J. Anim Sci. 2008. 86:511-514. doi:10.2527/jas.2007-0329
© 2008 American Society of Animal Science
Frequency of the HAL-1843 mutation of the ryanodine receptor gene in dead and nonambulatory-noninjured pigs on arrival at the packing plant1
M. J. Ritter2,
M. Ellis3,
G. R. Hollis,
F. K. McKeith,
D. G. Orellana,
P. Van Genugten,
S. E. Curtis and
J. M. Schlipf
Department of Animal Sciences, University of Illinois, Urbana 61801
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Abstract
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Four Midwestern packing plants (designated as plants A, B, C, and D) were visited on 53 occasions, and tissue samples were collected postmortem from a total of 2,019 pigs to determine the frequency of the HAL-1843 mutation of the ryanodine receptor gene in dead (DOA), nonambulatory-noninjured (NANI), and normal animals. The sampled pigs came from approximately 130,000 animals from 454 farms and were transported on 861 trailer loads, with an average of 152 pigs/load and an average pig live BW/load of 125 (SD 7.02) kg/pig. Frequency of animals with the HAL-1843 mutation was low, with only 2.7% of the pigs being either homozygous recessives (nn, 0.45%) or carriers (Nn, 2.3%) for the mutation and 97.3% of the pigs being homozygous for the normal allele (NN). The mutation was present in all 3 classes of pig, with 1.8% of normal, 1.8% of NANI, and 4.7% of DOA animals having at least 1 copy. Two of the plants (A and C) had a greater frequency (P < 0.05) of carrier (3.7 and 3.5 vs. 1.1 and 1.0 for plants A and C vs. B and D, respectively) and homozygous recessive (1.0 and 0.9 vs. 0.0 and 0.0, respectively) animals than the others (plants B and D). There was a greater frequency (P < 0.05) of carriers in DOA animals than in the normal or NANI pigs (3.7 vs. 1.7 and 1.5 for DOA vs. normal and NANI, respectively). The 55 pigs that had at least 1 copy of the mutation came from 53 farms; therefore, the mutation was relatively widespread, being present in approximately 11% of the farms sampled. Although the HAL-1843 mutation is still present in commercial pig populations in the United States, its low frequency in DOA and NANI pigs suggests that it is not a major cause of these transport losses.
Key Words: dead • HAL-1843 mutation • nonambulatory • pig • transport loss
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INTRODUCTION
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Losses of slaughter-weight pigs during transport to the slaughter plant (dead and nonambulatory animals) are of concern to the US swine industry from both a welfare and an economic perspective. Historically, the HAL-1843 mutation of the ryanodine receptor gene (commonly called the halothane or stress gene) was considered responsible for a substantial proportion of transport losses (Murray and Johnson, 1998
). The specific HAL-1843 mutation was identified in 1991 (Fujii et al., 1991), and a DNA-based test for this mutation has been widely available since that time. Subsequently, breeding stock suppliers have been able to select against this unfavorable mutation, and there are claims that it has been eliminated from most commercial swine populations. However, there is evidence that the mutation is still present in US commercial pigs (Ivers et al., 2002). Even at a low frequency, this mutation could be an important causal factor in transport losses. Quantifying the frequency of the HAL-1843 mutation of the ryanodine receptor gene in pigs that die (DOA) or become nonambulatory-noninjured (NANI) in contemporary commercial pig populations of the US is an essential first step to determining the extent of its involvement in transport losses.
The current study was designed to investigate the frequency of the HAL-1843 mutation of the ryanodine receptor gene in DOA and NANI pigs and in a random sample of contemporary normal animals arriving at Midwest packing plants of the United States.
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MATERIALS AND METHODS
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Animal Care and Use Committee approval was not obtained for this study because all samples were taken postmortem from federally inspected slaughter facilities.
Slaughter Plants
Four large commercial packing plants (designated as plants A, B, C, and D) located in the Midwest of the United States were used in this study. They were chosen on the basis that the pigs handled at these facilities would be representative of the contemporary US pig population.
Plant Visits and Samples Collected
The plants were visited on 53 occasions (25, 9, 10, and 9 visits to plants A, B, C, and D, respectively) over a 5-mo period (from January 2006 to May 2006) to collect tissue samples for DNA analysis. The target was to obtain approximately 650 samples of each class of pig, and this was achieved for NANI animals on 34 visits. However, because of the relatively low incidence of DOA observed, extra visits were required to complete the sampling of this class of animal. In total, samples from 2,019 animals were obtained postmortem, consisting of 511, 555, 459, and 494 samples from plants A, B, C, and D, respectively (Table 1). These represented samples from 726 NANI, 644 DOA, and 649 normal animals.
