CA2267347A1 - Assay for duroc muscle fibre type - Google Patents

Abstract

There is provided an assay to determine whether an animal has an allele for or exhibits muscle fibre composition characteristic of a Duroc pig. The assay comprises obtaining a tissue sample from said animal and subjecting said sample to genetic analysis and/or obtaining a muscle sample from said animal and determining by histochemical or immunochemical analysis whether said sample exhibits phenotypic traits indicative of said allele(s) and/or shows a muscle composition characterisitc to the Duroc pig. Suitable physical traits include the following: i. the percentage frequency of SO fibres present in said muscle; and/or ii. the number of SO fibres present per cluster; and/or iii. the number of muscle fibres present per cluster; and/or iv. the level of m calpain present per unit muscle; and/or v. the level of .mu. calpain present per unit muscle. The assay can be used to select animals having Duroc muscle type for breeding or for meat production. Anti-calpain antibodies are especially useful and may be employed as part of an ELISA style assay.

Description

3 The present invention relates to animals, especially 4 pigs, having improved meat quality.
6 In the United Kingdom, elsewhere in Europe and 7 increasingly throughout the world, pig producers are 8 selecting breeds to use on their farms which are 9 efficient producers of Iean meat and thus provide the farmer with the maximum possible economic return.

12 In the main, these pigs are from highly selected breeds 13 of pigs such as "Large White" and "Landrace". A breed 14 is defined as a group of animals that has been selected by man to possess a uniform appearance that is 16 inheritable and distinguishes it from other groups of 17 animals within the same species (Glutton-Brock 1981).
18 The most commonly used breed in the United Kingdom is 19 the Large White. This is defined in the World Dictionary of Livestock Breeds by Mason (Mason 1988) as 21 an English meat pig, white in colour and with prick 22 ears, originating from local Yorkshire with Chinese 23 {Cantonese) crosses in the late 18th century. A
24 Herdbook was formed for the breed in 1884: The breed has many synonyms in many countries, but the most 1 common synonym is 'Yorkshire'.

3 This breed is commonly used as a component of a hybrid 4 cross female also containing genes of a 'Landrace' breed, but the 'Large White' is also commonly used as a 6 terminal sire, i.e. the father of the generation of 7 pigs destined for slaughter.

9 As indicated, it is common for a 'Landrace' breed to be used as a component of a hybrid breeding female, but 11 such breeds can also be used as terminal sires.
12 'Landrace' includes improved native white lop-eared 13 (Celtic) breeds of North West Europe, for example 14 Danish Landrace and derivatives such as British Landrace (Mason 1988).

17 In recent years a high proportion of commercial pig 18 breeders have purchased their breeding animals from pig 19 breeding or pig genetics companies. These companies have applied intensive selective breeding to improve 21 the commercial value of the breeding pigs they sell to 22 their pig producer customers. Conventionally these 23 breeding companies maintain lines of 'Large White' and 24 'Landrace' pigs with other breeds which they cross to sell to commercial producers or sell as pure lines. In 26 some cases, these lines of "White" pigs are no longer 27 maintained as pedigree registered pigs and they may 28 even have trade names, but an averagely skilled 29 observer would recognise from their appearance lines of 'Large White' and 'Landrace' pigs. The term "White"
31 pigs is used herein to refer to Large White, Landrace 32 and similar breeds thereto and crosses of such breeds.

34 The 'Large White' and 'Landrace' breeds of pig especially those produced by pig breeding companies in 36 the United Kingdom are characterised by having a good 1 growth rate and producing carcases with a low 2 subcutaneous and i-ntermuscular fat level and thus a 3 high lean content. These characteristics also lead to 4 animals with a high feed conversion efficiency.
Considerable progress in improving the lean meat 6 content of these breeds of pig has been made in recent 7 years in the United Kingdom.

9 In 1971 when the Meat and Livestock Commission began classification of pig carcases in Great Britain, the 11 average back fat depth (PZ) was close to 20 mm, by I995 12 this had reduced to 11 mm (MLC 1996). This reduction is 13 in the context of increasing carcase weights, which 14 would normally increase fatness and therefore the reduction in the genetic potential of the pigs to 16 produce fat is even greater than it appears.

18 In recent years, pig industries, especially those in 19 the United Kingdom and Denmark have become increasingly concerned that the quality of the pig meat produced in 21 these modern versions of the 'Large White' and 22 'Landrace' is not as good as the quality desired by 23 consumers.

There are reasons to believe that this long-term 26 selection for lean content may have had the consequence 27 of coincidentally selecting for pigs with a biological 28 predisposition to poor meat quality. In particular, the 29 lean meat may be increasingly predisposed to a problem known as Pale Soft Exudative meat (PSE), and may have 31 eating quality problems such as toughness and dryness.

33 The meat defect PSE is known to have a strong genetic 34 component due to alteration in a single gene, the halothane gene. The halothane gene codes for a protein 36 in the calcium channels of the pigs muscle. The mutant 1 allele or alleles of the gene leads to leaky calcium 2 channels, pigs with an increased lean content, but also 3 with an increased predisposition to death from Porcine 4 Stress Syndrome and PSE in the muscle after slaughter.
6 Recently the precise DNA mutation of the halothane gene 7 which leads to PSE was discovered and is described in 8 WO-A-92/11387. This allows pig breeders to control the 9 incidence of the mutated version of the gene in their populations and control the incidence of PSE. However, 11 PSE can also be caused by the pig's response to its 12 pre-slaughter handling and thus PSE could remain a 13 problem where pre-slaughter handling in the abattoir is 14 not of the highest standard.
16 Although the use of 'Large White' and 'Landrace' breeds 17 of pig is increasing throughout the world, there are 18 still very many cases of other breeds being used for 19 meat production according to local tastes and the suitability of the pigs for local conditions. Important 21 characteristics may be tolerance of local climatic or 22 other conditions, or resistance to disease. In some 23 cases, meat quality may also be a characteristic 24 considered in the choice of breed.
26 Another important world breed of pig is the 'Duroc'.
27 This is a North American breed of meat pig, red in 28 colour and originating between 1822 and 1877 from 'Old 29 Duroc' of New York and 'Jersey Red' of New Jersey. A
breed society was formed in 1833 (Mason 1988). The 31 'Duroc' remains very popular in the United States and 32 has been imported into Europe a number of times this 33 century.

Within Europe, especially the United Kingdom, the 36 'Duroc' is characterised as being of reasonable growth WO 98I15837 PCTlGB97/02741 S
1 rate, but fatter and less efficient with regard to meat 2 production than 'Large White' and 'Landrace'. However, 3 it has been shown a number of times to have meat of 4 superior quality, especially colour and tenderness, than the "White" breeds (as defined above).

7 In Canada, Denmark, France and New Zealand, pigs 8 produced from "White" hybrid mothers and 'Duroc' sires 9 have produced pigs with a tenderness advantage ranging from 10 to 170 over similar but 'white' sired pigs 11 ((Martel, Minveille et al. 1988);(Barton-Gade 12 1989);(Gandemer and Legault 1990) and (Purchas, Smith 13 et al. 1990)).

