DOUBLE MUSCLING & MYOSTATIN:

Definition:

Double muscling or muscular hypertrophy is an inherited condition in cattle, characterised by hyperplasia (increase in number) and, to a lesser extent, hypertrophy (enlargement) of muscle fibres.

Characteristics of the Double Muscling Syndrome:

a....Muscle:

Double-muscled animals are characterized by an increase in muscle mass of about 20%, due to general skeletal muscle hyperplasia and, to a lesser extent, hypertrophy.
This relative increase in the number of muscle fibres (hyperplasia) occurs during intra-uterine development, such that double-muscled cattle possess nearly twice the number of muscle fibres at birth as do normal cattle.
The muscles of double-muscled cattle also have a significantly reduced amount of connective tissue (collagen).
Not only is collagen reduced in amount, but it is structurally different to normal collagen in that it has a lower proportion of stable, non-reducible, cross-links.
Muscular hypertrophy and hyperplasia is not uniform throughout the beast, being minimal around the neck and increasing as one moves to the hindquarters where it is maximal. This distribution results in the caecases of double-muscled animals having a higher proportion of "expensive" cuts of meat relative to carcases of normal cattle.

b....Bone:

The bone mass of double-muscled cattle tends to be around 10% less than that of normal cattle. This is primarily due to their long bones being shorter, more slender, and of lower density.
This reduced bone mass results in a significantly higher muscle : bone ratio in double-muscled cattle.

c....Fat:

Double-muscled cattle exhibit hypodevelopment of their fatty tissues. This is due to a reduction in the volume of fat cells rather than to a reduction in their numbers.
Not only is the total fat content reduced, but its composition is different, with double-muscled animals having a much higher percentage of polyunsaturated fats (11% compared with 5% in normal cattle).

d....Physiology:

During forced exercise, double-muscled cattle show signs of fatigue faster than normal cattle. This is thought to be due to a reduced capacity for aerobic metabolic activity by the exercising muscles.
Double-muscled cattle tend to have a reduced tolerance for heat stress. This is thought to be due to the increased heat production associated with their increased muscle mass.

e....Reproduction and Growth:

The syndrome of double muscling is associated with a number of reproductive problems. In the case of those animals where the syndrome is fully expressed there may be:
..........(i).....delays in puberty.
..........(ii)... reduced fertility due to an increased incidence of mortality in double-muscled embryos,
..........(iii).. increased incidence of dystocia,
..........(iv)...reduced milk production,
..........(v)....increased calf mortality.

Most double-muscled calves tend to have higher birth weights and higher pre-weaning growth rates than their normal contemporaries. Post-weaning, however, their growth rate tends to fall behind that of their normal contemporaries; this appears to be due to a lower feed intake.
If muscle weight gain per unit energy intake is taken into account, double-muscled cattle have better feed efficiency than normal cattle.

f....Carcase:

When compared with normals, the carcases of double-muscled cattle have many desirable characteristics:

Higher dressing percentage:
The carcases of double-muscled cattle dress out at between 65 and 70 percent. This is due to a combination of:
..........(i).....increased muscle mass,
..........(ii)....reduced body fat,
..........(iii)...reduced bone mass, and
..........(iv)...smaller internal organs.

Higher proportion of "expensive" cuts of meat:
This is due to the non-uniform distribution of the muscular hypertrophy and hyperplasia which is found in double-muscled cattle.

Reduced fat content with a higher proportion of polyunsaturated fats:
(See above)

Better meat quality:
Meat from double-muscled cattle is significantly more tender than that from normal cattle. Much of this is thought to be due to its lower collagen content and to the fact that what collagen is present is not as tough due to its lower proportion of stable, non-reducible, cross-links.

The significance for producers is that double-muscled animals produce a higher proportion of desirable cuts of lean meat with greater efficiency than do comparable, conventional cattle. For consumers, this meat is more tender and, being lean and having a higher polyunsaturated fat content, conforms more closely with current nutritional guidelines than meat from normal animals.

History:

The double muscling syndrome was first documented some 200 years ago in Durham cattle by the Englishman, George Culley (1804) (hence the synonym, culard), and was later described in detail by Kaiser in 1888. Right from these early days, it was considered that the condition was an inherited one and, in 1929, Wriedt proposed that the syndrome was due to a single gene defect. This proposal, however, did not receive universal support and little progress was made in the understanding of the genetics of double muscling until 1995 when researchers in the Faculty of Veterinary Medicine at the University of Liege, Belgium, mapped the genetic defect to the centromeric end of bovine Chromosome 2 (Charlier et al., 1995), and in so doing provided strong evidence that it was due to a single, autosomal, gene defect.

