Heterozygous Pigs Research Articles

The effect of the halothane gene on pork production and meat quality of pigs reared under commercial conditions

Production and meat quality traits were evaluated on 74 commercially raised heterozygous pigs (Nn) and 50 homozygous normal pigs (NN) at the halothane locus, with an average carcass weight of 79.7 kg. Eight-week weight, average daily gain and age to market, as well as measures of carcass quality (i.e., dressing percent, grade site fat and muscle depths and predicted lean yield) did not differ among pigs with Nn and NN genotypes. The longissimus dorsi muscle from Nn and NN pigs had similar subjective colour and structure scores. Reflectance meter readings (CIE L*-values) were significantly higher on chops from Nn than from NN pigs taken from the centre (57.4 vs. 55.6) and from the anterior portion (56.2 vs. 54.5) of the loin. Drip loss among Nn pigs was higher than among NN pigs for chops taken from the centre (4.8 vs. 3.8 g 100 g−1) and from the anterior portion (3.7 vs. 2.9 g 100 g−1) of the loin (P < 0.01). Soluble protein was 1.7% lower (P < 0.001) in Nn pigs compared with the NN pigs. Barrows attained market weight earlier (P < 0.05) than gilts, but they had a lower dressing percentage (P < 0.05) and greater grade fat depth (P < 0.001) and had 1.2% less predicted lean yield of carcass (P < 0.001) than gilts. Since meat quality declined without concomitant increases in growth rate or improvements in carcass quality, it was concluded that the intentional use of the halothane gene should be discouraged. Key words: Swine, production, carcass quality, meat quality, halothane gene

Cosegregation of porcine malignant hyperthermia and a probable causal mutation in the skeletal muscle ryanodine receptor gene in backcross families

A study of the inheritance of malignant hyperthermia (MH) in the British Landrace breed revealed the same substitution of T for C at nucleotide 1843 in the ryanodine receptor ( RYR1) gene that was previously shown to be correlated with MH in five Canadian swine breeds. Cosegregation of the mutation with MH in 338 informative meioses led to a lod score of 101.75 for linkage at O max = 0.0. The substitution was also associated with a HinPI − BanII + RasI − haplotype in this breed, as in the five breeds tested earlier, suggesting its orgin in a common founder animal. DNA-based detection of the MH status in 376 MH-susceptible heterozygous ( N n ) and homozygous ( n n ) pigs was shown to be accurate, eliminating the 5% diagnostic error that is associated with the halothane challenge test and flanking marker haplotyping procedures in current diagnostic use. These results strongly support the view that the substitution of T for C at nucleotide 1843 is the causative mutation in porcine MH and demonstrate the feasibility of rapid, accurate, noninvasive, large-scale testing for porcine MH status using DNA-based tests for the mutation.

Distinct immunopeptide maps of the sarcoplasmic reticulum Ca2+ release channel in malignant hyperthermia.

Sarcoplasmic reticulum isolated from malignant hyperthermia-susceptible (MHS) muscle exhibits abnormalities in the regulation of calcium release. To identify the molecular basis of this abnormality, the Ca2+ release channel from both normal and MHS sarcoplasmic reticulum was examined using proteolytic digestion followed by immunoblot staining with a polyclonal antibody against the rabbit Ca2+ release channel protein. Under appropriate conditions, trypsin digestion of isolated sarcoplasmic reticulum vesicles from the two types of pigs revealed a distinct difference in the immunostaining pattern of the Ca2+ release channel-derived peptides. An approximate 86-kDa peptide was the predominant fragment in normal sarcoplasmic reticulum while an approximate 99-kDa peptide fragment was the major peptide detected in MHS sarcoplasmic reticulum. Digestion of sarcoplasmic reticulum vesicles isolated from four normal and four MHS pigs showed that the differences were highly reproducible. Trypsin digestion of sarcoplasmic reticulum isolated from heterozygous pigs, which contain one normal and one MHS allele, showed an antibody staining pattern that was intermediate between MHS and normal sarcoplasmic reticulum. These results can be explained by a primary amino acid sequence difference between the normal and MHS Ca2+ release channels and support the hypothesis that a mutation in the gene coding for the sarcoplasmic reticulum Ca2+ release channel is responsible for malignant hyperthermia.

Halothane-sensitivity gene and muscle contractile properties in malignant hyperthermia.

Malignant hyperthermia (MH) results from the presence of the halothane-sensitivity gene and is characterized by abnormalities in muscle function. Populations of genetically defined pigs were used to determine the in vivo and in vitro expression of this gene in both the homozygous and the heterozygous condition. On exposure to halothane, isolated muscle bundles from the homozygous halothane-sensitive pigs exhibited decreased tetanus tension and increased tetanus half-relaxation time and contracture and were clearly distinguished from homozygous normal muscles. The heterozygous and homozygous normal muscles were similar in contractile responses except for the occurrence of halothane-induced contractures in the heterozygotes. The heterozygous halothane-negative pigs did not exhibit the characteristic signs of an MH episode in response to halothane succinylcholine, although some metabolic responses were significantly altered (e.g., increased venous partial pressure of CO2 and arterial and venous K+ concentration). Thus the heterozygous pigs were not MH susceptible but did represent a phenotype distinct from the homozygous normal pigs both in vitro and in vivo. These data provide the first convincing evidence for the expression of the halothane-sensitivity gene in heterozygotes.

