Porcine stress syndrome (PSS) is a hypermetabolic and hypercontractile syndrome triggered by anesthesia or various stressors that cause a sustained increase in sarcoplasmic calcium ion (Ca2+) [1]. The main consequence of PSS in humans and swine is the appearance of malignant hyperthermia (MH). Susceptibility to PSS is inherited as an autosomal dominant disease. Human MH is a life-threatening, acute pharmacogenetic disorder of the skeletal muscle cells. Its manifestations are normally silent and only made evident when susceptible patients receive general anesthesia with volatile anesthetic agents or succinyldicholine [2].
The
Malignant hyperthermia exhibits a hypermetabolic response with the release of lactic acid, potassium, and proteins (
Total plasma calcium concentrations increase during a PSS crisis. Sarcoplasmic proteins also leak from muscle fibers during an acute episode. For instance, it is well-known that myoglobin, which has a low molecular weight (
The conversion of lactate, alanine and aspartate to glucose and oxidation of each to carbon dioxide (CO2), are processes that can be influenced by PSS [14]. Also, the stress causes a depletion of adenosine triphosphate (ATP) and extensive glycolysis in muscles [15], which can affect the activity of alkaline phosphatase (AP) responsible for removing the phosphate groups from several molecules, including nucleotides. Another interesting parameter closely related to ATP conversion is creatine phospho-kinase (CPK) that is associated with PSS [16]. It was suggested that fewer proteins are catabolized during the alanine cycle in stress-susceptible pigs; thus, total proteins were also included in this study [14]. Regarding that fact the accumulation of degradation metabolites (creatinine and bilirubin) generated by increased metabolic activity could be expected as possible biochemical markers of MH.
The effects of PSS have been investigated on maternal performances. Heterozygous PSS (Nn) females farrowed more pigs in first pig birth than normal (NN) and homozygous (nn) females. The proportion of pigs born alive that survived was not different between females of differing PSS genotypes. Normal and Nn females had nearly identical adjusted number of live-born piglets [17]. The goal of this study was to determine the differences in nine biochemical parameters associated with PSS genotypes, and matching the PSS genotype with maternal traits in selected sows.
Blood was taken from 73 randomly chosen sows that were not exposed to stress. After the centrifugation step (1000 × g for 10 min. at room temperature), the plasma samples underwent further biochemical analyses, while the leucocytes served for DNA extraction. DNA extraction from leucocytes was done using a DNA purification kit (Cat. #A1125; Promega, Madison, WI, USA), according to the manufacturer’s instructions.
Genotyping of PSS was performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) by digestion of the amplified part of the
Quantitative records on newborn, stillborn and weaned piglets (piglets that became independent) required for the statistical analyses were obtained from the data kept on the farms. Statistical analysis for genotype distribution and allele frequencies were determined by individual and allele counting. For the quantitative parameters and productive traits, descriptive statistical parameters (mean, minimum, maximum and mean standard error (MSE) were calculated. Later, the differences in the genotypes were tested with the Student
An experiment was conducted with sows genotyped for PSS using DNA based methods. Using a PCR-RFLP approach, we determined the genotypes for PSS in 73 DNA samples, identifying 60 stress-free animals (NN), 11 heterozygots (Nn) and two homozygous stress-susceptible animals (nn). Allele frequency of the N and n alleles was 0.897 and 0.103, respectively. The PCR-RFLP characterization of PSS is shown in Figure 1.
The results from genotyping were matched with values of nine biochemical parameters that were examined as possible predictors of PSS susceptibility in non-stress induced animals (Table 1). The study also explored the relevance of different biochemical parameters that could be used as possible biochemical markers for PSS susceptibility. Maternity traits matched with the genotype for PSS are shown in Table 2.