Meanwhile, a team of scientists in the Department of Molecular Biology and Genetics at the Johns Hopkins University School of Medicine in Baltimore was researching a group of proteins that belong to what is collectively known as the Transforming Growth Factor Beta (TGF-ß) superfamily of growth factors. This group encompasses a large number of growth and differentiation factors that play important roles in regulating embryonic development and in maintaining tissue homeostasis in adult vertebrates. Using molecular genetic techniques to search for the genes responsible for these growth factors, the scientists discovered a hitherto unidentified gene which coded for a novel protein of 376 amino acids which they initially called growth/differentiation factor-8 (GDF-8) and which appeared to be produced specifically in developing and adult skeletal muscle. To investigate the biological function of this GDF-8, they disrupted the GDF-8 gene in mice by gene targeting, and found that in those animals where the functioning gene had been knocked out, there was a dramatic increase in muscle mass with individual muscles weighing twice as much as those of normal mice. The scientists therefore concluded that GDF-8 functioned specifically as a negative regulator of skeletal muscle growth and, because of this, they renamed GDF-8, myostatin (McPherron et al., 1997 a).

Because of the phenotypic simularity between double-muscled cattle and those mice whose myostatin gene had been disrupted, the above research teams at the University of Liege (Grobet et al., 1997) and at the Johns Hopkins University (McPherron et al., 1997 b) simultaneously analysed the myostatin gene in Belgian Blue double-muscled cattle and found it to be defective with an eleven (11) base-pair deletion which results in non-functional myostatin protein being produced.
Meanwhile, researchers at the U.S. Department of Agriculture, Clay Centre, Nebraska, were able to show that the bovine myostatin gene mapped to the area on Chromosome 2 (Smith et al., 1997), thus complementing the earlier work of the group at the University of Liege.

Subsequently, a further five loss-of-function (disruptive) mutations in the myostatin gene were identified in phenotypically double-muscled cattle (Grobet et al., 1998; Karim et al., 2000). These are summarised in the following diagram and table:

Diagram 1: In the boxes, the top line (red) is the name of the mutation. The second line (black) is the normal DNA nucleotide sequence
at this position. The letters in the third line (black) indicate the amino acids in the resultant myostatin protein. The fourth line displays the
altered DNA nucleotide sequence in the mutated gene, while the fifth line shows the change in the now altered myostatin protein.

Type of gene mutation (nucleotide position after the initiation codon)	Change in myostatin protein	Cattle breeds
nt419(del7-ins10) Deletion of 7 base pairs and replacement with an unrelated stretch of 10 base pairs (419)	Truncation of the protein due to a premature STOP codon	Maine-Anjou
Q204X C --> T transition (610)	Truncation of the protein due to a premature STOP codon	Charolais Limousin
E226X G --> T transversion (676)	Truncation of the protein due to a premature STOP codon	Maine-Anjou
nt821(del11) Deletion of 11 base pairs (821)	Results in a frameshift and subsequent premature termination in the bioactive carboxyterminal domain of the protein	Belgian Blue Blonde d'Aquitaine Limousin South Devon
E291X G --> T transversion (874)	Truncation of the protein due to a premature STOP codon	Marchigiana
C313Y G --> A transition (938)	Substition of cysteine by a tyrosine, leading to an alteration in the three dimensional structure of the protein	Piedmontese