In the United Kingdom, the 'Duroc' is used to some 16 extent in two situations. It has gained popularity as a 17 component of breeding females, typically at 50 or 250 18 'Duroc' genes content, for use in outdoor units or in 19 units where hardiness is an important characteristic.
Secondly, it is used, in purebreed form, as a sire or 2i as a component of a crossbred sire or dam of superior 22 meat quality, especially eating quality characteristics 23 such as tenderness. However, the widespread use of the 24 'Duroc' is hindered because of the higher cost of producing pig meat from the 'Duroc' and because of its 26 lower carcase value. Carcase value is diminished both 27 because of the increase in fatness and because the 28 'Duroc' crosses tend to have more coloured skin on the 29 carcase and more deep-seated dark hairs which are not easily removed in the abattoir.

' 32 The interest in the 'Duroc' breed in the United Kingdom 33 prompted the Meat and Livestock Commission to undertake 34 what is probably the most comprehensive evaluation of the breed ever done. Conventional 'White' British 36 commercial pigs ('Large White' sires crossed to 'Large 1 White' cross, 'Landrace' dams) containing zero percent 2 'Duroc' genes were compared with pigs containing 25, 50 3 or 75% 'Duroc' genes produced by various crosses (MLC
4 1992). Some results for 0% and 50% 'Duroc' pigs are presented in Table 1 and illustrate the relative merits 6 of the two pig types.

8Table 1 0% 50%

1 Daily live weight gain (g) 806 803 12Feed conversion ratio 2.70 2.83 13Lean tissue feed conversion 6.19 6.81 ratio 14PZ fat depth (mm) 9.3 10.9 15Lean % 58.8 56.6 16PSE carcases (%) 8.3 1.6 17Deep seated hair (% carcases) 1.1 17.6 1 Tenderness score* 4.96 S.32 19Pork flavour*

2 In lean 3.88 3.96 2 In fat 3.87 4.06 2 Pork odour in fat* 3.58 3.73 25* sensory scores are on a 1-8 here scale higher w scores 26indicate more tender, juic y results etc. are A11 for 27pigs fed ad-libitum but re strictedly feed pigs show 28similar results. MLC 1992.

5 Thus it can be seen that ' Duroc' s cros pigs have good 6 quality meat in comparison to pigs 'White' but this is 7 obtained at the expense of being efficient, less fatter 8 and having other carcase q uality lems.
prob 10 To date there is no clear explanation of what causes WO 98I15837 PCTlGB97/02741 1 the meat quality differences between the breeds. There 2 is a widely held belief that the level of fat in the 3 muscle (intramuscular) fat may be important (Bejerholm 4 1984) but there are contradictory views about the role of fatness and the 'Duroc' clearly differs from 'White' 6 pigs in more respects than just fatness.

8 One of the observations made in our own earlier studies 9 (MLC 1992) was that pigs containing 'Duroc' genes have a higher level of haem pigment. This observation and 11 the higher levels of intramuscular fat are an 12 indication of a higher oxidative capacity in the 13 muscle.

The muscle of the animal which constitutes the meat is 16 made up of a variety of different muscle fibre cell 17 types, which can be classified according to their 18 contractile and metabolic nature. The proportions of 19 the fibre types vary between muscles. It is known, for example according to one method of classification (see 21 Peter et al, 1972) that muscle comprises slow-twitch 22 oxidative (SO), fast-twitch glycolytic (FG), 23 fast-twitch oxidative/glycolytic (FOG) and fast-twitch 24 oxidative muscle fibre types.
26 These fibre types are common to most muscles from most 27 meat animals. Typically the different fibres are spread 28 throughout the muscle cross-section resulting in a 29 chequered pattern in the stained muscle biopsy slides.
However, the arrangement of these fibres is unusual in 31 the pig in that the different fibre types are arranged 32 with clusters or groups of adjacent SO fibres 33 surrounded by other fibre types (Szentkuti and Cassens 34 1978). This association of muscle cells of similar metabolic types was described as forming "metabolic"

1 clusters (Handel and Stickland 1987). The number of SO
2 clusters is believed to be proportional to the number 3 of primary fibres formed during myogenesis, the number 4 of primary fibres being fixed in the pig foetus by 70 days gestation.

7 There is evidence of differences in the proportions of 8 these different fibres among pig breeds (Iwamoto, 9 Kawaida et al. 1983) and (Ruusunen 1993). Differences in proportion of different fibre types have also been 11 shown to occur among different pig breeds when fibre 12 proportion is analysed for bundles of mixed fibre types 13 (Skvrjanc, Salehar et al. 1994). There has also been a 14 tendency for breed crosses including 'Duroc' to have more SO and more FOG fibres (Uhrin, Kuliskova et al.
16 1986). This latter observation is entirely consistent 17 with the proposed higher oxidative capacity as 18 indicated by higher haem content.

The clearest breed difference in SO frequency was that 21 seen by (Ruusunen 1993}. These workers examined the 22 fibre type composition of the Longissimus Dorsi of 38 23 pure 'Hampshire' (H), 52 'Finnish Landrace' (L) or 24 'Yorkshire' (Y) sires cross onto (L x Y females}, and 52 H sires crossed onto ( L x Y females) pigs. SO
26 frequency was 15.3%, 11.5% and 11.6% respectively. The 27 H had significantly more SO fibres than either cross.
28 The fibre composition of the H cross animals more 29 closely resembled the composition of the animals which did not contain H than the pure H animals.

32 In studies conducted in sheep, it was found that a 33 single gene, the callipyge gene (Cockett, Jackson et 34 al. 1994) was associated with an increased frequency and size of FG fibres and a corresponding decrease in 1 the proportion of SO and FOG fibres (Carpenter, Rice et 2 al. 1996). The studies demonstrated that an increase in 3 the proportion of FG fibres were associated with 4 increased toughness of the meat (Koohmaraie, Shackelford et al. 1995).

7 Studies in cattle have shown that increases in SO
8 frequency are associated with improved sensory scores 9 for meat tenderness (Ockerman, Jawore et al. 1984;
Catkins, Dutson et al. 1981; and Maltin et al, Animal 11 Science; in press). In contrast the results of Seideman 12 and Theer 1986 could be take to imply that a higher 13 proportion of SO fibres was associated with lower panel 14 tenderness scores. Similarly a higher proportion of SO
fibres has been associated with higher shear force 16 values (Catkins, Dutson et al. 1981). To add to the 17 confusion regarding a relationship between SO fibres 18 and meat tenderness in beef (Seideman and Crouse 1986) 19 found higher SO frequency to be associated with increased tenderness in steers but not in bulls.

22 The present invention is concerned with determining, by 23 immunochemical, histochemical or genetic analysis, 24 whether a particular individual animal has desirable muscle characteristics.

