Half of 23 Belgian dog breeds has a compromised genetic diversity, as revealed by genealogical and molecular data analysis.

The genetic diversity in 23 dog breeds raised in Belgium was investigated using both genealogical analysis and microsatellite markers. Some of these breeds are native breeds, with only small populations maintained. Pedigree and molecular data, obtained from the Belgian kennel club, were used to calculate the inbreeding coefficients, realised effective population size as well as probabilities of gene origin and average observed heterozygosity. Inbreeding coefficients ranged from 0.8 to 44.7% and realised effective population size varied between 3.2 and 829.1, according to the used method and breed. Mean observed heterozygosity ranged from 0.47 to 0.73. Both pedigree and molecular methods reveal low genetic diversity and presence of bottlenecks, especially in native Belgian breeds with small population sizes. Furthermore, principal component analysis on the set of investigated diversity parameters revealed no groups of breeds that could be identified in which similar breeding strategies could be applied to maintain genetic diversity.

Art meets science: The Cosmopolitan Chicken Research Project.

The Cosmopolitan Chicken Project is an artistic undertaking of renowned artist Koen Vanmechelen. In this project, the artist interbreeds domestic chickens from different countries aiming at the creation of a true Cosmopolitan Chicken as a symbol for global diversity. The unifying theme is the chicken and the egg, symbols that link scientific, political, philosophical and ethical issues. The Cosmopolitan Chicken Research Project is the scientific component of this artwork. Based on state of the art genomic techniques, the project studies the effect of the crossing of chickens on the genetic diversity. Also, this research is potentially applicable to the human population. The setup of the CC®P is quite different from traditional breeding experiments: starting from the crossbreed of two purebred chickens (Mechelse Koekoek x Poule de Bresse), every generation is crossed with a few animals from another breed. For 26 of these purebred and crossbred populations, genetic diversity was measured (1) under the assumption that populations were sufficiently large to maintain all informative SNP within a generation and (2) under the circumstances of the CCP breeding experiment. Under the first assumption, a steady increase in genetic diversity was witnessed over the consecutive generations, thus indeed indicating the creation of a "Cosmopolitan Chicken Genome". However, under the conditions of the CCP, which reflects the reality within the human population, diversity is seen to fluctuate within given boundaries instead of steadily increasing. A reflection on this might be that this is because, in humans, an evolutionary optimum in genetic diversity is reached. Key words.

The MC4R c.893G>A mutation: a marker for growth and leanness associated with boar taint odour in Belgian pig breeds.

Since surgical castration of male piglets without anaesthesia is under heavy societal pressure, finding a sustainable solution to reduce boar taint has become urgent. One way to circumvent this animal welfare violation is raising entire male pigs whilst selecting against the tainted phenotype through marker-assisted selection. Since slaughtering at a lower weight is often suggested to reduce boar taint, selection using a marker for that trait could be a promising strategy. Therefore, in this study a melanocortin 4 receptor (MC4R) mutation, frequently described in different pig breeds as marker for fat content, weight gain and feed intake, was examined in relation to boar taint in pig breeds used in Belgian pig farms. Although results suggest an association between this mutation and a boar taint odour score assigned by experts, no association was found between the mutation and the concentration of the individual chemical boar taint components androstenone, skatole and indole.

The effect of enterotoxigenic Escherichia coli F4ab,ac on early-weaned piglets: a gene expression study.

