The effect of age on distribution of skatole and indole levels in entire male pigs in four breeds: Yorkshire, Landrace, Hampshire and Duroc.

High skatole and to a lesser degree indole levels in some entire male pigs result in occurrence of off-flavor in meat, called boar taint. In this study, plasma samples from 117 Yorkshire, 134 Landrace, 184 Hampshire and 75 Duroc entire male pigs collected at different ages were analyzed for skatole and slightly fewer samples for indole concentrations. For each breed, a distinct age-related distribution of plasma skatole levels was observed. Skatole levels were increased at approximately 180-200 days of age, reaching very high levels is some individuals. Afterwards, the levels decreased; in Yorkshire and Landrace at approximately 240-260 and in Hampshire and Duroc at 310-360 days of age. The decrease of skatole levels was also observed in samples collected from 15 Landrace boars at two different ages: 16.8 µg/L (SD=17.36) vs. 5.9 µg/L (SD=3.48, p=0.016) in samples taken at the mean age of 210 and 314 days, respectively. Indole levels showed similar age- and breed-related variations to skatole. At the time of increased skatole concentrations (age from 180 to 360 days, depending on breed) 25.5% of Yorkshire, 31.6% of Landrace, 20.3% of Hampshire and 61.1% of Duroc entire male pigs had skatole levels above 12.6 µg/L of plasma, which corresponds to the threshold level of 0.20 µg/g of fat that is used for selecting tainted carcasses. The results indicate that increased levels of skatole in entire male pigs are related to puberty and taking skatole measurements at that age could be advantageous when considering genetic selection to reduce boar taint level in entire male pigs. Breed differences should be also taken into account.

Relationship between parental cardiac status in Cavalier King Charles spaniels and prevalence and severity of chronic valvular disease in offspring.

OBJECTIVE: To study the relationship between parental cardiac status in Cavalier King Charles Spaniels and development of chronic valvular disease (CVD) in offspring. DESIGN: Historical cohort. ANIMALS: 54 female and 53 male Cavalier King Charles Spaniel offspring. PROCEDURE: 7 sires, selected on the basis of their liability to develop CVD, were screened for clinical signs of CVD and assigned to 1 of 3 groups (late, intermediate, and early onset of CVD). The mates of these sires (30 dams) were selected and classified likewise, and 107 offspring produced in 1988 from matings between these parents were screened for clinical signs of CVD at a mean age of 5.3 +/- 0.3 years. RESULTS: 55% of the offspring were free from clinical signs of CVD, whereas 45% had cardiac murmurs of low or moderate intensity. The proportion of offspring with heart murmurs and the intensity of murmurs were significantly greater with increased parental classification. More males than females had developed murmurs, and murmurs of moderate intensity also were more prevalent in males. Results of multiple-regression analysis indicated that mean parental classification and sex had significant effects on proportion of offspring with murmurs and their intensity. Additionally, age affected disease prevalence and severity, despite the narrow range in age of offspring examined. CLINICAL IMPLICATIONS: Parental CVD status is an important factor influencing the probability of heart murmurs and their intensity in offspring. The results of this study indicate that CVD development is a polygenic threshold trait and that sex of the offspring influences threshold levels.

Effect of polymorphism in the porcine cytochrome b5 ( CYB5A) gene on androstenone and skatole concentrations and sexual development in Swedish pig populations.