27 The invention is founded upon the following novel 28 observations:

1. That the percentage frequency of SO fibres per 31 muscle, and likewise the proportional area of SO
32 fibres per unit muscle is increased in the Duroc 33 pig relative to the "White" pig;

2. That the number of SO fibres per cluster is 1 increased in the Duroc pig relative to the "White"
2 Pi9 4 3. That m calpain is preferentially localised in the 5 SO fibres of pigs. Therefore pigs with more SO
6 fibres (eg Duroc) have more m calpain in the 7 muscle as a whole. Thus the amount of m calpain 8 is increased per unit muscle in the Duroc pig 9 relative to the "White" pig;
11 4. That the amount of ~ calpain per fibre is 12 increased in the Duroc pig relative to the "White"
13 pig;

5. That the muscle fibre composition characteristic 16 of a Duroc pig (in particular the SO fibre 17 frequency) is controlled by a single gene or gene 18 cluster.

In more detail, we have observed that the percentage 21 frequency of SO fibres in the 'Duroc' is substantially 22 higher than in 'White' pigs. Separately we have found 23 that the proportional area of SO fibres per unit muscle 24 are increased in the "Duroc" pig relative to "White"
pigs.

27 In the pig populations tested we have found that 28 typically slaughter weight pigs (eg 50-100kg carcase 29 weight) of the Duroc breed have an SO fibre frequency (mean + standard deviation) of 15.60 (~2.1~s), whereas 31 non-Duroc pigs have a much lower SO fibre frequency, 32 generally 10.8g (~3.2%) (see Table 5, Examples). Our 33 observations have led us to conclude that such animals 34 having a muscle fibre composition with an SO fibre frequency of approximately 13~ or higher can be WO 98/15837 PCT/GB97/02?41 1 classified as having a muscle fibre composition of the 2 type characteristic of the Duroc pig. Consequently, it 3 is now possible to analyse a muscle of any particular 4 animal on the basis of percentage frequency of SO
fibres in order to determine whether or not that animal 6 has a muscle fibre composition characteristic of the 7 Duroc pig. Such measurements may be made directly (eg 8 by counting the number of SO fibres in a sample or by 9 determining the proportional area of SO fibres in a sample) or indirectly by measuring the percentage 11 frequency or proportional area of other fibre types, eg 12 FG and FOG fibres.

14 Further, we have also observed that in the Duroc pig, the number of SO fibres present per cluster is 16 significantly higher than in the non-Duroc pig. The 17 term "cluster" is defined herein as meaning those 18 fibres surrounding and touching a single central SO
19 fibre. Consequently, it is also possible to analyse the muscle of any particular animal on the basis of SO
21 fibres per cluster in order to determine whether or not 22 that animal has a muscle fibre composition 23 characteristic of the Duroc pig.

The data giving the number of SO fibres present per 26 cluster in Duroc and non-Duroc slaughter weight pigs is 27 set out below in Table 2:
Table 2 Total number of Number of SO

fibres/cluster fibres/cluster Oa Duroc 3.98 t0.8 2.65 0.5 100% Duroc 7.60 0.1** 3.66 0.3**

** P < 0.01 compared with 0~ Duroc 1 0~ Duroc - Large White, Landrace or Large 2 White/Landrace crosses.

4 Likewise we have found that in pigs of live weights of typically around 8kg, the number of SO fibres per 6 cluster (mean ~ standard deviation) is 2.5 + 0.2 for 7 the Duroc pig and 1.6 ~ 0.1 for non-Duroc pigs.

9 It is well documented that post mortem storage of animal carcases at below ambient temperature, but above 11 freezing, results in an improvement in meat tenderness.
12 This increase in tenderness is due to the enzymatic 13 breakdown of myofibrillar proteins and there is 14 evidence that calpains are responsible for 90~s of the tenderisation that occurs during post mortem storage 16 (Taylor et al 1994). Calpains are intracellular, 17 calcium activated/dependent thiol proteases present to 18 some extent in most body tissues. However, their exact 19 role in normal physiological conditions is still undefined. Several isoforms of calpain are known to 21 occur in various body tissues of birds and animals.
22 Two isoenzymes, ~ calpain and m calpain, with different 23 calcium requirements were originally isolated (Huston 24 and Krebs 1966, Mellgren 1980). More recently tissue specific calpains have been isolated from skeletal 26 muscle and stomach (Sorimachi et al 1989, Sorimachi et 27 al 1993). It is the actions of ~ calpain and m calpain 28 that are thought to be involved in post mortem 29 tenderisation of meat. In animal carcasses ~ calpain is most active during the first 15 hours post slaughter 31 whereafter its activity declines rapidly whilst the 32 activity of m calpain is much more persistent. The 33 activity of both a and m isoforms of calpain is 34 regulated by a natural inhibitor, calpastatin, which is also ubiquitously distributed in a11 body tissues.

1 Our studies have shown that m calpain is concentrated 2 in the SO fibres of pig muscle. As Duroc meat has a 3 greater proportion of SO fibres compared to meat from 4 other breeds the corresponding increase in m calpain levels could account for the tenderness of Duroc meat.

7 Surprisingly we have also found evidence that there is 8 an overall increased amount of a calpain per fibre in 9 the muscles of Duroc pigs. An increased concentration of ~ calpain per fibre could also explain the increased 11 tenderness of Duroc meat.

13 Moreover and surprisingly it has been found that the 14 cross-bred progeny of 'Duroc' crossed 'White' parentage have an SO frequency essentially the same as pure 16 'Duroc' animals. This observation indicates that there 17 is an apparently dominant genetic effect on muscle 18 fibre type which is likely to be the cause of the meat 19 quality advantages of the animals containing 'Duroc' genes seen in the studies described above. See Table 3 21 below (data for slaughter weight pigs).

23 Table 3 24 Total number of Number of SO

fibres/cluster fibres/ cluster 0~ Duroc 3.98 t0.8 2.65 0.5 26 50~ Duroc 5.15 1.1* 3.32 .9*

27 100o Duroc 7.60 t0.1** ++ 3.66 .3**

28 F1 5.16 0.5* 3.92 0.5*

*
P
<
0.05 31 compared with Oo Duroc **
P
<
0.01 32 compared with 50% Duroc or ++ F1 P
<
0.01 34 ge Landrace or Large Oa White, Duroc =
Lar Crosses White/Landrace WO 98/15837 PCTlGB97/02741 1 50~ Duroc - Duroc x (Large White x Landrace) 3 F1 - Duroc x Large White.

6 Furthermore we have shown that the SO fibre frequency 7 in animals containing only 250 'Duroc' genes shows a 8 variation among individuals consistent with a single 9 gene controlling SO frequency. This observation is consistent with the inheritance of a dominant gene 11 being inherited in a normal Mendelian manner. The 12 results for fibre frequency in our studies can be seen 13 in Table 5, Examples. The SO frequency seen in our 14 'White' pigs is similar to that found in Swedish Yorkshire ('Large White') at 8% (Karlsson, 16 Essen-Gustavsson et al. 1994).