Diarrhoea in neonatal and early-weaned piglets due to enterotoxigenic Escherichia coli-F4 (ETEC-F4) is an important problem in the pig farming industry. There is substantial evidence for a genetic basis for susceptibility to ETEC-F4 since not all pigs suffer from diarrhoea after an ETEC-F4 infection. A region on SSC13 has been found to be in close linkage to the susceptibility of piglets for ETEC-F4ab,ac. Potential candidate genes on SSC13 have been examined and although some polymorphisms were found to be in linkage disequilibrium with the phenotype, the causative mutation has not yet been found. In this study we are looking at the expression of porcine genes in relation to ETEC-F4ab,ac. With the aid of the Affymetrix GeneChip Porcine Genome Array we were able to find differentially expressed genes between ETEC-F4ab,ac receptor positive (Fab,acR(+)) piglets without diarrhoea and F4ab,acR(+) piglets with diarrhoea or F4ab,acR(-) animals. Since the susceptibility to ETEC-F4ab,ac was described as a Mendelian trait, it is not so surprisingly that only two differentially expressed genes, transferrin receptor (TFRC) and trefoil factor 1 (TFF1), came out of the analysis. Although both genes could pass for functional candidate genes only TFRC also mapped to the region on SSC13 associated with susceptibility for ETEC-F4, which makes TFRC a positional functional candidate gene. Validation by qRT-PCR confirmed the differential expression of TFRC and TFF1. In piglets without diarrhoea, the expression of both genes was higher in F4ab,acR(+) than in F4ab,acR(-) piglets. Similarly, TFRC and TFF1 expression in F4ab,acR(+) piglets without diarrhoea was also higher than in F4ab,acR(+) piglets with diarrhoea. Consequently, although both genes might not play a role as receptor for F4 fimbriae, they could be of great importance during an ETEC-F4 outbreak. An upregulation of TFRC can be a consequence of the piglets ability to raise an effective immune response. An elevation of TFF1, a protein involved in mucin formation, may also affect the piglet's capability to cope with ETEC bacteria, rather than being a receptor for its fimbriae.

Susceptibility of piglets to enterotoxigenic Escherichia coli is not related to the expression of MUC13 and MUC20.

Enterotoxigenic Escherichia coli (ETEC) is one of the most frequently isolated enteropathogens in production animals, especially pigs and calves. Economically, the swine industry is by far the most affected by infections with ETEC because of mortality, morbidity and decreased growth rate of newborn and early-weaned piglets. After ingestion by the animal, these bacteria attach themselves to specific receptors on the small intestinal epithelium by means of proteinaceous surface appendages, the fimbriae. The F4 fimbriae, which attach to the F4 receptor, are the most studied. The aim of our study was to investigate gene expression in the small intestine of piglets of MUC13 and MUC20 in relation to animals with a different treatment towards or a different reaction on ETEC-F4ac by means of quantitative reverse transcription chain reaction (qRT/PCR). MUC13 and MUC20 are positional candidate genes for this F4ac receptor and are located in the region on SSC13q41 that segregates with the susceptibility to ETEC-F4ac. The condition of the small intestine is crucial when examining expression differences between different samples. Therefore, the expression of two genes, fatty-acid binding protein 2, intestinal (FABP2) and pancreatitis-associated protein (PAP), now known as regenerating islet-derived 3 alpha (REG3A) in the small intestine was simultaneously checked. FABP2, a standard for epithelial content, reflects the state of damage, whereas REG3A is a measure for inflammation in the small intestine. The four different substudies presented here suggest that expression of MUC13 and MUC20 is not related to the susceptibility of piglets to ETEC-F4ac.

Effects of dietary supplementation of methionine and its hydroxy analog DL-2-hydroxy-4-methylthiobutanoic acid on growth performance, plasma hormone levels, and the redox status of broiler chickens exposed to high temperatures.