In the course of their research, Grobet et al. (1998) found a cytosine to adenine (C-->A) transversion at nucleotide position 282 in the myostatin gene, which results in the phenylalanine at amino acid position 94 in the myostatin protein being replaced by leucine. This missence variant, known as the F94L mutation, did not disrupt the myostatin protein as did the six loss-of-function mutations described above, but it did interfere with its function, rendering it less effective in controlling muscle growth; as a result, animals with this variant do not exhibit typical double muscling, but they do have an increased muscle mass due an increase in the size of the muscle fibres (but not in their number). This mutation is common in Limousin cattle, but also occurs in South Devon cattle.
In 2000, Grobet's team described two further missence mutations (Miranda et el. 2000), which give rise to an increase in muscle mass similar to the F94L mutation:
..........• S105C: ......cytosine to guanine (C-->G) transversion at nucleotide position 314 in the myostatin gene, which results in the serine at amino acid position 105 in the myostatin protein being replaced by cysteine.
..........• D182N: .... guanine to alanine (G-->A) transition at nucleotide position 544 in the myostatin gene, which results in the aspartic acid at amino acid position 182 in the myostatin protein being replaced by asparagine.
More recently, a fourth missence variant, known as the L64P mutation, has been described (Dierks et al. 2015). This mutation involves a thymine to cytosine (T-->C) transition at nucleotide position 191 in the myostatin gene, which results in the leucine at amino acid position 64 in the myostatin protein being replaced by proline.

As well as the six loss-of-function (disruptive) mutations and the four missense mutations just described, there are at least a dozen other mutations which are "silent", i.e. they do not result in any reduction in function of the myostatin protein (Dunner et al. 2003).

For recent reviews of the subject, the reader is referred to papers by Bellinge et al. (2005), and Rodgers & Garikipati (2008). Up to date information is to be found on the Online Mendelian Inheritance in Animals (OMIA) website.

In summary:
Tthe phenotype of double-muscled cattle is largely due to the loss of functional myostatin which, in turn, is due to a disruptive mutation in the myostatin gene which is located on Chromosome 2.

Implications of mutations in the Myostatin gene:

The bovine karyotype (i.e. the number and type of chromosomes in somatic cells) consists of 29 pairs of autosomes, together with one X and one Y chromosome. As Chromosome 2 is an autosome, each cell contains two copies of it, and thus two copies of the myostatin gene. For the full expression of double-muscling to occur, there has to be a complete absence of functional myostatin protein which requires that both copies of the myostatin gene be defective. What happens, however, when an animal is heterozygous for the myostatin gene, i.e. it has one normal and one abnormal copy?

Arthur (1995) has summarised a number of early (i.e. prior to 1997) studies which have examined the progeny of matings between double-muscled bulls (i.e. presumably homozygous for disruptive myostatin genes) and phenotypically normal cows (i.e. presumably homozygous for the normal myostatin gene). The progeny of these matings should be heterozygous for the myostatin gene, receiving a defective copy from their sire and a normal copy from their dam. Arthur's summary is as follows:
By using a double-muscled bull (DM) on normal females (N), reproductive problems associated with the double muscled syndrome are reduced to the degree that in some studies no significant differences were obtained in comparisons between DM x N and N x N matings... Growth rates and liveweights of DM x N animals are either similar to, or slightly lower than those of N x N animals. DM x N animals retain some of the superior carcase characteristics for which double-muscle animals are renowned. At the same age, DM x N cattle have 1-3% higher dressing percentage, about 3-10% more lean meat (%muscle or meat yield) and 13% less fat (%fat) relative to N x N cattle. The magnitude of these differences is maintained when assessed at the same carcase weight. Meat from DM x N carcasses is more tender than that from N x N carcases, in relation to both overall tenderness (shear force) and tenderness due to reduction in meat toughness from connective tissue (adhesion force).

Following the discovery of the myostatin gene, a series of studies on heterozygous animals were conducted by researchers at the U.S. Department of Agriculture, Clay Centre, Nebraska (Casas et al., 1998 & 1999; Wheeler et al., 2001). In these studies the animal's myostatin gene status was confirmed by genetic testing.

Trait	Homozygous vs Heterozygous (Kilograms or Residual Standard Deviations)
Birth Weight	+ 3.2 Kg
200 day Weight	+ 9.1 Kg
365 day Weight	+ 4.5 Kg
Eye Muscle Area	+ 1.35 sd
Retail Beef Yield	+ 1.60 sd
Subcutaneous Fat	- 0.84 sd
Marbling	- 1.01 sd

Meat quality, as assessed by tenderness, ease of fragmentation, and amount of connective tissue, was significantly higher in homozygote and heterozygote carcases as compared to those carcases with two copies of the normal myostatin gene.

A recent study from the Roslin Institute in Scotland (Gill et al, 2009) examined the carcase characteristics of Angus-sired cattle that were heterozygous for the eleven base pair deletion in the myostatin gene that is found in South Devon cattle, and compared these with animals with the normal myostatin allele. They found that one copy of the mutant myostatin gene significantly increased carcase weight, muscle conformation score and eye muscle area, but had no effect on the fat traits.