18 For the first time therefore we can conclude that the 19 increased proportion of SO muscle fibres found in the Duroc pig is due to a genetic effect, namely a single 21 gene {which term also includes a gene cluster) which 22 controls SO fibre formation. Control of and/or 23 selection for this gene will enable pigs having an 24 increased proportion of SO fibres in their musculature to be preferentially bred and/or raised for meat 26 production.

28 For the first time therefore it is possible to 29 determine (for example using muscle biopsy and suitable histochemical analysis) whether any particular animal 31 has a muscle fibre composition characteristic of the 32 Duroc pig.

34 The present invention provides an assay to determine whether an animal has a muscle fibre composition 36 characteristic of a Duroc pig, said assay comprising:

1 a. obtaining a tissue sample from said animal and 2 subjecting said sample to genetic analysis to 3 determine whether genetic features typical of an 4 animal having a muscle fibre composition 5 characteristic of a Duroc pig are present; and/or 7 b. obtaining a muscle sample from said animal and 8 determining by histochemical or immunochemical 9 analysis:

11 i. the percentage frequency of SO fibres present 12 in said muscle; and/or 13 ii. the proportional area of SO fibres per unit 14 muscle; and/or 15 iii. the number of SO fibres present per cluster;

16 and/or I7 iv. the number of muscle fibres present per 18 cluster; and/or 19 v. the level of m calpain present per unit muscle; and/or 21 vi. the level of ~ calpain present per unit 22 muscle.

24 The results of the assay will indicate whether or not the animal tested has a muscle fibre composition 26 characteristic of the Duroc pig and this information 27 can be used in selecting animals for breeding and/or 28 for slaughter and use for provision of meat.

The present invention also provides an assay to 31 determine whether an animal has an allele or alleles 32 for a muscle fibre composition characteristic of the 33 Duroc pig, said assay comprising:

a) obtaining a tissue sample from said animal, 1 extracting genetic information therefrom and 2 analysing said genetic information to determine 3 whether the genotype of said animal includes said 4 allele(s); and/or 6 b) obtaining a muscle sample from said animal, and 7 analysing said sample by histochemical or g immunochemical techniques to determine whether 9 said sample exhibits phenotypic traits indicative of said alleles) (for example the phenotypic 11 traits set out above under paragraphs i to vi).

13 The results of the assay will indicate whether or not 14 the animal tested has genetic information from the Duroc breed, specifically at least one copy of the 16 (dominant) alleles) determining Duroc muscle fibre 17 type. This information can be used in selecting 18 animals for breeding and/or for slaughter and use for 19 provision of meat.
21 With reference to the genetic analysis referred to 22 above (see paragraphs a) a number of different 23 techniques may be used to give a genetic "fingerprint"
24 of the test animal. This "fingerprint" can then be compared to known standards (eg typical "Duroc" and 26 "non-Duroc" standards). Whilst the present invention 27 is not limited to any particular technique, mention may 28 be made of techniques such as RAPD, AFLP, RFLP, SSCP
29 and other mini-satellite or micro-satellite techniques or hybridisation techniques. Sequencing of genetic 31 information can also be useful, when the test sequence 32 can be compared to a known standard sequence. Thus, 33 using techniques such as RFLP (restriction fragment 34 length polymorphism), AFLP (amplified fragment length polymorphism), and RAPD (random amplification of 1 polymorphic DNA) it would be possible to identify 2 suitable Duroc genotype marker or markers that 3 constitute a genetic fingerprint and which associate 4 with the improved eating quality of pork derived from this breed of pig.

7 RFLPs are detected in restriction enzyme digested 8 genomic DNA which has been size fractioned by 9 electrophoresis, Southern blotted to a membrane support then hybridised to a labelled probe. The probe used in 11 RFLP analysis is usually a single gene locus of 12 interest to the researcher. PCR (polymerase chain 13 reaction) based techniques can also be used to detect 14 single locus RFLP, eliminating the need for Southern blotting and hybridisation. In this instance the 16 amplified DNA is digested with a restriction 17 endonuclease prior to gel electrophoresis.
18 Polymorphisms are evident as differences in the 19 resulting DNA fragment sizes.
21 An alternative technique is AFLP. AFLP is based on the 22 PCR amplification of genomic DNA after digestion with 23 restriction enzymes) and ligation of oligonucleotide 24 adapters. The technique is facilitated by the use of PCR primers that span a region of the oligonucleotide 26 adapters and extend into the DNA restriction fragment.
27 Only those fragments of DNA in which PCR primer 28 extensions have a perfect match are amplified and the 29 resultant mixture of PCR products can be analysed by electrophoresis. This technique could be used to 31 identify a marker of the Duroc factor associated with 32 the improved eating quality of pork derived from this 33 breed of pig. A description of AFLP is given by Vos et 34 al, 1995.

1 Particular mention may be made of analysing the genes 2 of the calpain/calpastatin system and comparing the 3 results to a known standard.

Genetic analysis is preferred for both determination of 6 meat quality and in respect of devising breeding 7 programs.

9 With reference to histochemical and immunochemical analysis referred to above (see paragraphs b) any 11 technique able to analyse the number of SO fibres per 12 cluster or the frequency of SO fibres and/or the amount 13 of m calpain per unit muscle and/or the amount of 14 calpain per unit muscle may be used.
16 For example, where the number of SO fibres per cluster 17 is to be determined the muscle sample may be prepared 18 and stained so that the muscle fibres can be viewed 19 (for example using a microscope) and counted. Details of a suitable protocol are given in Example 1. Similar 21 techniques can be used for determining the frequency of 22 the SO fibres in the sample.

24 We have found, for example that in slaughter weight pigs (carcase weight 50-100kg) an animal having over 26 13o SO fibre frequency and/or 3 or more SO fibres per 27 cluster and/or 5 or more fibres per cluster (see Table 28 3) indicates a muscle fibre composition characteristic 29 of the Duroc pig. It should be noted that muscle fibre type and frequency (and in the pig SO fibre cluster 31 size) will vary with the weight and age of the animal, 32 but that a distinct difference will be observed between 33 animals of Duroc muscle fibre composition and non-Duroc 34 muscle fibre composition. Thus, for example an animal having a live weight of approximately 8kg and having a 1 mean of 2 SO fibres per cluster or more can be 2 characterised as having a muscle fibre composition 3 characteristic of the Duroc pig.

Immunochemical techniques may be useful to aid 6 visualisation of the SO fibres, by exposing the muscle 7 sample to labelled antibodies which bind preferentially 8 to SO fibres, eg MI-iCs (myosin heavy chain slow isoform) 9 of Novocastra Laboratories Limited UK.
11 With regard to determining the amount of m calpain or 12 calpain immunochemical techniques may be used, for 13 example an ELISA assay. Anti-m calpain, anti-~
14 calpain, (anti-calpastatin) and anti-myosin (heavy chain slow isoform) antibodies are available 16 commercially. Examples include MAB3082 (anti-~e calpain 17 antibody), MAB3084 (anti-calpastatin antibody), AB1625 18 (anti-m caipain antibody), a11 of Chemicon 19 International, Inc (Temecula, CA 92590, USA). These antibodies (and other similar antibodies) can be used 21 as described in the manufacturer's instructions or 22 according to known protocols. Reference is also made 23 to the description of immunocytochemical locations of 24 the calpain proteolytic system in porcine muscle described in Example 4.