Identification of Dead and Nonambulatory Animals
After trailers were unloaded, packing plant employees identified dead and nonambulatory pigs, and University of Illinois investigators selected the NANI individuals from among the nonambulatory animals. Non-ambulatory-noninjured animals were defined as noninjured pigs showing physical symptoms of stress (open-mouthed breathing, skin discoloration, muscle tremors, abnormal vocalization, or any combination of these symptoms) that either could not walk or were having difficulty in walking and could not keep up with the remainder of the group.
Additional Information Collected
Information collected on either the animals that were sampled or the trailer loads from which they came included the sex of the animal, the number of pigs per load, the average pig live BW per load, trailer design, and the number of dead and nonambulatory pigs per load.
Sampling Procedures
Ear tissue samples were obtained postmortem by using an ear punch. Samples from DOA pigs were obtained in the unloading area at the plant after the trailer had been unloaded. For trailer loads that had a DOA or NANI animal or both, a contemporary normal pig (identified by the common load tattoo number) was randomly selected from the same trailer load. Subsequently, ear tissue samples from the NANI and contemporary normal animals were obtained postmortem on the slaughter line before the head was removed from the carcass. Immediately after collection, the samples were placed in a plastic bag that was sealed, stored on ice, and subsequently transferred to a freezer (– 20° C) for storage until preparation for shipping to the laboratory for DNA analysis. The sample storage bag was labeled with the animal identification, the class of the animal (DOA, NANI, or normal), the slaughter plant, and the date of sample collection.
HAL-1843 Genotyping
All genotyping was conducted by a commercial laboratory (Genalysis Laboratory Inc., Lakeside, OH). A thin slice of tissue from each frozen ear sample was placed in a prelabeled, 0.65-mL microcentrifuge tube and shipped to the laboratory. The DNA was extracted according to the laboratory’s standard operating procedures, and genotyping was carried out according to the procedure described by Fujii et al. (1991).
The genotypes were defined as follows: 1) homozygous dominant (NN), 2) heterozygous carrier (Nn; monomutant with 1 copy of the mutation), or 3) homozygous recessive (nn; dimutant with 2 copies of the mutation).
Statistical Analysis
The total number and frequency (percentage) of the 3 genotypes (i.e., NN, Nn, and nn) were determined for each class of animal (normal, NANI, and DOA), each plant (A, B, C, and D), and each class of animal within each plant by using the FREQ procedure (SAS Inst. Inc., Cary, NC). Frequency means were compared by using a 
2 test and the RANK procedure of SAS.
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RESULTS
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The number of trailer loads of pigs and the number of farms from which DOA, NANI, or normal animals, or their combination, were sampled are summarized by plant in Table 1
. Across the 4 plants, pigs from 861 trailer loads, representing 454 different farms, provided samples of DOA, NANI, and normal animals for this study. The number of pigs per load averaged approximately 152, with an average live BW of approximately 125 kg. Approximately half of the trailers from which pigs were sampled were of the potbelly design (51%), with the remainder being of the straight-deck design (49%). The 2,019 pigs sampled consisted of 56% barrows and 44% gilts that came from a total of approximately 130,000 animals (861 trailer loads with approximately 152 pigs/load); therefore, the pigs that were sampled represented 1.6% of all pigs that were transported.
The frequencies of the 3 genotypes broken down by plant and class of animal are presented in Tables 2 and 3, respectively. Overall, the frequency of animals with the HAL-1843 mutation was relatively low, with 2.7% of the animals having at least 1 copy of the mutation (i.e., either Nn or nn) and 97.3% of the animals being homozygous for the dominant allele (NN). Of the animals with the mutation, 46 (2.3% of all pigs tested) were carriers (Nn) and 9 animals (0.45% of all pigs tested) were homozygous recessive (Table 2). Fifty-three farms had animals with the mutation; 51 farms had only 1 animal represented with 1 or 2 copies of the mutation, and 2 farms each had 2 pigs represented with 1 or 2 copies of the mutation. The sample of pigs used in this study came from 454 farms (Table 1); therefore, the mutation was present in animals from approximately 11% of the farms sampled, with 2% of the farms having homozygous recessive animals and 9% of the farms having carrier animals.
The frequency of homozygous dominant pigs was greater (P < 0.05) for plants B and D compared with plants A and C (Table 2). Two of the plants (A and C) had a higher (P < 0.05) frequency of both carrier and homozygous recessive animals than the others (plants B and D). The frequency of homozygous dominant pigs was lower and that of carrier animals was higher (P < 0.05) in DOA pigs than in either normal or NANI animals (Table 3).