Heat stress is known to impair performance and to induce oxidative stress in poultry. The aim of the present study was to compare the effects of dietary supplementation of dl-methionine (dl-M) or the synthetic analog 2-hydroxy-4-methylthiobutanoic acid (dl-HMTBA) on broiler growth performance, plasma hormone levels, and some oxidative stress-related parameters under conditions of chronic exposure to high temperatures (HT). From 2 to 6 wk of age, male broiler chickens were reared under either a constant temperature of 32°C until 6 wk of age or a normal temperature scheme (gradual decrease to 18°C at 5 wk of age). Chicks in both the normal and HT treatments were provided with a commercial grower diet supplemented with either 1.0 or 1.2 g/kg of dl-M or 1.0 or 1.2 g/kg of dl-HMTBA. Because there were no effects of supplement dose, data were pooled over both doses within each temperature treatment. The chronic HT treatment impaired feed intake and BW gain, but these negative effects were less pronounced when the chickens received dl-HMTBA. Exposure to HT was also associated with decreased (P < 0.001) plasma thyroid hormones and increased (P < 0.0001) plasma corticosterone levels. At 4 wk of age, and irrespective of the supplemental source, chickens subjected to HT were characterized by significantly lower plasma TBA-reactive substance levels. In contrast, at 6 wk of age, plasma TBA-reactive substance levels were significantly increased by HT, but this effect was observed only for the chickens receiving dl-M and not for those receiving dl-HMTBA. High temperatures induced a significant increase in hepatic total glutathione (GSH) and oxidized GSH levels, regardless of the supplemental source. However, the hepatic ratios of reduced GSH to total GSH and reduced GSH to oxidized GSH were highest in chickens supplemented with dl-HMTBA. In conclusion, dl-HMTBA supplementation partially prevented the growth-depressing effects of chronic heat exposure compared with dl-M supplementation. It can be inferred that dl-HMTBA is more efficient in alleviating HT-induced oxidative damage because of a more favorable reduced GSH-to-total GSH ratio.

Serum amyloid A3 (SAA3), not SAA1 appears to be the major acute phase SAA isoform in the pig.

The acute-phase serum amyloid A (SAA) protein family comprises two main circulating (systemic) isoforms, SAA1 and SAA2, synthesised in liver and one local isoform, SAA3, produced in extrahepatic tissues. Systemic and local SAA show structural differences, which suggests different functions. In the pig, AA-amyloidosis is extremely uncommon, and the structural protein in swine has characteristics of systemic SAA. The only pig SAA sequences published so far, either derived form hepatic or extrahepatic sites have been designated SAA2, but the translated protein shows the properties of SAA3 proteins. The aim of this study was to characterise all the porcine SAA isoforms by sequencing from cDNA and genomic DNA obtained form multiple porcine tissues. Primer pairs were designed to amplify presumably all isoforms of SAA firstly and then specifically for each isotype. Results show that the only isotype isolated and sequenced both from hepatic and extrahepatic tissues correspond to a SAA3-like amino acid sequence. No SAA1-like sequences were identified, which could be indicative of the gene being very rare and consistent with the observed resistance to AA-amyloidosis. Finally, it is concluded that the pig is unique among other species in that the main circulating hepatic SAA isotype shows the characteristics of local highly alkaline SAA. This likely precludes a function as apolipoprotein.

Mutations in the myostatin gene leading to hypermuscularity in mammals: indications for a similar mechanism in fish?

The transforming growth factor ß (TGF-ß) superfamily encodes secreted factors that are important in regulating embryonic development and tissue homeostatis in adults. Myostatin (MSTN, encoded by MSTN) or 'growth and differentiation factor 8', a member of this superfamily, is a negative regulator of skeletal muscle growth and is highly conserved among animal species. In 1997, a mutation associated with the so-called double-muscling phenotype in cattle was found in the MSTN gene. During the years following the discovery of the first MSTN mutation, other mutations were found in cattle and other mammalian species, and MSTN became one of the most thoroughly studied genes in animals. The aim of this review is mainly to describe the functional mutations located in the MSTN genes of several mammalian species, leading to double muscling in these animals. Furthermore, in light of the increasing importance of fish genetics, the possibility of functional mutations in piscine MSTN with a similar effect as in mammals, and a genetic model for MSTN research in fish, will also be discussed.

Indirect effect of IGF2 intron3 g.3072G>A mutation on prolificacy in sows.