27 In a further aspect, the present invention provides a 28 method of determining meat quality, said method 29 comprising determining whether an animal has an allele for or exhibits a muscle fibre composition 31 characteristic of the Duroc pig as described above and 32 segregating these animals found to have said allele or 33 said composition from the other animals.

The method may conducted in vitro or in vivo using a 1 sample from a living animal or post mortem following 2 the death of the animal tested.

4 In a further aspect, the present invention provides a 5 method of selecting animals for use in breeding 6 programs, said method comprising determining whether an 7 animal has an allele or alleles for, or exhibits a 8 muscle fibre composition characteristic of the Duroc 9 pig as described above and selecting those animals 10 found to have said alleles) in their genotype or said 11 composition for use in the breeding program.

13 The method may conducted in vitro or in vivo using a 14 sample from a living animal or post mortem following 15 the death of the animal tested. If the assay is 16 conducted post mortem, the information may be of use 17 for the siblings, parents or other close relatives of 18 the animal tested.

20 In one preferred embodiment of the invention the animal 21 is a pig, although other mammalian species are also 22 included.

24 In a further aspect the present invention provides a mammalian animal having increased proportions of SO
26 fibres in its musculature. Generally, the animal will 27 be the progeny of animals) selected for breeding by 28 the method given above.

By "increased proportions of SO fibres" is meant the 31 frequency of SO muscle fibres is elevated above the 32 common incidence of SO muscle fibres found in wild type 33 animal of a particular breed or species.

The increased proportion of SO fibres may lead to WO 98115837 PCT/GB97/02?41 1 improved meat quality, less pale and more tender 2 muscle.

4 In a further aspect, the increased proportion of SO
fibres in the animal of the present invention may be 6 due to introduction of a genetic polymorphism affecting 7 the frequency of SO fibres.

9 Our observations regarding the Mendelian inheritance of SO fibre number in Duroc pigs and Duroc pig crosses 11 support the view that a single gene is responsible for 12 the improved tenderness observed in meat quality 13 relative to animals not possessing this polymorphism.

Alternatively, other genes and/or controlling sequences 16 may be involved, especially the genes controlling the 17 calpain/calpastatin system.

19 According to another aspect of the present invention there is provided a method of enhancing tenderness 21 and/or colour of the muscle of a mammalian animal, said 22 method comprising influencing said animal or its 23 parents to increase the proportion of SO fibres present 24 in the muscles.
26 The present invention also provides a method of 27 enhancing the eating quality of musculature in a 28 mammalian animal, said method comprising enhancing the 29 proportion of SO fibres in the skeletal muscle of said animal.

32 In a further aspect the present invention provides meat 33 from a mammalian (non-human) animal, said meat having 34 improved meat quality wherein said animal has been selected for or influenced to increase the proportion 1 of SO fibres present in the muscle which forms said 2 meat.

4 In a further aspect the present invention provides a means of detecting the presence of a higher frequency 6 of SO fibres in an animal, especially a pig.

8 The means of detecting each of the above can be chosen 9 from the group of means consisting of genetic mapping, the detection of the restriction fragment polymorphism, 11 fibre typing (ie number of SO fibres and/or number of I2 SO fibres per cluster) and antibody linked assays such 13 as ELISA.

The invention further provides a.kit for the 16 identification of animals having an increased frequency 17 of SO fibres and/or animals having a muscle fibre 18 composition characteristic of the Duroc pig.

Preferably the kit comprises means for identifying SO
21 fibres or a pre-disposition in an individual animal to 22 developing SO fibres based on a test as outlined above.

24 For example, the means of detecting a higher frequency of SO fibres could be any of the following:

27 i. analysing genes of the calpain/calpastatin system 28 and comparing the results to a known standard;
29 and/or 31 ii. analysing the m calpain activity (eg in muscle 32 and/or in SO fibres); and/or 34 iii. analysing the ~ calpain activity.

1 Analysis of the m calpain activity in muscle tissue or 2 in SO fibres could be carried out using anti-m calpain 3 antibodies. Suitable antibodies are available 4 commercially. Examples include these AB1625 of Chemicon International, Inc (Temecula, CA 92590, USA).

7 In a further aspect, the present invention provides the 8 use of anti-m calpain antibodies or anti-~ calpain ' 9 antibodies to select for animals having the ability to produce tender meat. The selected animals may be used 11 directly for meat production or may be used for 12 breeding purposes.

14 The invention will now be described with reference to the following examples and figures in which:

17 Figure lA
18 ATPase from 8kg Duroc pig showing clusters of SO
19 fibres.
Figure 1B
21 ATPase from 8kg Large White pig showing clusters of SO
22 fibres.

24 Figure 2A
ATPase from slaughter weight Duroc pig showing clusters 26 of SO fibres 27 Figure 2B
28 ATPase from slaughter weight Large White pig showing 29 clusters of SO fibres.
31 Figure 3A
32 Section reacted to demonstrate the presence of m-33 calpain in 8kg Duroc pig. The clustered fibres are SO
34 type.
Figure 3B

1 Section reacted to demonstrate the presenceof m-2 calpain in 8kg Large White pig. The clustered fibres 3 are of SO type.

Figure 4A

6 Section reacted to demonstrate the presenceof /.c-7 calpain in 8kg Duroc pig. The overall brightness is 8 compared with that in Fig 4B.

9 Figure 4B

Section reacted to demonstrate the presenceof fc-11 calpain in 8kg Large White pig . The overall brightness 12 is less than that in Fig 4A.

14 Figure 5A

Section reacted to demonstrate the presenceof myosin 16 heavy chain slow isoform in the clusters Skg Duroc in I7 pig.

18 Figure 5B

19 Section reacted to demonstrate the presenceof myosin heavy chain slow isoform in the clusters 8kg Large in 21 White pig.

23 All figures are transverse sect ions throughlongissimus 24 dorsi muscle of pigs.

1 Example 1 3 Variation in fibre type associated with increasing 4 Duroc genotype 6 Background and Introduction 8 Evidence from various trials (for example MLC, 1992) 9 indicates that Duroc genes enhance the eating quality 10 of pork, in particular tenderness. Whilst Duroc cross 11 pigs tend to be fatter with higher levels of 12 intramuscular fat, it is not clear whether this is the 13 cause of the enhanced eating quality. Durocs also have 14 redder muscle with a higher concentration of the muscle 15 pigment, haem. This indicates a higher oxidative 16 capacity and therefore, fibre types were expected to 17 differ from "WhiteW genotypes. It is possible that 18 differences in fibre type may be related to eating 19 quality differences between Duroc crosses and "White"
20 pigs.