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DISCUSSION
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In this study, the animals that were sampled came from 454 different farms and can therefore be considered to represent a relatively large producer base in the Midwest of the United States. Samples were obtained from the vast majority of DOA and NANI pigs that were present at the packing plants on the days of the visits. Thus, the samples of DOA and NANI animals used in this study are likely to be representative of the respective populations. However, the 861 trailer loads that were sampled transported approximately 130,000 pigs to the plants; therefore, the contemporary normal animals sampled in this study represented less than 0.5% of all normal animals delivered to the plant in the loads that were sampled for DOA and NANI pigs.
The frequency of pigs with at least 1 copy of the HAL-1843 mutation, at 2.7% of animals tested, was relatively low. No recently published studies have estimated the frequency of this mutation in contemporary US pig populations. Murray and Johnson (1998) published the results of a survey of the frequency of this mutation in pigs arriving at 2 packing plants in Western Canada. This survey showed that the frequency of the homozygous recessive (nn), carrier (Nn), and homozygous normal (NN) genotypes for animals that were DOA or died before slaughter at the plant was 27.7, 25.2, and 47.1%, respectively. Obviously, the frequency of the HAL-1843 mutation in dead pigs was much higher in the Canadian survey than in the current study. The pigs sampled in the Canadian study were likely to be from different genetic suppliers than those represented in the current study. In addition, that study was carried out at the time when genetic suppliers were still offering HAL-1843 carrier sire lines, and this could have been one factor related to the higher frequencies observed. In addition, a significant time-period had elapsed since the study of Murray and Johnson (1998), during which the frequency of the mutation could have changed, particularly if breeding stock suppliers have actively selected against the mutation, as has been claimed.
Overall, these results suggest that, although the HAL-1843 mutation occurs at a relatively low frequency in contemporary pigs in the United States (less than 3% of pigs tested in this study), it is still relatively widespread, being present in approximately 11% of farms that were represented. Historically, most commercial programs that aimed to exploit the potential benefits of the HAL-1843 mutation (i.e., improved feed efficiency and increased carcass yield and lean content; Leach et al., 1996) were based on a sire line that was a carrier of the mutation (i.e., Nn) and a negative dam line (i.e., NN). However, the fact that homozygous recessive animals were present in the pigs sampled suggests the mutation was present on both the sire and dam side of the pedigree on some farms.
There were differences among plants in the frequency of animals with the mutation; however, these differences were relatively modest. The observed differences in HAL-1843 frequencies among plants could be due to random sampling effects.
There was a greater frequency of animals with at least 1 copy of the mutation (nn or Nn) in DOA pigs (4.7%) than in NANI (1.9%) or normal animals (1.8%). In all classes, however, the frequency of the mutation was low, which suggests that, although this mutation may be a factor in transport losses for individual animals, it is not a major cause of losses. Consequently, efforts to reduce the incidence of transport losses should focus on other causes, both genetic and nongenetic. This is not to say that members of the industry should discount efforts to eliminate this mutation from pig populations. However, given the relatively low frequency of the mutation observed in this study, the cost compared with the benefit of further testing needs to be considered.
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Footnotes
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1 The authors wish to acknowledge the financial support of the National Pork Board (Des Moines, IA) and the assistance of various staff at the 4 slaughter plants involved in this study. 
2 Current address: Elanco Animal Health, 56776 241st Street, Suite 200, Ames, IA 50010
3 Corresponding author: mellis7{at}uiuc.edu
Received for publication June 6, 2007.
Accepted for publication October 29, 2007.
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LITERATURE CITED
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Fujii, J., K. Otsu, F. Zorzato, S. De Leon, V. K. Khanna, J. E. Weiler, P. J. O’Brien, and D. H. MacLennan. 1991. Identification of a mutation in porcine ryanodine receptor associated with malignant hyperthermia. Science 253:448–451.[Abstract/Free Full Text]
Ivers, D. J., L. F. Richardson, D. J. Jones, K. L. Watkins, K. D. Miller, J. R. Wagner, R. Seneriz, A. Z. Zimmermann, K. A. Bowers, and D. B. Anderson. 2002. Physiological comparison of downer and non-downer pigs following transportation and unloading at a packing plant. J. Anim. Sci. 80(Suppl. 2):39. (Abstr.)
Leach, L. M., M. Ellis, D. S. Sutton, F. K. McKeith, and E. R. Wilson. 1996. The growth performance, carcass characteristics, and meat quality of halothane carrier and negative pigs. J. Anim. Sci. 74:934–943.[Abstract]
Murray, A. C., and C. P. Johnson. 1998. Impact of the halothane gene on muscle quality and pre-slaughter deaths in Western Canadian pigs. Can. J. Anim. Sci. 78:543–548.
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Abstract
INTRODUCTION