A similar study, again using Angus cattle, has been done by the Beef CRC in Australia (Cafe et al, 2006). This study found that, as a percentage of cold carcase weight, steers heterozygous for the myostatin mutation had a greater retail yield (67% v 63%) with less fat trim (15%v 18%) and bone (18% v 19%). Eye muscle area was significantly greater (85cm² v 73cm²), but there was no difference in P8 and rib fat depths, nor in marbling scores. The authors concluded that a breeding program that targets one non-functional myostatin mutant allele (heterozygotes) can increase retail beef yield by about 3.5%.

Opponents of breeding cattle heterozygous for a myostatin mutant allele argue, falsely it appears, that the improved carcase qualities come at the cost of poor reproductive performance. Recent research from the New South Wales Department of Primary Industries’ research station at Glen Innes ( McKiernan, 2009) has shown this assumption to be false. Cows heterozygous for a myostatin mutation had similar conception rates, calving and weaning percentages, and milk production, when compared with those known to have two copies of the normal (wild type) myostatin gene.

Thus, there is an increasing body of evidence to suggest that:
There are significant advantages to be gained from breeding heterozygote animals due to their the increased retail beef yield and meat quality.

Is the Myostatin gene the only gene involved in the Double Muscling Syndrome ?

Probably not.

Of all the cattle breeds, the expression of the double muscling syndrome is perhaps most pronounced in the Belgian Blue breed which exhibits an increase in muscle mass of 25-30%. In mice, on the other hand, the same genetic defect, namely the presence of two defective myostatin genes, leads to an increase in muscle mass of 200-300%. Even between breeds within the Bos taurus species, there is not always a clear relationship between the myostatin genotype and its resulting phenotype; for example, Grobet et al. (1998) found some Limousin and Blond d'Aquitaine animals which appeared to have the double muscling phenotype but which on testing had a normal myostatin genotype. Conversely, Smith et al. (2000) observed that not all South Devons with two defective myostatin genes were overtly double-muscled. This caused them to speculate that there must be other genetic factors influencing the expression of the double muscling phenotype. A similar conclusion was reached by Dunner et al (2003), and by researchers at the U.S. Department of Agriculture, Clay Centre, Nebraska (Casas et al., 2001) who, following their analysis of cross-bred progeny sired by double-muscled Belgian Blue and Piedmontese sires, concluded that although the myostatin gene has a considerable effect, other loci with more subtle effects are involved in the expression of the phenotype.

The status of the Myostatin gene in South Devon Cattle:

In 1962, Dr. J.C. McKellar, a Tavistock veterinary surgeon and a past president of the British Veterinary Association, wrote an article in the Veterinary Record describing several instances of the double muscling syndrome in South Devon cattle. He concluded that a degree of muscular hypertrophy was a highly desirable commercial factor giving an improved killing-out percentage of lean, tender meat. In response to this article, but being mindful of the problems associated with double muscling in its more extreme forms, the British South Devon Herd Book Society advised its members to breed from well muscled bulls, and to avoid undesirable extremes (Horsman, 1991).

Following the discovery of the myostatin gene, initial attention was directed towards the muscular European breeds in Europe and North America. In 2000, however, a team from the Roslin Institute near Edinburg, Scotland, (Smith et al., 2000) reported on their findings in ten beef breeds commonly used in the UK; as was to be expected, loss-of-function myostatin gene defects were found in some of the European breeds (Belgian Blue and Limousin) but, interestingly, not in any of the British breeds examined (Angus, Galloway, Hereford, and South Devon), with the sole exception of the South Devon. In this breed, the defect in the myostatin gene was the same one as had been described in Belgian Blue cattle, namely an 11 base pair deletion in the gene's DNA ( nt821(del11) ) which causes a non-functional myostatin protein to be produced. The authors were quick to point out that while these two breeds shared the same mutant gene, its effects appeared very different when judged by the resulting phenotype. They therefore concluded that:
Strong selection in breeds such as the Belgian Blue for the double-muscled phenotype may have resulted in extensive linkage disequilibrium for alleles in other genes controlling conformation and muscling traits together with the myostatin deletion. However, in the South Devon which carries the same myostatin mutation, selection has been for good confirmation rather than double-muscling. Thus the combination of alleles across all the loci involved is not found.