22 Materials and Methods 24 Animals 26 Samples from 0, 25~ and 50~ Duroc animals were sourced 27 from an MLC University of Newcastle-upon-Tyne trial 28 designed to examine the influence of lean tissue growth 29 rate on the eating quality of pork. These were taken from pigs fed ad libitum from weaning to slaughter.
31 100o Duroc animals were sourced separately from a 32 commercial abattoir and no control over rearing or 33 slaughter was exercised for these.

1 Slaughter 3 Pigs were slaughtered on reaching 85kg liveweight.
4 Transport, lairage and slaughter was carried out in accordance with MLC's Blueprint for pork.

7 Carcase Handling and Chilling 9 Carcases were chilled according to normal plant practice. Carcases were transported to MLC
11 at Winterhill following overnight chilling. Loin 12 samples were frozen after a five day ageing 13 period.

Sample Transport 17 The samples were transported from Milton Keynes by air 18 in insulated boxes and maintained in the frozen state.
19 The chops were then stored at -70~C.
21 Histochemistry 22 Blocking and sectioning chops 23~
24 Before the initiation of this study a novel method was developed which allowed the retrospective examination 26 of blast frozen meat. This method is simple, and relies 27 on slowly thawing the chops overnight at +4~C.
28 Subsequently, approximately 1 cm2 blocks were cut from 29 the centre of the longissimus dorsi muscle. Care was taken to ensure that the same area was sampled from 31 each of the chops. These cubes of muscle were 32 orientated for transverse sectioning, mounted on a 33 piece of cork with optimal cutting temperature compound 34 (OCT), covered with more OCT and with unperfumed talcum powder and frozen in liquid nitrogen with constant 1 agitation. Twelve blocks were taken from each chop and 2 once frozen, were stored in aluminium tins submerged in 3 liquid nitrogen. Throughout the period of the study the 4 blocks were maintained in the liquid phase of the nitrogen dewar to limit any freeze drying. The tins 6 were removed from the liquid nitrogen storage and 7 placed in the cryostat at -20~C 2 hours before 8 sectioning. Serial transverse sections were cut at 10m 9 using a Frigocut 2800 cryostat with motor driven cutting stroke to reduce variation in section 11 thickness.

13 The sections were allowed to air dry at ambient 14 temperature for 2 hours and then frozen overnight for staining the following day.

17 Fibre typing 19 The characterisation of fibre typing adapted in this study is based upon the reaction of individual fibres 21 to a minimum of three stains. The stains used were 22 chosen to demonstrate the activities of Caz+ activated 23 myofibrillar adenosine triphosphatase (ATPase), 24 nicotinamide adenine dinucleotide diaphorase (NADH), and a-glycerophosphate dehydrogenase (GPOX), which then 26 allowed the characterisation of the fibres based on 27 their contractile and metabolic activities as follows 28 and as illustrated in Table 4; ATPase - contractile 29 activity (fast or slow twitch); NADH - oxidative activity; GPOX - glycolytic activity.

2 Table 4 The histochemical basis of 3 characterisation of muscle fibre types in pig meat.
FIBRE TYPE STAIN

ATPASE NADH GPOX

FOG ++(+) +++ +++

FG +++ + +++

SO + +++ +

3 Quantification of fibre type and size, Quantitative assessments of fibre type and size were 6 made from the stained muscle preparations using a Torch 7 computer based image analysis system (Vision Dynamics, 8 Hemel Hempstead, Herts). Measurements of fibre size 9 were made on the sections reacted to demonstrate the activity of ATPase. For each animal, fibre size 11 estimation was carried out on eight blocks with two 12 fields per block being analysed.

14 The ATPase stained sections were examined under a light microscope fitted with a Sony video camera, the output 16 of which was applied to the image handling software of 17 the Torch computer. The use of the ATPase stain 18 generates an image in which three fibre types can be 19 distinguished based on their grey levels. Fibre type was confirmed through examination of printed images of 21 the NADH and GPOX stains to give information on the 22 metabolic character of each fibre. The three fibre 23 types were analysed separately, and thresholding was 24 altered to detect a11 fibres of the same type. Where adjacent fibres were thresholded and detected as a 26 single unit, manual editing operations were undertaken 27 to separate the fibres through the use of a 1 superimposed 'live' camera image to visualise the 2 sarcolemmal membranes accurately. The data for size, 3 frequency and percentage area was computed for each 4 animal. Approximately 1600 fibres were analysed for each pig.

7 Results 9 The results were clear and showed that possession of 500 or more Duroc genes was associated with a 11 significant increase in both the frequency and 12 percentage area of SO fibres, and a significant 13 reduction in the frequency of FG fibres; there was also 14 a tendency towards a reduction in FG percentage area (Table 5). In addition, pigs possessing 25% Duroc genes 16 showed an increased SO frequency over 0% pigs, with a 17 mean frequency value lying half way between the value 18 for 50% and 0% pigs (Table 5). Close inspection of the I9 individual values showed that half the animals had SO
frequencies similar to those seen the 50 and 100% Duroc 21 animals (mean frequency 14.8 (~2.2 (sd), n= 6), while 22 the remaining animals had SO frequencies which 23 resembled those seen in the 0% pigs (mean frequency 24 10.9 (t1.3 (sd), n=6)).
26 Sample results are shown in Figs lA and 1B.

28 The experiment was repeated using 8kg pigs (live 29 weight); sample results are shown in Figs 2A and 2B.

WO 98l15837 PCTlGB97l02741 1 Table 5. The fibre type distributions in muscle 2 from pigs containing different proportions of Duroc 3 genes .
FOG FG gp AREA ZFREQ ZAREA AREA ZFREQ ZAREA AREA ZFREQ ZAREA

OX 2079 28.5 26.9 2352 60.6 64.5 1740 1Q.8 8.5 Duroc (401) (2.1) (2.8) (478) (3.6) (3.8) (243) (3.2) (2.2) 25Z 2137 32.2 3I.5 2338 54.8 S8.4 1716 12.8 9.8 Duroc (388) (1.9) (4.1) (426) (3.7) (4.6) (266) (2.7) (1.0) 50Z 2510 28.2 28.0 2760 S6.2 61.6 1675 15.6 10.3 Duroc (S07) (1.8) (2.8) (438) (3.6) (3.5) (19S) (3.6) (1.9) I

100Z Z006 32.8 27.4 2867 51.S 61.7 1628 15.6 10.8 Duroc t440) (1.8) (1.8) (508) (3.2) (1.7) (103) (2.1) (I.4) 4 Discussion 6 Pigs containing Duroc genes have more SO fibres. The 7 results show clearly that animals with 500 or more 8 Duroc genotype have a significantly increased number of 9 SO fibres and decreased numbers of FG fibres compared 10 to Oo genotypes. The observations of the SO frequencies 11 in the 25o Duroc suggests independent segregation of 12 genes and supports the concept that the Duroc factor is 13 a heritable trait inherited in a normal Mendelian 14 manner.
16 Consequently, the present data provide a basis that 17 there islare inheritable muscle specific Duroc genes) 18 which confer properties beneficial to the eating 19 quality of Duroc pig meat.

