Early Development, Identification of Mode of Action, and Use of Dantrolene Sodium

Dantrolene—a nitrofurantoin derivative—was developed by Snyder et al. in 1967. After initial discovery of its muscle relaxation potential, investigations in a number of species demonstrated dose-dependent reductions in skeletal muscle tone that were long lasting, relatively nontoxic, and free of adverse effects such as respiratory impairment. Ellis et al. then published a number of papers investigating the means by which dantrolene produced these effects. Using a series of classic physiologic models, Ellis investigated potential sites of action for the new drug, eventually narrowing this down to the intracellular calcium-release mechanism. Ellis went on to play a pivotal role in the discovery of dantrolene’s effectiveness for the treatment of malignant hyperthermia, after reading a scientific bulletin about muscle rigidity in pigs affected by porcine stress syndrome, contacting Gaisford Harrison and sending dantrolene to him for trial.

Associations of biochemical changes and maternal traits with mutation 1843 (C>T) in the RYR1 gene as a common cause for porcine stress syndrome

Stress syndrome is usually caused by a mutation in theryanodine receptorgene(ryr1) and it is widely studied in humans and swine populations. The protein product of this gene plays a crucial role in the regulation of calcium transport in muscle cells. A G>T mutation in the humanryr1gene, which results in the replacement of a conserved arginine at position 614 where a leucine occurs at the same position as the previously identified Arg→Cys mutation reported in all cases of porcine stress syndrome (PSS). Porcine stress syndrome affects biochemical pathways in stress-susceptible individuals during a stress episode and some biochemical parameters that were used as markers for diagnostic purposes. Also, PSS has remarkable influence on the maternal characteristics of sows. This study dealt with different genotypes for PSS and its association with possible biochemical changes and maternal traits of sows. Seventy-three reproductive sows genotyped for PSS by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) were included in this survey. Sixty of them were stress-free (NN), 11 were heterozygous carriers (Nn) and two animals were homozygous (nn) for the 1843 (C>T) mutation. Significant differences in non stress induced animals with different PSS genotypes were found in the values of creatine phoshokinase (CPK), lactate dehydrogenase (LDH), alkaline phosphatase (AP) and aspartate aminotransferase (AST). Regarding the maternal traits, our study showed that stress susceptible animals (nn) have an increased number of stillborn piglets and a reduced number of newborn piglets compared with heterozygous and normal animals.

Porcine stress syndrome (PSS) in mangalitsa pigs

Porcine stress syndrome (PSS) is one kind of molecular genetics defect which will cause malignant hyperthermia syndrome in pigs. It was reported that mutation of pig rynodine receptor (RYR1) gene is the main reason for PSS. The aim of this study was to test the RYR1 genotype of 10 Mangalitsa pigs using a polymerase chain reaction-restriction endonuclease fragment length polymorphism (PCR-RFLP) technique, which is a reliable and simple method for RYR1 gene testing. Extraction of DNA was done by using hair follicles. The results showed that the RYR1 genotype of all the 10 porcine cases were negative. These results suggested that Mangalitsa pig could be one of the porcine breeds selectively bred for medical and clinically experiments.

An improved method for characterization of the mutation associated to porcine stress syndrome by PCR amplification followed by restriction analysis

A mutation in the gene coding for the ryanodine receptor 1 (RYR1), also known as halothane (hal) gene or swine stress gene, is associated to the porcine stress syndrome (PSS). Detection of the mutation is normally accomplished by PCR amplification of an 81bp fragment of the hal gene, followed by digestion with the HhaI restriction endonuclease. Wild-type allele (N) is cut in two fragments, whereas the mutant allele (n) is not digested by the restriction enzyme. Electrophoresis of the digested DNA on agarose gel and ethidium bromide staining allows the reading of the result. The correct interpretation is difficult due to the small size of the DNA fragments. In this study we designed a new set of primers for amplification of a 144bp fragment that facilitates the reading of the result. In addition, we optimized the PCR reaction to allow amplification from a single hair bulb, added directly into the PCR mix without previous treatment. This improved method was used to genotype 165 sows and boars used in a breeding program. Forty-nine percent of the animals had the NN genotype, whereas 50% were Nn and only 1% was nn.

Markers of acute stress in pigs

This study explores the biological validation of markers of acute stress in pigs subjected to transportation for slaughter. The stress markers selected for monitoring were neopterin and cortisol. Their levels in pig serum were measured for two porcine stress syndrome genotypes, NN and Nn, after a 30-min transport to a slaughterhouse. Blood samples were withdrawn before transport (control group) and immediately after the animals' arrival (experimental group). The values of neopterin and cortisol measured before the transport were 5.60+/-1.65 nmol/l and 273.54+/-66.17 nmol/l respectively. After the transfer, the concentration of cortisol rose significantly compared to the control (355.69+/-85.13 nmol/l, p<0.01). Neopterin concentrations in the serum (8.25+/-1.60 nmol/l) were also significantly higher (p<0.01) after transportation. The elevated concentrations of both analytes were found to be independent of the genotype. These results document the stimulation of the endocrine system and the immune system that develops in animals undergoing transportation for slaughter.

Application of DNA markers in animal industries

The current animal industry is both technology-intensive and globalised. Efficient molecular tools, such as DNA markers, are in demand to strengthen competitive power by maximising the improvement of livestock and obtaining the trust of customers by the verification of product origins. This review describes the present techniques applying DNA markers in the animal industry, with a focus on beef cattle and pigs. Preliminary data from an individual traceability assay for Hanwoo (Korean cattle) using 20 microsatellite markers is described. The potential uses of the assay are demonstrated for several key markers of different traits: for the porcine stress syndrome gene using the RYR mutation; for acid meat using the PRKAG3 mutation; for intramuscular fat using the FABP3 mutation and for fixing the Dominant white allele using KIT duplication. In addition, a possible strategy is suggested to discriminate between pig breeds using mutations of KIT, MC1R, ND2 and the 11-bp insertion in the D-loop of mitochondrial DNA. The industrial application of DNA techniques is limited at present, however, it is expected that DNA markers originating from trait genes, especially those of low-heritability and difficult-to-measure traits, may contribute to maximising the improvement of the major economic traits of animals in the future.