Biochemical data of plasma samples from animals with different porcine stress syndrome genotypes. Reference values from the Research Animal Resources, University of Minnesota, Minneapolis, MN, USA ( A A A A A A A A A NN: stress-free; Nn: heterozygous carrier; nn: homozygous carrier; Ca2+: calcium ion; AP: alkaline phosphatase; AST: aspartate aminotransferase; ALT: alanine aminotransferase; LDH: lactate dehydrogenase; CPK: creatine phosphokinase.Biochemical Parameter Genotype Reference Values NN Nn nn Ca2+ (µmol/L) 2.62 2.68 2.71 2.50-2.95 1.14 Creatinine (µmol/L) 142.10 157.30 174.60 88.40-265.20 9.97 Total proteins (g/L) 72.40 75.70 74.90 68.00-89.00 1.42 Bilirubin (µmol/L) 1.65 2.54 4.12 0.00-12.00 12.79 AP (U/L) 21.80 45.30 62.40 9.00-70.00 14.55 AST (U/L) 29.20 58.30 125.50 17.00-45.00 16.32 ALT (U/L) 35.30 51.70 48.20 9.00-60.00 9.14 LDH (U/L 398.00 655.00 894.00 159.00-475.00 17.02 CPK (U/L) 276.00 591.00 764.00 67.00-489.00 15.29
Association of the porcine stress syndrome genotype with maternal traits (newborn, stillborn and weaned piglets). A A A NN: stress-free; Nn: heterozygous carrier; nn: homozygous carrier.PSS Genotype Newborn Piglets NN 10.92 0.22 Nn 11.73 0.52 2.59 nn 11.50 1.50 Stillborn Piglets NN 0.15 0.06 Nn 0.09 0.09 2.53 nn 1.5 0.50 Weaned Piglets NN 8.20 0.19 Nn 9.00 0.46 0.05 nn 7.50 0.50
We did not find a significant correlation between the level of Ca2+ and stress susceptibility [12,19]. The values of plasma Ca2+ showed no significant differences among the different PSS genotypes. All values for concentrations of Ca2+ were in the reference range. The biochemical data for total protein and ALT did not show significant association with the PSS genotypes.
The potential link between creatinine and bilirubin values with the PSS genotype has been previously presented [20]. The levels of creatinine, bilirubin and AP were in the frame of their reference ranges although the PSS-susceptible animals had higher values of creatinine, bilirubin and AP compared to those from stress-free and heterozygous animals. Nevertheless, the differences in the three groups of animals in some parameters were highly significant. Taking into account the role of AP in energy metabolism, creatinine as a final product of increased energy level and the releasing of myoglobin as one of the bilirubin precursors, those differences are explainable.
Lactate dehydrogenase was used as a biochemical marker for the identification of stress susceptible animals before development of molecular tools [12,16]. Our data confirmed that only the values of the enzyme from non induced stress-free animals (398.00 U/L) were in the frame of reference values, while the values of heterozygous (Nn) animals were slightly out of that range (655.00 U/L). The values of stress-susceptible animals were 2-fold higher (894.00 U/L) than the maximum values of reference range (159.00-475.0 U/L) (
Creatine phosphokinase was reported to be affected in PSS animals [21]. Our results showed that the values of the enzyme from stress-free animals (276.00 U/L) were within the normal range, the CPK values from the heterozygous pigs (591.00 U/L) were out of range, and the values of stress-susceptible animals were higher (764.00 U/L) than the maximum values of the reference range. Those differences matched with the PSS genotype were highly significant (
Deviation of AST values were observed in the animals with the different PSS genotype. We observed the highest deviation between the values we measured and the reference values. Only the data of stress-free animals (29.20 U/L) were in the frame of reference values, the values of heterozygous animals were higher (58.30 U/L), while the values of the homozygous animals (125.50 U/L) were 3-fold higher than those in the reference range (17.00-45.00 U/L) (
Regarding maternal traits, we found that the heterozygous PSS sows with genotype Nn expresses the best reproductive ability, the highest number of newborn and the lowest number of stillborn piglets compared with normal (NN) and stress susceptible sows (nn) (