1 Examele 2 3 Antibodies raised against cc calpain, m calpain and 4 calpastatin are now commercially available (MAB3082, AB1625 and MAB3084 respectively, a11 of Chemicon 6 International Inc, Temecula, CA USA). Data from the 7 supplier of these antibodies show that these antibodies 8 bind to the calpain proteins of a number of different 9 species and there was no reason to believe that they would not bind to the porcine calpain and calpastatin 11 epitopes as at the amino acid sequence level the 12 polypeptide products of the calpain and calpastatin 13 genes are highly conserved from species to species.
14 Using standard immunocytochemical techniques, transverse sections of Longissimus dorsi muscle from 16 Large White new-born and lOkg pigs were prepared and 17 developed using the panel of antibodies described 18 below. For Duroc animals transverse sections of 19 Longissimus dorsi muscles from new-born and lOkg pigs were also prepared as were transverse sections of 21 semimembranous and biceps femorus muscles from lOkg 22 animals. In addition, longitudinal sections were cut 23 from a block of semimembranosus muscle. The antibody 24 to m calpain was a rabbit polyclonal which was developed and visualised with an anti rabbit FITC
26 conjugate. Both anti calpastatin and anti fc calpain 27 were murine monoclonal antibodies that were developed 28 using an appropriate anti mouse conjugate. For some of 29 these samples serial sections were cut and stained using both standard histochemical and immuno-31 cytochemical techniques. Pooled information from these 32 serial sections were then subjected to analysis and 33 interpretation. Sample results are shown in Figs 3A, 34 3B, 4A and 4B. Alternative staining using antibodies to myosin heavy chain slow isoform (eg NCL-MHCs of 1 Novocastra Laboratories Ltd, Newcastle, UK) are shown 2 in Figs 5A and 5B.

4 ExamQle 3 6 RAPD Assay 8 120 primers were purchased from Genosys Biotechnologies 9 Inc. One h undred primers had a G+C
content of 50, 60, 70 and 80~ and had sequences as follows:
the 12 50~ G+C con tent AGGATACGTG TACATCAGCG

GGAAGACAAC TTTACGGTGG

24 60~ G+C content CGCAGTACTC GAGTCTGTCG GAGTGTCTGC

CTACTACCGC GGCGATATGG GCAGCTCATG

1 70~ G+C content CGCATTCCGC GTATGCCGCG

TGCAGCACCG CGCACTCGTC

13 80~ G+C con tent CGCCCAAGCC CGACGCGTGC ACTCGGCCCC

GCACGCCGGA GCAGGTCGCG GGCAAGCGGG

A further were purchased into which twenty primers a 26 restriction endonuclease site had been incorporated.

27 This facilitates of any amplified products cloning 28 after digestion appropriate restriction with the 29 endonuclease ably cut cloning vector.
into a suit 31 CGGGATCCGC BamHl 32 GGCTGCAGCG Pstl 33 GCGGTACCCG Kpnl 34 CCCTCGAGGC Xhol CCAGATCTGC Bgll 1 CCAAGCTTGC HindIII
2 GCATCGATCG Clal 3 GGCTGACGCG Sall 4 GCGTTAACGC EcoRl CGAATTCGGC EcoRl 6 CGGATCCCGC EcoRl 7 CGCGATGCGC Sphl 8 GGGATCCGCC BamHl 9 GCCAAGCTTC HindIII
CGATCGATGC Clal 11 GCGAGCTCTG Sacl 12 GGAAGCTTCG HindIII
13 CGTCTAGAGC Xbal 14 CCCTGCAGGC Pstl CGCGAGCTCG Sacl 17 RAPD Assay Development 19 Oligonucleotide primers were dissolved in sterile distilled water at a concentration of 25 pico moles per 21 microlitre and used to optimise the PCR based RAPD
22 reaction. To make the RAPD test as sensitive and 23 reproducible as possible nine different buffer regimes 24 were assessed with a panel of the primers chosen at random. In these buffers the pH, salt concentration, 26 and buffer component were varied. Reactions performed 27 in a 50,cc1 volume containing 20mM Tris HC1 pH 8.75, lOmM
28 KC1, lOmM (NH3)ZS04, 0.75mM MgClZ, 0.75mM MgS04, 29 O.l~Triton, O.Ol~Tween, 0.001$gelatin, 200 mM dNTPS, (1X PCR assay buffer) using 25pmoles of appropriate 31 oligonucleotide primer, 200ng of template DNA and 2.5 32 units of Taq DNA polymerase were found to be 33 consistently better than any of the others tried in the 34 assay. Amplifications were performed using an APPLIGENE/ONCOR crocodile III microprocessor controlled 1 incubation system programmed for 25 or 30 cycles of 2 94~C for 45 sec, 37~C for 3 min, 72~C for 3 min. The 3 products of these reactions were analysed by 4 electrophoresis in gels containing 2.5~ agarose in 1X
5 TAE buffer. Using this technique 120 primers of known 6 arbitrary sequence have been assessed and 12 which show 7 differences when DNA templates isolated from pure bred 8 Duroc or Large White pigs were examined.

10 Pools of Duroc and Large White DNA were screened in 11 RAPD tests using the commercially available primers and 12 this has allowed the identification of markers that can 13 distinguish the two breeds of pig when pooled DNA is 14 used in the test. The use of pooled DNA samples 15 prevented the isolation of a sex or breed specific 16 individual polymorphic marker. Individual DNA samples 17 were from pure bred Duroc and Large white pigs or from 18 samples from 50~ Duroc/Large White crosses. Genetic 19 material can also be prepared from non Duroc or Large 20 White pigs both pure bred and crossed so that a Duroc 21 breed specific test that is indicative of superior meat 22 quality can be developed.

24 The following is a list of the oligonucleotides that 25 gave DNA fingerprints which showed differences between 26 the Large White and Duroc genotype when used in the 27 RAPD test.

GCGGTACCCG CGGAATTCCG GGAAGCTTCG GGTCGACGCG

WO 98I15837 PCTlGB97/02741 1 GCCCCATGCG.

3 Example 4 Despite recent progress in the identification and 6 characterization of calcium activated proteases 7 (calpains) and the genes that encode them, their 8 precise biological role and that of their endogenous 9 inhibitor, calpastatin, is still unclear. In skeletal muscle, a calpain and m calpain appear to be 11 ubiquitously expressed and are implicated in myoblast 12 fusion (Brustis et al 1994), the degradation of 13 cytoskeletal proteins (Elamrani et al 1995) and enzymes 14 (Hong et al I995 et al 1995) Calpastatin is also thought to play a role in myoblast differentiation and 16 fusion (Barnoy et al 1996).

18 Materials and methods Longissimus dorsi muscles were removed rapidly from 21 lOkg pigs, orientated for either longitudinal or 22 transverse sectioning and frozen in liquid nitrogen.
23 Standard immunocytochemical techniques were employed 24 using commercially available antibodies raised against a calpain, m calpain and calpastatin (Chemicon 26 International Inc, Temecula, CA 92590). Localization 27 of ~ and m calpain was carried out in sections serial 28 to those used for localization of calpastatin.