The present study investigated the presence of a single-nucleotide polymorphism (G > T) at base -8 upstream of ATG in 5' untranslated region of cytochrome b5 (CYB5A) gene in Swedish pig populations and evaluated the significance of this polymorphism for androstenone and skatole levels, sexual development and performance parameters in pigs. Frequencies of the T allele were 6.7% for Swedish Yorkshire × Landrace crossbred pigs (n = 245), 6.5% for Swedish Yorkshire (n = 99) and 12.8% for Landrace breed (n = 74). No deviations from Hardy-Weinberg equilibrium were observed in the investigated populations. In Swedish Yorkshire × Landrace crossbred entire male pigs (n = 193), plasma samples were analysed for skatole, androstenone, testosterone and oestrone sulphate, and fat samples were analysed for androstenone, skatole and free oestrone. Additionally, testis weight and bulbourethral gland length for crossbred pigs were recorded. Plasma androstenone levels were significantly lower in the G/T genotype at 90 kg live weight compared with the wild G/G genotype at the same live weight (P = 0.006). In heavier pigs, plasma androstenone levels did not differ between genotypes (P = 0.382). Fat androstenone levels were not affected by CYB5A genotype (P = 0.252). Skatole levels in the G/T genotype at 115 kg live weight were lower compared with those in the G/G genotype in plasma (P = 0.048) and fat (P = 0.028), although no differences were observed in lighter pigs. Testis weight, bulbourethral gland length, testosterone and oestrone sulphate levels in plasma, and oestrone levels in fat were not affected by genotype. We concluded that the presence of the T allele in the CYB5A gene resulted in lower androstenone levels in plasma, and lower skatole levels in fat and plasma; this reduction, however, was dependent on the live weight of the animals. Reproductive hormones and growth rate did not differ between the pigs of different genotypes, whereas a higher lean meat content was found in the G/T genotype in comparison with the G/G genotype. The practical application of those results in Sweden is doubtful because of lack of the effect on androstenone in fat and the low frequency of the T allele in the studied Swedish pig populations.

Two-dimensional gel electrophoresis of sheep plasma proteins: genetic polymorphism of an alphal-protease inhibitor and a post-transferrin.

Two-dimensional electrophoretic analysis of sheep plasma proteins was performed by a first-dimension separation in agarose gel (pH 5.0) followed by a second-dimension one in horizontal polyacrylamide gel (pH 9.0). This method resulted in improved and reproducible separation of many alpha- and beta-globulins. Two groups of alpha 1-globulins, designated Pi-1 and Pi-2, were found to be protease inhibitors. These two inhibitors differed from each other in protease inhibitory spectra. Genetic polymorphism was observed for the Pi-2 protein and another unidentified protein, tentatively designated as post-transferrin (Ptf). Family data supported the hypothesis that Pi-2 and Ptf types were controlled by codominant, autosomal alleles. Three Pi-2 alleles and two Ptf alleles were observed in one population of the Gotland breed of sheep. The analysis of data from 50 informative matings showed no evidence of genetic linkage between the Pi-2, Ptf and transferrin (Tf) loci in the population of sheep studied.

Two-dimensional gel electrophoresis of cattle plasma proteins: genetic polymorphism of an alpha 1-protease inhibitor.

Two-dimensional electrophoretic analysis of cattle plasma proteins was done by a first dimension separation in agarose gel (pH 5.0), followed by a second dimension in horizontal polyacrylamide gel (pH 9.0). This method resulted in improved and reproducible separation of many alpha- and beta-globulins. Three groups of alpha-globulins, designated Pi-1, Pi-2 and Pi-3, were found to inhibit the esterolytic activity of bovine trypsin and bovine chymotrypsin. Pi-2 showed appreciable inhibition only for trypsin and genetic polymorphism was observed for this protein. Family data supported the hypothesis that the three Pi-2 types observed were controlled by two codominant, autosomal alleles. The occurrence of a third Pi-2 allele was also postulated in some animals studied. The frequency of the most common allele, Pi-2s, ranged from 0.5-0.8 in the different breeds of cattle studied (Swedish Red and White, Friesian, Jersey, Charolais and Simmental). The post-transferrins Ptf-1 and Ptf-2 in cattle plasma were shown to be two different genetic systems.

Genetic variation at pig plasma protein locus PLP1 and its assignment to chromosome 5.

A new genetic polymorphism of an unidentified plasma protein (PLP1) in pigs was described by using a method of two-dimensional gel electrophoresis and protein staining. Two codominant alleles, with frequencies of 0.83 and 0.17, were found in the Swedish Yorkshire breed. The PLP1 marker was typed in a three-generation pedigree and tested for linkage against a set of 128 markers. The PLP1 locus showed significant LOD score values with three different microsatellite markers (S0092, DAGK and S0005), previously assigned to chromosome 5.

