normal) exhibited an abnormal response to halothane anesthesia. More recently, Allison et al. (2005) reported that the incidence of halothane-sensitive pigs in several commercial HAL-1843-normal lines ranged from 0 to 62%. It is unclear if pigs that are HAL-1843-normal but exhibit adverse sensitivity to halothane (blotchy red or purple skin, muscle rigidity, tremors) have a lower tolerance for stress than animals that are HAL-1843-normal and exhibit a normal response to halothane anesthesia. The experiment described herein tested the hypothesis that HAL-1843-normal pigs that respond abnormally to halothane anesthesia would be more prone to becoming NA due to fatigue when subjected to a rigorous handling test than pigs that exhibit a normal response to halothane.
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MATERIALS AND METHODS
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Animals and Halothane Testing
Pigs used in this study were selected from those halothane tested by Allison et al. (2005). Briefly, 4 commercially available HAL-1843-normal sire lines were used to inseminate a single dam line. When progeny were approximately 9 wk of age they were subjected to 3% halothane for 3 min. The response to halothane was scored by visually evaluating limb rigidity on a scale of 1 to 4 and skin discoloration and tremors on a scale of 1 to 3. In each case, the higher number indicated a more severe response. The average limb rigidity was added to the discoloration and tremor score to calculate the halothane score for each pig. This score was then used to categorize pigs into 3 groups: halothane sensitive-low (HS-L; <4.0); HS-intermediate (HS-I; 4.01 to 5.49); and HS-high (HS-H; >5.49). Of the 4 commercial lines evaluated by Allison et al. (2005), 2 of the lines exhibited <2% HS-H pigs, whereas the remaining 2 lines exhibited >25% HS-H pigs. Therefore, HS-L (n = 33), HS-I (n = 10), and HS-H (n = 37) were selected from the 2 lines possessing >25% HS-H pigs.
Animal Transport, Sampling, and Handling Test
Pigs were loaded into trailer compartments in their test groups of 8, with additional nontest pigs filling the remainder of the compartment (0.39 m2/pig). Pigs were transported from New Ulm, MN, to Greenfield, IN (~1,100 km), in late January with sidewalls of the trailer closed and shavings provided. On arrival, pigs were allowed 3 h of rest with access to water to mimic marketing conditions. The 8 pigs that comprised a test group were housed 4 to a pen, in 2 pens. All test groups were balanced for live weight, sex, and line. The halothane sensitivity of the first 3 groups included 2 to 3 HS-L, 2 to 3 HS-I, and 2 to 3 HS-H. Remaining test groups were comprised of 4 HS-L and 4 HS-H pigs. The temperature of the building was maintained at approximately 18.3°C.
The animal-handling test used for this study was based on a model previously described (Benjamin et al., 2001). All procedures for the handling test were performed in accordance with the Eli Lilly Animal Care and Use Committee. After a 3-h rest and before testing, skin discoloration and open-mouth breathing were visually evaluated on a binary scale. After snare restraint, rectal temperature was measured using a portable handheld thermometer (Model 216, GLA Agricultural Electronics, San Luis Obispo, CA), and a blood sample was collected from the vena cava (pretest). Blood (10 mL) was removed from each animal as quickly as possible (generally <1 min) to reduce erroneous results in metabolites. The handling course was constructed to be 36.6 m long and 2.1 m wide at each end. The middle 18.3 m was reduced to 0.6 m wide to mimic a single-file chute. Ten groups of 8 pigs each were moved down and back (1 lap) 4 times. The animal handler was provided a sort board and an electric prod and was instructed to move the pigs at a fast walking pace. In addition to the handler-imposed prods, an additional person was stationed in the middle of the aisle and was instructed to prod each pig on each pass (8 times total). Electric prods were approximately 0.5 s in duration. After the completion of the fourth lap, pigs were returned to their pen, where rectal temperature, skin discoloration, and open-mouth breathing were recorded. A blood sample was collected within 10 min after the handling test (posttest) from all 8 pigs as just described. One hour after the test, rectal temperatures were recorded, and skin blotchiness and open-mouth breathing were visually assessed (1 h posttest). After the 1 h posttest observations, the pigs were allowed access to feed and water.
A pig was classified as NA by an on-site veterinarian if the pig was unwilling to move or had a rectal temperature 
41°C and showed multiple signs of distress (open-mouth breathing, skin discoloration, or muscle tremors). One pig became NA while moving through the course. This pig was not required to finish the course and was gently moved to the nearest pen and allowed access to water. Posttest observations of this pig began immediately on removal from the course. Two pigs were deemed by an on-site veterinarian to be in distress after the handling test and were immediately euthanized using a captive bolt gun. Two additional pigs were euthanized because of visual signs of distress before post-test transport.