Results 32 Calpastatin was localized around, but not in, the 33 nucleus. In addition to this perinuclear localization, 34 in transverse sections a granular dispersion of stained material scattered throughout the cytoplasm was 1 observed but with no fibre type specific distribution.
2 Longitudinal sections showed that calpastatin 3 immunoreactivity was also present down the inner 4 surface of the sarcolemma, and in association with some component of myofibril ultrastructure. There was no 6 fibre specific distribution of ~ calpain which was 7 localized around the sarcolemma with a variable level 8 of cytoplasmic staining. Examination of longitudinal 9 sections however, showed that ~ calpain was localized in a regular banded pattern, indicating a highly 11 ordered localization of this protease in muscle 12 cytoplasm, and suggesting some association with 13 contractile proteins in the A or I bands. In contrast 14 to both calpastatin and ~ calpain, m calpain localization did appear to be associated with specific 16 fibre types. Immunoreactivity for m calpain was 17 primarily associated with slow twitch fibres; much less 18 intense staining being noted in a11 other fibre types.

Discussion 22 Mellgren 1991, proposed a role for the calpains in the 23 turnover of nuclear proteins, hence a perinuclear site 24 for calpastatin might be predicted. However, this apparent compartmentation raises some questions as to 26 its role in the inhibition of the calpains at the 27 sarcolemma and in the cytoplasm. The presence of 28 calpain at the sarcolemma is consistent with its role 29 in the regulation of the activity of enzymes and cytoskeletal protein degradation. The fibre type 31 localization of m calpain in slow fibres may relate to 32 the higher rate of protein turnover in these fibres 33 types (Garlick et al l989). Overall, the results 34 suggest that the calpains and calpastatin have an ordered localisation in porcine skeletal muscle 1 suggesting a specialised role for each of these 2 proteins in skeletal muscle.

4 Example 5 Breeding Program 7 Method 8 Large White and Duroc pedigree pigs were used in a 9 breeding programme to produce an F1 population which were 50% Duroc and 50% Large White. Specifically two 11 crosses were set up. In the first cross Duroc boars 12 were mated to Large White sows and in the other Duroc 13 sows were served by Large White boars. The resulting 14 F1 populations showed the Duroc muscle phenotype indicating a dominant Duroc gene or gene cluster. Sows 16 from the F1 population were then crossed with Large 17 White boars to generate an F2 backcross population.
18 Classical Mendelian genetics suggests that in such 19 crosses there should be a 50% 50% segregation of any given genetic trait. In this F1 backcross population 21 there is such a segregation of muscle phenotype into 22 Duroc and Large White types (SO fibres per cluster).

24 Results The number of SO fibres per cluster (mean + SD) in the 26 F2 population (8kg live weight) fell into two distinct 27 groups as follows (see Figs 5A and 5B wherein the SO
28 fibres are stained using the myosin heavy chain slow 29 isoform antibody NCL-MHCs):
31 Group A (n=3; non-Duroc phenotype): 1.6 + 0.1 32 Group B (n=5; Duroc phenotype): 2.5 + 0.2 34 At least 12 samples were taken per animal and the SO
cluster sizes pooled.

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Claims (20)

1. An assay to determine whether an animal has an allele for or has a muscle fibre composition characteristic of the Duroc pig, said assay comprising:
a. obtaining a tissue sample from said animal and subjecting said sample to genetic analysis to determine whether i) the genotype of said animal includes said allele(s); and/or ii) genetic features typical of an animal having a muscle fibre composition characteristic of a Duroc pig are present; and/or b. obtaining a muscle sample from said animal and determining by histochemical or immunochemical analysis whether said sample exhibits phenotypic traits indicative of said allele(s) and/or shows a muscle composition characteristic to the Duroc pig.

2. An assay as claimed in Claim 1 wherein genetic analysis of a tissue sample is carried out.

3. An assay as claimed in Claim 2 wherein said genetic analysis involves a RAPD, AFLP, RFLP, SSCP or similar technique, or a nucleotide hybridisation or sequencing technique.

4. An assay as claimed in any one of Claim 1 to 3 wherein the genes of the calpain/calpastatin system are analysed.

5. An assay as claimed in Claim 1 wherein histochemical or immunochemical analysis of a muscle sample is carried out and at least one of the following factors factors is measured:
i. the percentage frequency of SO fibres present in said muscle; and/or ii. the proportional area of SO fibres per unit muscle; and/or iii. the number of SO fibres present per cluster;
and/or iv. the number of muscle fibres present per cluster;
and/or v. the level of m calpain present per unit muscle;
and/or vi. the level of a calpain present per unit muscle.

6. An assay as claimed in either one of Claims 1 and 5 wherein the level of m calpain and/or the level of µ
calpain is analysed.

7. An assay as claimed in either one of Claims 5 and 6 wherein said level(s) of m calpain and/or a calpain is determined by using an ELISA style assay.

8. An assay as claimed in either one of Claims 1 and 5 wherein the percentage frequency of SO fibres present in said sample is analysed.

9. An assay as claimed in either one of Claims 1 and 5 wherein the proportional area of SO fibres present per unit muscle is analysed.

10. An assay as claimed in either one of Claims 1 and 5 wherein the number of SO fibres present per cluster is analysed.

11. An assay as claimed in either one of Claims 1 and 5 wherein the number of muscle fibres present per cluster is analysed.

12. A method of selecting animals for use in breeding programs, said method comprising determining whether an animal has an allele for, or exhibits a muscle fibre composition characteristic of the Duroc pig by providing the animals to be tested, subjecting those animals to an assay as claimed in any one of Claims 1 to 11 and selecting those animals found to have said allele(s) in their genotype or said muscle composition for use in the breeding program.

13. A method of determining meat quality, said method comprising determining whether an animal has an allele for or exhibits a muscle fibre composition characteristic of the Duroc pig by providing the animals to be tested, subjecting those animals to an assay as claimed in any one of Claims 1 to 11 and selecting those animals found to have said allele(s) in their genotype or said muscle composition.

14. A method as claimed in either one of Claims 12 and 13 which is conducted post mortem.

15. A method as claimed in either one of Claims 12 and 13 which is conducted in vitro or in vivo whilst the animal tested is alive.

16. A mammalian animal having increased proportions of SO fibres in its musculature and obtained through a breeding program wherein at least some of the animlas were selected using the method of Claim 12.

17. Meat from a mammalian animal selected by the method as claimed in Claim 13.

18. A kit to identify an animal having a muscle fibre composition characteristic of a Duroc pig, said kit containing means to identify whether said animal has an allele for or exhibits a muscle fibre composition characteristic of the Duroc pig.

19. A kit as claimed in Claim 18 comprising anti-µ
calpain antibodies and/or anti-m calpain antibodies.

20. Use of anti-m calpain antibodies or anti-µ calpain antibodies to select for animals having a muscle fibre composition characteristic of the Duroc pig.