Simultaneous phenotyping of pig plasma alpha-protease inhibitors (PI1, PO1A, PO1B, PI2) and four other proteins (PO2, TF, CP, HPX) by a simple method of 2D horizontal electrophoresis.

A simple and rapid method of 2D agarose gel (pH 5.4)-horizontal polyacrylamide gel (pH 9.0) electrophoresis was developed for the simultaneous phenotyping of pig plasma alpha-protease inhibitors (protease inhibitor-1 and -2; postalbumin-1A and -1B), postalbumin-2, transferrin, ceruloplasmin and haemopexin. These eight plasma proteins were clearly visible on gels stained with Coomassie Brilliant Blue G250. The 2D patterns and mobilities of several variants of alpha-protease inhibitors were described. By using two agarose gels and 10 polyacrylamide gels, 120 samples were easily analysed in a day. Since alpha-protease inhibitors show extensive polymorphism and as the gene for postalbumin-2 is closely linked to the halothane sensitivity locus Hal, this method is a useful tool for conducting parentage control and for predicting Hal genotypes of individual pigs.

Extensive genetic polymorphism of four plasma alpha-protease inhibitors in pigs and evidence for tight linkage between the structural loci of these inhibitors.

Two-dimensional horizontal gel electrophoresis of pig plasma samples (under non-denaturing conditions) using Immobiline pH gradient gels 4.0-6.0 for the first dimension separation, resulted in clear resolution of the variants of four different alpha-protease inhibitors (protease inhibitor -1 and -2, PI1 and PI2; post-albumin -1A and -1B, PO1A and PO1B). All these variants were readily visualized by general protein staining. About 900 families each of Swedish Landrace (SL) and Yorkshire (SY) breeds were studied. The extensive inheritance data, including the recombinants encountered, indicated that each of these four inhibitors is controlled by a separate, autosomal locus and that the four loci are tightly linked (spread over a distance of 1-1.5 cM) with the order as Pi1-Po1A-Po1B-Pi2. The alleles observed were two of Pi1, 14 of Po1A, 11 of Po1B and 8 of Pi2. About 40 haplotypes were observed in each of the two breeds. The allele frequencies at Po1A, Po1B and Pi2 loci were remarkably different in the two breeds; the alleles at these three loci showed a very strong linkage disequilibrium (0.8-1.0). The females showed much higher recombination frequencies than the males in the Po1A-Pi2 interval, suggesting that gene conversion-like events may be occurring at these loci. This linkage in pigs and similar ones comprising some plasma alpha-protease inhibitor genes in humans and in rodents, reported recently in the literature, indicate evolutionary conservation of a homologous linkage group in these species.

Genetic polymorphism and close linkage of two plasma protein loci in dogs.

By using a simple method of two-dimensional horizontal electrophoresis, phenotypes of an unidentified plasma protein (PA4) were determined in 967 dogs belonging to 43 different breeds. Two codominant, autosomal alleles (F and S) of PA4 were reported. While many of the breeds of middle and north-eastern Asia (akita inu, Alaskan malamute, chow chow, samoyed, Siberian husky and Tibetan terrier) showed a substantial frequency (0.1 to 0.6) of the S allele, a majority of the European breeds had only the F allele. Evidence was provided that the PA4 locus is closely linked to the plasma pretransferrin 1 locus (PRT1). No recombinant was observed in 45 informative offspring studied. In nearly all breeds, the PA4 S allele was almost always in coupling phase with the PRT1 F allele.

Polymorphic plasma postalbumin (Po) of llamas and alpacas identified as Gc protein.

Immunoblotting with antiserum specific to human Gc protein was used to identify Gc protein as the previously reported polymorphic plasma postalbumin (Po) of llamas and alpacas. This is the first report of Gc polymorphism in camelid species. One Gc variant appeared to be identical in llamas, alpacas, dromedaries and bactrian camels.

Genetical control of phosphoglucose isomerase isozymes in the Japanese quail erythrocytes.