A QTL located in the paternally expressed insulin-like growth factor 2 (IGF2) gene is known to increase muscle growth and reduce fat deposition in pigs. This makes the QTL in IGF2 a good marker for use in pig breeding programmes. However, care has to be taken as it is postulated that increased leanness and lowered fat deposition may have a negative effect on the prolificacy and longevity of sows. Selection of sire and dam lines for different alleles of the mutation in the paternally imprinted IGF2 gene could actually provide a solution to this problem. Therefore, in this study, the effect of the IGF2 QTL on prolificacy-related traits in sows was investigated. It was found that the paternal IGF2 wild-type allele was associated with higher reproduction performance in the sow. Moreover, it was also examined whether the difference in prolificacy in sows could be a consequence of differential IGF2 expression in the ovarian follicles of the sow or whether it is mainly a secondary effect caused by differences in fatness traits. Therefore, IGF2 expression was measured in follicles of different sizes from sows with different genotypes for the paternal IGF2 allele. It was observed that, however, while the size of the follicles was associated with follicular IGF2 expression level, the IGF2 genotype was not. It could be concluded that the difference in prolificacy of sows with a different paternal IGF2 genotype could be a secondary effect, resulting from differences in fat deposition.

Interactions between genes involved in growth and muscularity in pigs: IGF-2, myostatin, ryanodine receptor 1, and melanocortin-4 receptor.

In the swine breeding industry, two economical traits are of particular importance in sires, namely, muscle growth and average daily gain (ADG). These traits are quantitative, which implies that they are under the control of multiple genes. Mutations in these genes, associated with either muscularity or growth, are useful quantitative trait nucleotides (QTN) for unraveling genetic variation of these traits and can be used in marker-assisted selection. Until now, QTN involved in muscle growth and/or ADG in pigs were identified in porcine ryanodine receptor 1 (RYR1), insulin-like growth factor-2 (IGF-2), and melanocortin-4 receptor (MC4R). Recently, a fourth possible QTN was found in porcine myostatin (MSTN). All four QTN have an influence on muscle growth and/or somatic growth, so an influence of one mutation on one or more of the other mutations should not be excluded. However, although the polymorphisms in the RYR1 and the MC4R gene affect the function of the respective protein, the polymorphisms of the IGF-2 and MSTN gene influence the mRNA expression of the respective gene. Therefore, this study investigated possible interactions between the genotypes of MSTN, IGF-2, and MC4R (population 1) or the RYR1, IGF-2, and MSTN QTN (population 2) on IGF-2 and MSTN expression in different muscle types in pigs. In both skeletal muscle and heart muscle growth, the IGF-2:MSTN ratio seems to play an important role. Also, the RYR1 genotype had a significant effect on IGF-2 expression in m. longissimus dorsi. No effect of the MC4R QTN could be seen.

Characterization of the complete porcine MSTN gene and expression levels in pig breeds differing in muscularity.

Myostatin (MSTN), a transforming growth factor beta superfamily member, is an essential factor for the growth and development of muscle mass. The protein functions as a negative regulator of muscle growth and is related to the so-called double-muscling phenotype in cattle, where a series of mutations renders the gene inactive. One particular breed of pigs, the Belgian Piétrain, also shows a heavily muscled phenotype. The similarity of muscular phenotypes between the double-muscled cattle and Piétrain pigs indicated that MSTN may be a candidate gene for muscular hypertrophy in pigs. In this study, we sequenced and analysed the complete MSTN gene from 45 pigs of five different breeds, including the heavily muscled Piétrain breed at one extreme and the Meishan and Wild boar breeds at the other extreme. In total, 7626 bp of the porcine MSTN gene were sequenced, including the 5' and 3' UTR. Fifteen polymorphic loci were found, three of which were located in the promoter region, five in intron 1 and seven in intron 2. Most mutations were found when comparing the obtained MSTN sequence with porcine MSTN sequences already published. However, one polymorphism located at position 447 of the porcine MSTN promoter had a very high allele frequency in the Piétrain pig breed and disrupted a putative myocyte enhancer factor 3 binding site. Real-time PCR using Sybr Green showed that this mutation was associated with expression levels of the MSTN gene in m. longissimus dorsi at an age of 4 weeks.

The effect of mutations in the insulin-like growth factor-II and ryanodine receptor-1 genes on biochemical and histochemical muscle fibre characteristics in pigs.

