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.

Role of proline and hydroxyproline in N-nitrosamine formation during heating in cured meat.

N-Nitrosamines are formed in a multi-step reaction of nitrite with free amino acids and amines in the meat products. The aim of this study was to determine the role of proline and hydroxyproline in N-nitrosamines formation during heating of cured meat. A lean meat model was used with different nitrite concentrations (0, 120, and 480mg/kg), and addition of proline and hydroxyproline (1000mg/kg), followed by heating at different temperatures. Volatile nitrosamines were analyzed with GC-TEA. The nitrosamine content never exceeded 10µg/kg and stayed 120°C.

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.