Separation of swine plasma LDL from Lpb2/3 heterozygotes into two apoB allelic haplotypes, Lpb2 and Lpb3, with apoB epitope specific antibodies.

Studies were performed to investigate the separation of Lpb (lipoprotein B) species present in plasma of heterozygous swine bearing the Lpb2 and Lpb3 apoB mutant genes. Low density lipoprotein (LDL) fractions from Lpb2/2 and Lpb3/3 homozygotes were coupled to a matrix and used to isolate affinity-purified antibodies anti-Lpb2 and anti-Lpb3 from swine alloimmune sera, one with specificity for the Lpb2 epitope(s) and the other for Lpb3. These antibodies in turn were used to construct two immunosorbers, anti-Lpb2 and anti-Lpb3 Sepharose columns. To separate the two Lpb haplotype populations present in LDL, a density gradient ultracentrifuge subfraction (d 1.032-1.043 g/ml) obtained from Lpb2/3 heterozygous pigs was applied to the specific immunosorbers. The retained fraction from the anti-Lpb2 column reacted in the double immunodiffusion test with anti-Lpb2 and anti-Lpb13 immune sera but not with either anti-Lpb3 or anti-Lpb12, while the unretained fractions reacted with anti-Lpb3 and anti-Lpb12 but not with either anti-Lpb2 or anti-Lpb13. The reaction patterns obtained with the two sets of alloimmune sera indicate the existence of two separate lipoprotein populations in LDL: one lipoprotein carrying the Lpb2 and Lpb13 epitopes corresponding to the Lpb2 apoB allele, and the other carrying the Lpb3 and Lpb12 allotypes specified by the Lpb2 gene. Immunoblotting with anti-Lpb2 and anti-Lpb3 and silver staining showed that the epitopes of both isolated LDL subpopulations are associated with apoB-100. Neutral lipid analyses showed no differences between the isolated Lpb2 and Lpb3 lipoprotein species from the Lpb2/3 heterozygotes. These studies demonstrate that plasma LDL subfractions from Lpb heterozygous swine can be separated into two haplotype populations, each corresponding to the product of one apoB gene, and reveal a new insight into the phenotypic expression of plasma LDL, and the LDL phenotype-genotype relationship. Furthermore, this approach will facilitate studies on metabolic differences of two structurally distinct LDL, unaffected by in vitro manipulation, exposed to the metabolic milieu of one individual.

Platelet thrombus formation on collagen type I. A model of deep vessel injury. Influence of blood rheology, von Willebrand factor, and blood coagulation.

Collagen type I is a major component of atherosclerotic vessel wall that is exposed on deep vessel injury, such as in balloon angioplasty or plaque rupture. Collagen type I from pig Achilles tendon was mounted in a tubular perfusion chamber placed within an extracorporeal circuit (carotid artery to jugular vein). The material was exposed to blood from normal pigs (n = 13), severe homozygous von Willebrand factor (vWF)-deficient pigs (vWF less than 3%) (n = 6), and heterozygous vWF-deficient pigs (vWF = 24%) (n = 2). Thrombus formation was measured by autologous 111In-platelet labeling and by ultrastructural morphology. Heparinized and native blood from these pigs was perfused over the substrate for 3 and 5 minutes at local shear rates from 212 to 3,380/sec. On collagen type I exposed to nonanticoagulated blood, for all exposure times studied, thrombus formation in the absence of vWF was significantly reduced at high shear rate typical of stenotic areas but not at low shear rate typical of unobstructed medium-size arteries. A similar inhibition in thrombus formation due to vWF deficiency was observed in both heparinized and native blood; however, thrombus formation was significantly more reduced (p less than 0.05) in the presence of heparin, presumably due to the lack of stability of the accumulated platelets in the absence of fibrin formation. Intermediate levels of vWF, as in heterozygous von Willebrand's disease (vWD), support platelet deposition to extents not significantly different from normal conditions. Therefore, on collagen type I, both the activation of blood coagulation proteins and the presence of vWF contribute significantly to the platelet-platelet interactions necessary for thrombus formation. The effect of vWF occurs primarily at high shear conditions typical intravascularly of flow at the apex of advanced stenotic lesions; thus, these findings may suggest that the absence of vWF may be protective against the development of acute thrombosis in these regions.