Three phenotypes of phosphoglucose isomerase (PGI) from the Japanese quail erythrocytes were observed by horizontal starch gel electrophoresis. Population and family data from one laboratory population of quail was consistent with the theory that PGI polymorphism was controlled by two codominant, autosomal alleles designated PGIF and PGIS with gene frequency values 0.25 and 0.75, respectively. The study supported the earlier view that the Japanese quail is highly polymorphic with regard to biochemical variation.

Prediction of the halothane (Hal) genotypes of pigs by deducing Hal, Phi, Po2, Pgd haplotypes of parents and offspring: results from a large-scale practice in Swedish breeds.

Results from a large-scale study, comprising 75 different breeding herds, are reported on predicting the halothane (Hal) genotypes of individual pigs by making use of the known close linkage between Hal and three electrophoretic blood marker loci (Phi, Po2, Pgd). The parents haplotypes (involving Hal and marker loci) were determined from the HAL phenotypes (halothane test results) and marker loci phenotypes of their offspring in the first one or two litters studied. In subsequent litters of the Hal-marker loci haplotyped parents, the offspring's expected Hal genotypes could be predicted on the basis of the marker loci haplotypes inherited by them. By comparing the expected and observed HAL phenotypes of offspring in subsequent litters, the predicted Hal genotype was found to be correct in 90-95% of the 4000 offspring (from Nn X Nn and Nn X nn matings) of Swedish Landrace and Yorkshire breeds studied. The order of the three marker loci was confirmed as Phi-Po2-Pgd but the position of Hal with regards to Phi could not be resolved. The recombination frequencies between the most distant loci in this region, viz. Hal-Pgd and Phi-Pgd, were estimated to be 3-4.5% and 4-6%, respectively. The easy and rapid electrophoretic techniques described in the study to phenotype PHI, PO2, PGD, also allowed phenotyping of six other polymorphic protein systems on the same gels. Thus Hal genotyping and effective parentage control can be conducted simultaneously.

Polymorphic serum prealbumin (Pa) of pig, identified as and alpha1-protease inhibitor.

Pig serum proteins were analysed by horizontal polyacrylamide gel electrophoresis, with a discontinuous buffer system (pH 9.0). A 12% acrylamide concentration in the separation gel was used. Each of the two paralbumin (Pa) alleles gave rise to two closely migrating fractions. The polymorphic Pa was identified as an alpha1-protease inhibitor as the Pa fractions inhibited the esterolytic activity of both bovine trypsin and chymotrypsin. Therefore, it has been proposed that the locus symbol for this prealbumin be changed to Pi-1. The protease inhibitory spectra and electrophoretic mobility of the Pa (Pi-1) fractions suggested that this protein was probably the same as the pig serum alpha1-protease inhibitor described in some earlier studies and that it corresponds to human serum alpha 1-protease inhibitor (Pi).

Close linkage between the albumin and Gc loci in the horse.

Evidence for close linkage between the structural loci for albumin and Gc protein in the horse was presented. A recombination frequency (c) of 0.009 +/- 0.006 (95% confidence limits: 0.001 less than c less than 0.032) was estimated. These results were based on a study of a large sire family comprising 223 offspring from informative matings. No evidence of linkage disequilibrium was observed in one horse population studied.

Polymorphic post-albumin of cattle and horse plasma identified as vitamin D binding protein (Gc protein).

Cattle and horse plasma samples of known post-albumin types were radiolabelled with 14C-vitamin D3. These samples were then analysed by polyacrylamide gel electrophoresis, followed by autoradiography. The patterns observed were identical to those of post-albumin variants. The polymorphic post-albumin protein of cattle and horse was thus identified as the vitamin D binding protein and homologous to the Gc protein of human plasma.

Linkage between the loci for transferrin and ceruloplasmin in pigs.

Evidence for genetic linkage between the loci for transferrin (Tf) and ceruloplasmin (Cp) in pigs was presented. The results were based on a study of a single sire family comprising 35 informative offspring. No recombinants were observed. The recombination frequency was estimated to be in the range of 0 to 8%. This indicated that the recombination frequency between Tf and Cp loci in pigs may be much lower than that reported previously between these two loci in cattle and in human.

Genetic polymorphism of human plasma alpha 1B-glycoprotein: phenotyping by immunoblotting or by a simple method of 2-D electrophoresis.