Blood Metabolites
Blood samples were allowed to stand at room temperature for 1 h and were then centrifuged at 1,000 x g for 15 min. Serum was collected and stored at –20°C until analyses were performed. Serum samples were analyzed using the Monarch Chemistry System (Allied Instrumentation Laboratory, Lexington, MA) for acetoacetate, ß-hydroxybutyrate (BHB), creatine phosphokinase (CPK), glucose, glycerol, lactate, NEFA, blood urea nitrogen (BUN), ammonia, phosphorus, triglycerides, and total protein. In all cases, samples were assayed in duplicate and standards were assayed with each group. Acetoacetate and BHB were analyzed using an enzymatic method quantifying D(-)-BHB and acetoacetic acid in serum (Williamson, et al., 1962). Creatine phosphokinase was analyzed by monitoring the conversion of NADH to NAD+ spectrophotometrically (Rosalki, 1967). Glucose was determined using the hexokinase method coupled to glucose-6-phosphate dehydrogenase (Kunst, et al., 1984). Glycerol was quantified using an enzymatic method reported by Wieland (1984). Lactate was measured via enzymatic conversion described by Olsen (1962). Nonesterified free fatty acid concentration was determined using the colorimetric method of Shimizu et al. (1980). Blood urea nitrogen and ammonia concentration were determined by enzymatic analysis (Kerscher and Ziegenhorn, 1985; Bergmeyer and Beutler, 1985). Serum phosphorus concentration was measured by using a colorimetric assay (Fiske and Subbarrow, 1925) and triglyceride levels were determined indirectly via an enzymatic method that measures the glycerol released from triglycerides on hydrolysis by lipase (Esders and Goodhue, 1980). Total protein was determined by measuring total Kjeldahl nitrogen (Doumas, 1975). The interassay coefficient of variation for acetoacetate, BHB, glucose, phosphorous, protein, triglycerides, and urea were 2.8, 1.1, 3.0, 2.3, 1.7, 0.8, and 3.6%, respectively. The remaining assays were compared with standard curves that were run with each group of samples. All samples for a given metabolite were assayed within 1 day.
Statistical Analysis
Differences in the number of NA pigs, percentage of discolorations, and the number of pigs exhibiting open-mouth breathing within different classifications of halothane sensitivity were analyzed by logistic regression using the GEN MOD procedure of SAS (SAS Inst., Inc., Cary, NC). The statistical model included the dependent variable mobility status, discolorations, or open-mouth breathing and the independent effect of halothane category, line, gender, and mobility status. Mobility status was not included as an independent effect when it was evaluated as a dependent effect.
Prods per pig was also analyzed by logistic regression using the GEN MOD procedure of SAS. The statistical model included the dependent variable of prods per pig with the independent effects of gender, line, and halothane category. Rectal temperature was analyzed using a protected least significant difference test utilizing the mixed model procedure of the SAS software. The statistical model included the fixed effects of gender, line, and halothane category with the random effect of group. Blood metabolites were compared between ambulatory and NA pigs using the fixed effects of gender, line, halothane category, and mobility status with the random effect of group. Blood parameters were also compared between halothane categories using the fixed effects of gender, line, mobility status, and time with the random effect of group.
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RESULTS AND DISCUSSION
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This study was designed to determine if halothane-sensitive pigs are more prone to becoming NA when subjected to rigorous handling than pigs that respond abnormally to halothane. A controlled handling test has previously been shown to increase the frequency of NA pigs compared with that typically seen in the swine industry, thereby allowing for comparisons to be made between ambulatory and NA pigs (Benjamin et al., 2001). Using this test system also reduced the inconsistencies of previous handling and transport observed in commercial slaughtering facilities, and allowed relationships between handling inputs and metabolic effects on individual animals to be quantified.
No pigs became NA during transportation or during the lairage rest before the handling test. However, 9 animals became NA as a result of the rigorous handling (11.3%; Table 1), and all of these animals became NA as a result of fatigue, with no apparent physical injury. The higher proportion of NA pigs observed in this study compared with that typically seen in the swine industry likely resulted from more rigorous handling of the entire group, as opposed to slow-moving pigs only. Pigs classified as HS-H were more prone to becoming NA compared with those classified as HS-L (P < 0.02; Table 1). All but 1 of the 9 pigs became NA after returning to their pens after movement through the test. The only animal that became NA during the course did not exhibit discoloration of the skin or an elevated rectal temperature, but appeared to be experiencing labored breathing. However, this HS-L pig was classified as NA because it was unwilling to move and was resting in a sitting position.