The finding that South Devon cattle had the same defective myostatin gene as did Belgian Blues, but had been bred for a different phenotype, prompted a closer look at the South Devon breed by the researchers at the Roslin Institute in Scotland (Wiener et al., 2002). Examining some 320 animals from 20 British studs they found that approximately 15% were homozygous for the defective myostatin gene (i.e. had two copies), approximately 50% were heterozygous (i.e. one normal copy and one defective copy), while the remainder (35%) were homozygous for the normal myostatin gene (i.e. two normal copies). Thus, nearly two-thirds of all pure-bred South Devons carry at least one copy of the defective myostatin gene, but relatively few are obviously double-muscled. Does this mean that the defective myostatin gene has no effect on the phenotype of South Devons? No. Wiener's group (2002) at the Roslin Institute, having determined the genotype of their South Devons, looked at the effect of having zero, one, or two copies of the defective myostatin gene on the following traits: 200-day weight, 400-day weight, muscle score, subcutaneous fat, eye muscle area, and calving difficulty. While the mutant gene had no effect on the weight at either 200 or 400 days, it had a significant additive effect on both muscle score and subcutaneous fat; in the former, visual muscling was increased while, in the latter, subcutaneous fat was decreased in proportion to the number defective genes (one or two) present. The mutant gene also had an effect on eye muscle area, leading to an increase in its size, but this effect did not reach statistical significance (Wiener, personal communication). When it came to calving difficulty, heterozygous calves did not experience any problems, but there was a significant increase in the incidence of dystocia with homozygous calves. Even when these potential calving problems were taken into account, Wiener et al. (2002) concluded that the presence of a mutant myostatin gene was of economic benefit in South Devon cattle. Similar conclusions were reached by Casas et al. (2004).

Conclusions:

Identification of the myostatin gene has been one of the most important findings in beef cattle genetics of the last two decades, since it represents the first gene to have been identified with a large effect on an economically important trait, namely meat yield and quality.

Meat Yield:
The carcases of double-muscled cattle dress out at between 65 and 70 percent.
This is due to a combination of:
..........(i).....increased muscle mass,
..........(ii)....reduced body fat,
..........(iii)...reduced bone mass, and
..........(iv)...smaller internal organs.
When muscle weight gain per unit energy intake is taken into account, double-muscled cattle have better feed efficiency than normal cattle.

Meat Quality:
Meat from double-muscled cattle tends to be of better quality.
This is due to a combination of:
..........(i).....a higher proportion of "expensive" cuts of meat,
..........(ii)....increased tenderness,
..........(iii)...reduced fat content with
..........(iv)...a higher proportion of polyunsaturated fats.

Significance for Producers:
Double-muscled animals produce a higher proportion of desirable cuts of lean meat with greater efficiency than do comparable, conventional cattle.

Significance for Consumers:
This meat is more tender and, being lean and having a higher polyunsaturated fat content, conforms more closely with current nutritional guidelines than meat from normal animals.

The physical consequences (phenotype) of the defective myostatin gene can be quite variable with some breeds (e.g. Belgian Blue and Piedmontese) exhibiting exaggerated muscling while others (e.g. South Devon) do not. Breeders need to be aware that there are significant problems associated with these phenotypic extremes, ranging from unacceptable levels of calving difficulty, through to inability to fatten, and reduced heat tolerance.

The reason for the variability in phenotypic expression lies in the fact that the effects of the myostatin gene appear to be influenced to a greater or lesser extent by a number of other, as yet unidentified, genes. Thus, in selecting animals for the presence (and benefits) of a defective myostatin gene, the breeder needs to select for those animals in whom these other genes are modifying the exaggerated phenotype and thus minimising the disadvantages inherent in double muscling.

While the defective myostatin gene has been found in a number of the European beef breeds, the only British beef breed in which it occurs with any frequency is the South Devon. In this breed, it appears that over the last century or so, breeders, while selecting for muscularity, have been careful to avoid the extreme double-muscled phenotypes. Thus, almost by accident, they have selected not only for the defective myostatin gene, but also for those other modifying genes, with the result that significant numbers of the breed (two-thirds) now carry a defective myostatin gene (with its associated advantages) but do not present the problems posed by those breeds with more extreme double muscling. Thus those breeders wanting the advantages of a defective myostatin gene without its disadvantages should consider using South Devon cattle.

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