Genetic polymorphism of human plasma (serum) alpha 1B-glycoprotein (alpha 1B) was observed using one-dimensional horizontal polyacrylamide gel electrophoresis (PAGE) pH 9.0 of plasma samples followed by Western blotting with specific antiserum to alpha 1B. A simple method of two-dimensional agarose gel electrophoresis (pH 5.4)-horizontal PAGE (pH 9.0) of plasma samples, followed by general protein staining, was reported as an alternative method for alpha 1B typing. The three different phenotypes of alpha 1B observed (designated 1-1, 1-2, and 2-2) were apparently identical to those reported by Altland et al. (1983), who used double one-dimensional electrophoresis. Family data supported the hypothesis that the three alpha 1B phenotypes are determined by two codominant alleles at an autosomal locus, designated A1B. Allele frequencies in a Swedish population were: A1B1, 0.937; A1B2, 0.063; PIC, 0.111. For clues on linkage relationships of human A1B, the previously known linkages of A1B in pigs and horses, including the one between A1B and the gene that determines susceptibility to malignant hyperthermia in pigs were discussed.

A fourth allele in the plasma esterase-1 (Es-1) system of the domestic fowl.

Plasma samples of fowl were analysed by horizontal polyacrylamide gel electrophoresis (pH 9.0). Evidence was presented for the subdivision of an earlier reported esterase-1 allele (Es-1A) into two alleles designated Es-1A1 and Es-1A2. Family data were consistent with the hypothesis that the Es-1 phenotypes were controlled by four codominant, autosomal alleles Es-1C, Es-1A1, Es-1A2 and Es-1B). The White Leghorn samples showed high frequency of Es-1A1 (about 0.7) and also had considerable frequency of Es-1A2 (0.2) and of Es-1B (0.1). The three meat-type breeds studied (White Plymouth Rock, Rhode Island Red and New Hampshire) showed a very high frequency of Es-1B (0.8-1.0).

Polymorphic plasma postalbumins of some domestic animals (pig PO2, horse Xk and dog Pa proteins) identified as homologous to human plasma alpha 1B-glycoprotein.

Pig, horse and dog plasma proteins, separated by horizontal polyacrylamide gel electrophoresis (pH 9.0) and electrophoretically transferred to nitrocellulose membranes, were tested for cross-reaction with antiserum to human plasma alpha 1B-glycoprotein (alpha 1B). The results showed that one previously reported polymorphic plasma postalbumin in each of these species (pig PO2, horse Xk and dog Pa protein) was homologous to human plasma alpha 1B. In the light of the previously known genetic linkages in these species, this implied: (1) alpha 1B gene is close linked to Phi, Pgd and Hal (halothane sensitivity locus) loci in pigs; and (2) alpha 1B gene is linked to ME1 and Phi loci in horses. This suggested that the alpha 1B gene may also be found to be closely linked to gene(s) controlling susceptibility to malignant hyperthermia in humans and other mammals.

Genetic polymorphism of horse serum protein 3 (SP3).

Two-dimensional agarose gel (pH 8.6)-horizontal polyacrylamide gel (pH 9.0) electrophoresis of horse serum samples, followed by general protein staining, revealed genetic polymorphism of an unidentified protein tentatively designated serum protein 3 (SP3). The SP3 fractions appeared distinctly when a 14% concentration of acrylamide was used in the separation gels. The 2-D mobilities of SP3 fractions were quite similar to that of albumin. Family data were consistent with the hypothesis that the observed SP3 phenotypes were controlled by four co-dominant, autosomal alleles (D, F, I, S). Evidence was provided that the F allele can be further divided into two alleles (F1 and F2); the mobilities of F1 and F2 variants were very similar. Each of the SP3 alleles gave rise to one fraction and each of the heterozygous types showed two fractions. More than 600 horses representing five different breeds (Swedish Trotter, North-Swedish Trotter, Thoroughbred, Arab and Polish Tarpan) were typed for SP3, and allele frequency estimates were calculated. SP3 was highly polymorphic in all breeds studied.