No differences were observed due to halothane sensitivity in the number of animals exhibiting skin discoloration or in rectal temperature assessed over the 3 evaluation times (Table 1
). However, a lower percentage of HS-H pigs exhibited open-mouth breathing compared with the HS-L or HS-I pigs (P < 0.05; Table 1). These observations suggest that some halothane sensitive pigs may become conditioned to stressors or are better able to adapt to stressful experiences (van Putten, 1982). A greater proportion of pigs exhibited open-mouth breathing and skin discolorations after the handling test (posttest) than at the pretest or 1 h posttest observations within halothane status (P < 0.05; Table 1). However, no differences were observed in these visual characteristics between pretest or 1 h posttest observations. Rectal temperatures were highest in pigs immediately after the handling course (39.7°C) compared with pre- or posttest temperatures (38.6°C vs. 39.0°C, respectively; P < 0.05). Temperatures were still elevated 1 h posttest compared with pretest values (39.0°C vs. 38.6°C, respectively) but were lower than those observed immediately after the handling (39.7°C vs. 39.0°C; P < 0.05).
Electric prods were used in this study to compound the effects of rigorous handling. These prods were of a short duration (0.5 s) and recorded individually for each pig. Pigs classified as HS-H received fewer prods per pig than those classified as HS-L (P < 0.05; Table 2). This observation is opposite of anticipated results. Pigs classified as HS-H were expected to be more stress susceptible and subsequently more likely to fatigue faster. This would place the pig closer to the animal handler and would result in these pigs receiving more prods. Interestingly, when mobility status is accounted for in the statistical model, HS-H pigs that remained ambulatory received fewer prods per pig than HS-L pigs (P < 0.05; Table 2), and HS-H tended to receive fewer prods than HS-I pigs (P = 0.10). These observations suggest that HS-H pigs might have generally been in front of the group, or farther away from the animal handler than HS-L or HS-I pigs. Pigs that were HS-H and became NA received almost 7 more prods per pig compared with the ambulatory pigs (P < 0.05; Table 2). It is important to note that no HS-H pigs became NA during the handling course. Those that became NA and exhibited fatigue did so during the posttest period. It is unclear if the pigs in the current study became NA as a result of the increased number of prods or if these pigs were slowed by physical or metabolic limitations associated with halothane sensitivity and thus received more prods from the handler.
No differences were observed in the blood metabolites between HS categories (Table 3). This is in contrast to previous reports demonstrating HS pigs are more susceptible to stress and have a greater autonomic response to stimulation than halothane nonsensitive pigs (Gregory and Lister, 1981). In a classical stress response situation, HS pigs have been shown to be in a hypermetabolic state and have elevated levels of blood metabolites (Jones et al., 1972; Veum et al., 1979; Heinze and Mitchell, 1989). The increase in blood metabolites after the rigorous handling indicates that these animals were being physically challenged, regardless of halothane sensitivity category (P < 0.001; Table 3).
Pigs that became NA in this study had elevated levels of blood CPK, glycerol, lactic acid, NEFA, ammonia and BUN before initiation of the handling test (P < 0.05; Table 4). This would suggest that animals that became NA during the test were in a hypermetabolic state before the test. It is possible that elevation of these metabolites before the test could be an indication of a chronic excitability of these pigs (Barnett and Hemsworth, 1986). After the rigorous handling, the blood metabolites increased, and CPK, glycerol, NEFA, ammonia, and BUN continued to be different between mobility categories (P < 0.05; Table 4). These data suggest that the increased number of prods received by NA pigs resulted from a preexisting condition(s) that impaired the movement of these pigs through the course.
The elevation of blood metabolites is characteristic of a metabolic challenge or physical stress (Warriss et al., 1992). The elevated blood lactate indicates an increase in the work being done by these muscles. Lactate and hydrogen ions are the end product of anaerobic glycolysis and increase proportionately to the energetic needs of the cell. Elevated blood lactate is often associated with animals that are in a state of metabolic acidosis. Elevated blood CPK levels are typically associated with muscle cell damage and damage to the cells would be expected with increases in lactic acid and hydrogen ion concentration as a result of an increase in glycolytic metabolism (Heinze and Mitchell, 1989). In contrast to most of the metabolites measured, including lactate, blood CPK levels did not change after the handling course. However, blood CPK activity was elevated in NA pigs compared with ambulatory pigs, both before and after the handling course (Table 4). This suggests that muscle damage may have existed before the handling course in pigs that became NA after handling.