Mutations in IGF-II (insulin-like growth factor-II) and RYR1 (ryanodine receptor-1) increase lean meat content in pigs but with different effects on meat quality. Differences in biochemical and histochemical muscle fibre characteristics between the two mutations can thus be suspected. Therefore, the aim of this study was to investigate the effect of the IGF-II mutation (Apat vs. Gpat) on biochemical and histochemical muscle fibre characteristics in relation to the RYR1 genotype (Nn vs. NN). A prenatal effect of the IGF-II mutation could not be excluded but the increased leanness in both mutations was clearly related to postnatal muscle hypertrophy due to an increase in muscle fibre diameter and a higher proliferative capacity in animals carrying the IGF-II mutation. No effect of the IGF-II or RYR1 mutation was found on fibre type composition and metabolic enzyme activities. Interactions between IGF-II and RYR1 genotypes suggest that the mechanism involved in increased leanness due to the impaired RYR1 receptor might influence the underlying mechanisms of the IGF-II mutation.

Effect of the insulin-like growth factor-II and RYR1 genotype in pigs on carcass and meat quality traits.

Recently, a new QTN (quantitative trait nucleotide), which is located in the regulatory sequence of the imprinted IGF-II gene was discovered in the pig and is associated with a significant increase in IGF-II mRNA expression in skeletal muscle during postnatal growth. The aim of the current study was to investigate the effect of the IGF-II paternal allele (Apat and Gpat animals that inherited, respectively, the mutant and wild type paternal allele of interest) on carcass and meat quality traits in Nn and NN RYR1 genotypes. A total of 141 animals were measured, almost equally distributed over the IGF-II and RYR1 genotypes and gender. The Apat allele increased carcass lean meat percentage with approximately 4.5% (P<0.001) as a result of decreased backfat thickness. Average live weight daily gain was not affected, hence average daily lean meat gain was significantly higher for Apat compared to Gpat animals. The IGF-II mutation had no noticeable effect on meat quality in contrast with the RYR1 mutation. No interaction effects of both mutations on meat quality were noticed.

Effect of age, muscle type, and insulin-like growth factor-II genotype on muscle proteolytic and lipolytic enzyme activities in boars.

Recently, a paternally expressed quantitative trait nucleotide (QTN) in the regulatory sequence of the IGF-II gene with effects on muscle growth and fat deposition was discovered in the pig. This QTN is also known as the IGF-II intron3 G3072A mutation. The aim of the current study was to determine the effects of age, muscle type, and IGF-II genotype (Apat, mutant allele vs. Gpat, wild-type allele) on muscle proteolytic and lipolytic enzyme activities. At approximately 4, 8, 16, and 26 wk of age, boars (n = 6 to 15 per genotype x age group) were slaughtered and mu- and m-calpain (CALP), calpastatin (CAST), cathepsins (CATH) B+L and H, acid lipase, and phospholipase activities were measured in Longissimus thoracis et lumborum, Semimembranosus, and Triceps brachii muscle samples taken soon after slaughter. Activities of CATH B+L and H, mu- and m-CALP, and acid lipase were not affected by the IGF-II genotype. Activity of CAST was greater (P < 0.005) and m-CALP:CAST was less (P < 0.05) in Apat animals. Because CAST activity and m-CALP:CAST are known to be related to protein degradation, satellite cell fusion, or both, it is likely that differences in proteolytic enzyme activities are involved in the greater percentage of muscle mass in Apat animals. Age and muscle type influenced proteolytic and lipolytic enzyme activities (P < 0.05), except for mu- and m-CALP (no effect of muscle) and acid lipase (no effect of age). The same pattern in mu-CALP, CAST, and m-CALP:CAST with age was found during growth for the 3 muscles, although clear differences (P < 0.05) between muscles existed. In general, and in agreement with previous reports, greater enzyme activities were found in the more oxidative Triceps brachii muscle compared with the other 2 muscles. A remarkable increase (P < 0.05) from 16 to 26 wk of age in mu-CALP, CAST, mu-CALP:CAST, and CATH H and a large decrease (P < 0.05) in acid phospholipase and m-CALP:CAST was found. For m-CALP and CATH B+L, a gradual decrease (P < 0.05) was found with age. Although age effects on enzyme activities could only partly be interpreted biologically in relation to the muscle growth rate, this study showed that proteolytic and lipolytic enzyme activities change during growth.