DNA polymorphism in the major histocompatibility complex of man and various farm animals*

In the past few years it has been possible by combining enzymatic cleavage of genomic DNA and the Southern blot hybridization technique to explore the endonuclease recognition site polymorphism of the MHC. HLA class I and DR and DQ alpha and beta class II specific probes as well as human C4 and Bf class III probes were used. All these probes were shown to cross-hybridize with DNA from pigs, cattle, sheep and horses. Hybridization of human genomic DNA with a class I probe showed 15-25 bands per genome depending on the enzyme used. Distinct endonucleases generated clusters of restriction fragments (RF) in HLA-informative families which correlated with HLA specificities. While numerous clusters were found associated with HLA-A alleles almost no cluster was related to HLA B or C specificities. Similarly, class II probes provided a large number of clusters. The existence of these clusters suggested that some polymorphic restriction sites are found in strong linkage disequilibrium and that the underlying mechanism might be gene conversion with heteroduplex correction. Since the degree of polymorphism detected by RF appears to be greater than the polymorphism defined by more traditional methods stronger associations between RF and pathological conditions are to be expected. Southern blot analysis was applied to unrelated pigs and sheep, as well as to families. Preliminary studies have also been performed on a few unrelated cattle and horses. Depending on the endonuclease used the HLA class I probe hybridized with around 15 bands in MHC heterozygous pigs and ruminants while up to 20 bands were found in horses. Therefore, a several-fold greater number of potential class I genes exist compared to those actually expressed. With the class II beta probe, cattle and sheep showed around 10 bands whereas 15 were observed in pigs and around 20 in horses. Based on limited results obtained with DQ alpha and beta probes and with the DR alpha probe there appeared to be fewer of these respective genes. Only one C4 gene has been detected in pig and this gene maps within the SLA region. Hybridization with the human C4 probe in cattle, sheep and horses revealed two to four bands which could possibly account for two C4 genes. To date their linkage to the MHC has not been established. The Southern blot hybridization technique represents a powerful tool for future immunogenetic studies.(ABSTRACT TRUNCATED AT 400 WORDS)

Von Willebrand's disease prevents occlusive thrombosis in stenosed and injured porcine coronary arteries.

We studied the role of von Willebrand factor in coronary thrombosis in normal, heterozygous, and homozygous von Willebrand's disease pigs by producing coronary stenosis with a Goldblatt clamp positioned around the left anterior descending coronary artery. Flow velocity was assessed by a 20-MHz Doppler velocity probe distal to the Goldblatt clamp. Myocardial extracellular potassium levels were measured by potassium-sensitive electrodes in myocardium supplied by the left anterior descending artery. Whereas stenosis sufficient to block reactive hyperemia to a 20-second occlusion produced an elevation of myocardial extracellular potassium, it produced neither spontaneous cyclic flow reductions nor permanent cessation of coronary blood flow velocity. Injury of the coronary artery at the stenosis site with spring-loaded forceps produced cyclic flow reductions or permanent cessation of flow in eight of nine phenotypically normal pigs. On the other hand, flow variations occurred in none of the 10 von Willebrand's disease pigs, including four given purified von Willebrand factor at a dose that failed to correct the bleeding time (p less than 0.001, chi 2 test). Permanent cessation of flow was caused by an occlusive platelet-fibrin-red-blood-cell thrombus. Scanning electron micrographs from pigs with cyclic flow variations and from von Willebrand's disease pigs showed injured endothelium covered by adherent platelets, red and white blood cells, and fibrin. These data suggest an important role of native von Willebrand factor in sudden occlusive arterial thrombosis following stenosis and intimal injury.

Linkage of C4 and C4 deficiency to BF and GPLA

The C4, Bf, and GPLA phenotypes of homo- and heterozygous C4-deficient guinea pigs were studied. The electrophoretic patterns suggest that the deficiency in circulating C4 results from an impaired structural gene, allelic to the C4F, C4S, and C4S1 alleles at the C4 locus. In family studies, support for linkage of C4 and Bf to theGPLA system was obtained. The defective gene appears to be the fourth allele, which is rare, in the polymorphism of the fourth component of guinea pig complement.

GENETIC CONTROL IN GUINEA PIGS OF IMMUNE RESPONSE TO CONJUGATES OF HAPTENS AND POLY-L-LYSINE.

Random-bred Hartley strain guinea pigs which do not respond immunologically to conjugates of hapten and poly-L-lysine mere mated with heterozygous guinea pigs which do. These responders were considered heterozygous for this trait since their mating resulted in at least one nonresponder offspring. Of 31 offspring from 10 breeding pairs (nonresponder x heterozygous responder) 14 were responders. There was no evidence that this trait is sex-linked. This finding confirms the view that, in guinea pigs, development of an immune response to the aforementioned conjugates is a genetically transmitted autosomal, unigenic Mendelian dominant trait.

Survival of Guinea Pigs after Lethal Irradiation and Homologous Bone Marrow Injections

The literature concerning whole-body irradiation of laboratory animals has reached voluminous proportions. However, one commonly used laboratory animal, the guinea pig, has been used relatively infrequently in irradiation experiments, owing probably to the general unavailability of inbred strains of guinea pigs. In a few experiments, inbred guinea pigs receiving normally lethal doses of radiation have manifested prolonged survival after isologous bone marrow transfusions (1, 2). The following studies were undertaken in an attempt to determine whether heterozygous guinea pigs could be similarly protected by homologous marrow after receiving graded doses of lethal whole-body radiation. The ability of these irradiated animals to accept homologous skin grafts was also investigated.