Increases in glycerol and NEFA (P < 0.01; Table 3 and 4) reflect an acute lipolytic response, in which triglycerides are mobilized from fat to provide energy needed to accommodate both physical and psychological stress. In addition, increases in ATP use would also initiate muscle glycolysis to replenish the energy needed for the cell to survive. Previous work has demonstrated that ammonia production is proportional to the work that is being done by muscle (Dudley et al., 1983). The observed increase in ammonia is most likely from the catalysis of adenosine monophosphate to inosine monophosphate by the enzyme adenosine monophosphate deaminase. Increases in ammonia can be toxic to the muscle cells if allowed to accumulate (Lowenstein, 1972). To prevent accumulation, the ammonia is processed through the urea cycle, which would increase BUN values, and is then excreted from the body. It is also possible that some ammonia is generated from the mobilization of amino acids from proteins. However, under the short-term stress described in this study, it is unlikely that ammonia generated from protein catabolism contributes significantly to the BUN values.
We speculate that HAL-1843-normal pigs that respond abnormally to halothane exhibit differences in calcium release from the sarcoplasmic reticulum, similar to those previously demonstrated in halothane-sensitive Pietrains (Mickelson et al., 1988). It is reasonable to expect that some halothane-sensitive pigs have become conditioned to stressors as a survival mechanism. This may explain the decreased number of HS-H animals exhibiting open-mouth breathing and the lower number of prods received by HS-H pigs that remained ambulatory after handling. However, we observed a higher incidence of NA pigs in the HS-H category after the handling, which suggests that some of the HS-H pigs have a lower tolerance for stress. Clearly, there is variability in the threshold level of stress required to induce fatigue among pigs.
After the handling test, pigs from each halothane sensitivity category were transported to 2 slaughtering facilities. The meat quality data from these pigs have been reported elsewhere (Allison et al., 2005). Briefly, no differences were observed in the initial pH, but HS-I and HS-H pigs had a lower ultimate pH than HS-L (5.93 vs. 5.77, HS-L vs. HS-H, respectively, at Plant B and 5.94 vs. 5.77 and 5.80, HS-L vs. HS-I and HS-H, respectively, at Plant C). The lower ultimate pH was associated with approximately 58% more purge loss from HS-H and HS-I loins compared with HS-L loins. These observations support the notion that halothane-sensitive pigs were able to recover from the rigorous handling more quickly and thus had more muscle glycogen available for conversion to lactate and hydrogen ions during postmortem anaerobic glycolysis.
Collectively, these data suggest that HAL-1843-normal HS pigs are more prone to becoming NA when subjected to rigorous handling. Some pigs appear to exhibit chronic elevation of key blood metabolites, and these pigs are more susceptible to becoming NA. Further work is needed to understand the biological link between halothane sensitivity and stress susceptibility or meat quality. It is of utmost importance to the swine industry to better understand the cascade of events that result in an animal becoming NA to develop intervention or prevention strategies.
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IMPLICATIONS
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Although nonambulatory pigs occur at a low frequency, approximately 1 million market pigs may become nonambulatory each year. These pigs represent substantial economic losses for the swine industry. Research to understand the mechanisms responsible for pigs becoming nonambulatory under commercial conditions is of great importance. These data suggest that HAL-1843-normal pigs, which exhibit halothane sensitivity, are more prone to becoming nonambulatory when subjected to rigorous handling. Furthermore, blood metabolites measured in pigs that became non-ambulatory were elevated before the test. This suggests that these pigs have preexisting metabolic condition(s) that make them more prone to becoming nonambulatory. Understanding the biological cause(s) of the metabolic condition that renders pigs nonambulatory is essential for the development of effective strategies to reduce the incidence of nonambulatory pigs.
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Footnotes
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1 The authors thank Elanco Animal Health for partial funding of this project and for assistance with the handling test. We are also grateful for support from the Michigan Agricultural Experiment Station. 
2 The HAL-1843 is a registered trademark owned by The Innovations Foundation, Toronto, Ontario, Canada.
3 Corresponding author: doumitm{at}msu.edu
Received for publication October 12, 2004.
Accepted for publication November 2, 2005.
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Abstract
INTRODUCTION