The RYR1 g.1843C>T mutation is associated with the effect of the IGF2 intron3-g.3072G>A mutation on muscle hypertrophy.

Muscle growth is a complex phenomenon regulated by many factors, whereby net growth results from the combined action of synthesis and turnover. In pigs, two quantitative trait nucleotides (QTN) are known to have an important influence on muscle growth and fat deposition: one QTN is located in the ryanodine receptor 1 (RYR1) gene (RYR1 g.1843C>T) and the other, a paternally expressed QTN, is in the insulin-like growth factor 2 (IGF2) gene (IGF2 intron3-g.3072G>A). The mutation in IGF2 abrogates in vitro interaction with a repressor, which leads to a threefold increase of IGF2 expression in post-natal muscle. The family of the calpains, a family of Ca(2+)-sensitive muscle endopeptidases, and their specific inhibitor calpastatin play an important role in post-natal protein degradation, also influencing muscle and carcass traits. This study investigated the possible interactions between the genotypes of the RYR1 and IGF2 QTN on IGF2 expression. Samples were taken from several muscles and from pigs at several ages, and messenger RNA expression levels were measured using a real-time quantification assay. IGF2 expression in m. longissimus dorsi of animals with mutations in both IGF2 and RYR1 was significantly lower than in animals that inherited the IGF2 mutation but were homozygous wildtype for RYR1.

The Asp298Asn missense mutation in the porcine melanocortin-4 receptor ( MC4R) gene can be used to affect growth and carcass traits without an effect on meat quality.

A promising tool to improve daily gain in pigs is the missense mutation (Asp298Asn) in the melanocortin-4 receptor (MC4R) gene, especially in the Belgian pig industry where the slow-growing Piétrain breed is very frequently used as the sire breed. The MC4R is expressed in the appetite-regulating region of the brain where it regulates feed intake and energy balance. The mutation has been associated with differences in fatness, daily gain and feed intake. However, less information on the correlated effects on meat quality is available. In order to evaluate the influence of the MC4R mutation on carcass and meat quality parameters, a total of 1155 pigs of a four-way cross were slaughtered at an average live weight of 109 kg, and data about daily live-weight gain, carcass and meat quality were collected. Allelic frequencies were 0.69 for the G-allele (298Asp variant or well-conserved variant) and 0.31 for the A-allele (298Asn variant or the mutated variant). Barrows and gilts were almost equally distributed in this population with, respectively, 49.9% and 50.1%. Moreover, independent of this mutation, the relationship between average daily gain (ADG) and carcass on the one hand and meat quality traits on the other hand was evaluated in this population. A significant positive influence of the MC4R mutation on ADG (P < 0.001) was found, accompanied by a higher fat thickness (P < 0.05) and a lower carcass lean meat content (P < 0.01), whereas muscle thickness and carcass conformation traits were not affected. The effects on meat quality traits were not significant, except for a lower shear force (P = 0.054) and a higher intramuscular fat content (P = 0.052) in AA animals. In the longissimus, pH and pork quality meter (PQM) values were not influenced, and effects on drip loss and colour were not apparent. Residual correlation coefficients between ADG and carcass lean meat content on the one hand and meat quality traits on the other hand were generally very low (|r|>0.1). Higher ADG, higher carcass fat thickness and lower carcass lean meat content were correlated with slightly lower shear force values (|r|∼0.1, P < 0.05). In conclusion, in the studied population, the Asp298Asn mutation in the MC4R gene was associated with improved daily gain, higher carcass fatness and almost no effect on meat quality traits.