The present invention relates to animals, especially pigs, having improved meat quality.

In the United Kingdom, elsewhere in Europe and increasingly throughout the world, pig producers are selecting breeds to use on their farms which are efficient producers of lean meat and thus provide the farmer with the maximum possible economic return.

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

common synonym is 'Yorkshire'.

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

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

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 In some cases, these lines of "White" pigs are no longer maintained as pedigree registered pigs and they may even have trade names, but an averagely skilled observer would recognise from their appearance lines of 'Large White'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

The 'Large White' and 'Landrace' breeds of pig especially those produced by pig breeding companies in the United Kingdom are characterised by having a good

growth rate and producing carcases with a low subcutaneous and i-ntermuscular fat level and thus a high lean content. These characteristics also lead to animals with a high feed conversion efficiency. Considerable progress in improving the lean meat content of these breeds of pig has been made in recent years in the United Kingdom.

In 1971 when the Meat and Livestock Commission began classification of pig carcases in Great Britain, the average back fat depth (P ₂ ) was close to 20 mm, by 1995 this had reduced to 11 mm (MLC 1996) . This reduction is in the context of increasing carcase weights, which would normally increase fatness and therefore the reduction in the genetic potential of the pigs to produce fat is even greater than it appears.

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

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

The meat defect PSE is known to have a strong genetic component due to alteration in a single gene, the halothane gene. The halothane gene codes for a protein in the calcium channels of the pigs muscle. The mutant

allele or alleles of the gene leads to leaky calcium channels, pigs with an increased lean content, but also with an increased predisposition to death from Porcine Stress Syndrome and PSE in the muscle after slaughter.

Recently the precise DNA mutation of the halothane gene which leads to PSE was discovered and is described in WO-A-92/11387. This allows pig breeders to control the incidence of the mutated version of the gene in their populations and control the incidence of PSE. However, PSE can also be caused by the pig's response to its pre-slaughter handling and thus PSE could remain a problem where pre-slaughter handling in the abattoir is not of the highest standard.

Although the use of 'Large White' and 'Landrace' breeds of pig is increasing throughout the world, there are still very many cases of other breeds being used for meat production according to local tastes and the suitability of the pigs for local conditions. Important characteristics may be tolerance of local climatic or other conditions, or resistance to disease. In some cases, meat quality may also be a characteristic considered in the choice of breed.

Another important world breed of pig is the 'Duroc'. This is a North American breed of meat pig, red in colour and originating between 1822 and 1877 from 'Old Duroc' of New York and 'Jersey Red' of New Jersey. A breed society was formed in 1833 (Mason 1988) . The 'Duroc' remains very popular in the United States and has been imported into Europe a number of times this century.

Within Europe, especially the United Kingdom, the 'Duroc' is characterised as being of reasonable growth

rate, but fatter and less efficient with regard to meat production than 'Large White' and 'Landrace' . However, it has been shown a number of times to have meat of superior quality, especially colour and tenderness, than the "White" breeds (as defined above) .

In Canada, Denmark, France and New Zealand, pigs produced from "White" hybrid mothers and 'Duroc' sires have produced pigs with a tenderness advantage ranging from 10 to 17% over similar but 'white' sired pigs ((Martel, Minveille et al . 1988) ; (Barton-Gade 1989) ; (Gandemer and Legault 1990) and (Purchas, Smith et al. 1990) ) .

In the United Kingdom, the 'Duroc' is used to some extent in two situations. It has gained popularity as a component of breeding females, typically at 50 or 25% 'Duroc' genes content, for use in outdoor units or in units where hardiness is an important characteristic. Secondly, it is used, in purebreed form, as a sire or as a component of a crossbred sire or dam of superior meat quality, especially eating quality characteristics such as tenderness. However, the widespread use of the 'Duroc' is hindered because of the higher cost of producing pig meat from the 'Duroc' and because of its lower carcase value. Carcase value is diminished both because of the increase in fatness and because the 'Duroc' crosses tend to have more coloured skin on the carcase and more deep-seated dark hairs which are not easily removed in the abattoir.

The interest in the 'Duroc' breed in the United Kingdom prompted the Meat and Livestock Commission to undertake what is probably the most comprehensive evaluation of the breed ever done. Conventional 'White' British commercial pigs ('Large White' sires crossed to 'Large

White' cross, 'Landrace' dams) containing zero percent 'Duroc' genes were compared with pigs containing 25, 50 or 75% 'Duroc' genes produced by various crosses (MLC 1992). Some results for 0% and 50% 'Duroc' pigs are presented in Table 1 and illustrate the relative merits of the two pig types.

Table 1

DUROC CONTENT oz 50Z

Daily live weight gain (g) 806 803

Feed conversion ratio 2.70 2.83

Lean tissue feed conversion ratio 6.19 6.81

P ₂ fat depth (mm) 9.3 10.9

Lean Z 58.8 56.6

PSE carcases (Z) 8.3 1.6

Deep seated hair (Z carcases) 1.1 17.6

Tenderness score* 4.96 5.32

Pork flavour*

In lean 3.88 3.96

In fat 3.87 4.06

Pork odour in fat* 3.58 3.73

* sensory scores are on a 1-8 scale where higher scores indicate more tender, juicy etc. All results are for pigs fed ad-libitum but restrictedly feed pigs show similar results. MLC 1992.

Thus it can be seen that 'Duroc' cross pigs have good quality meat in comparison to 'White' pigs but this is obtained at the expense of being less efficient, fatter and having other carcase quality problems.

To date there is no clear explanation of what causes

the meat quality differences between the breeds. There is a widely held belief that the level of fat in the muscle (intramuscular) fat may be important (Bejerholm 1984) but there are contradictory views about the role of fatness and the 'Duroc' clearly differs from 'White' pigs in more respects than just fatness.

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

The muscle of the animal which constitutes the meat is made up of a variety of different muscle fibre cell types, which can be classified according to their contractile and metabolic nature. The proportions of the fibre types vary between muscles. It is known, for example according to one method of classification (see Peter et al, 1972) that muscle comprises slow-twitch oxidative (SO), fast-twitch glycolytic (FG), fast-twitch oxidative/glycolytic (FOG) and fast-twitch oxidative muscle fibre types.

These fibre types are common to most muscles from most meat animals. Typically the different fibres are spread throughout the muscle cross-section resulting in a chequered pattern in the stained muscle biopsy slides. However, the arrangement of these fibres is unusual in the pig in that the different fibre types are arranged with clusters or groups of adjacent SO fibres surrounded by other fibre types (Szentkuti and Cassens 1978). This association of muscle cells of similar metabolic types was described as forming "metabolic"

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

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

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

In studies conducted in sheep, it was found that a single gene, the callipyge gene (Cockett, Jackson et al . 1994) was associated with an increased frequency and size of FG fibres and a corresponding decrease in

the proportion of SO and FOG fibres (Carpenter, Rice et al. 1996) . The studies demonstrated that an increase in the proportion of FG fibres were associated with increased toughness of the meat (Koohmaraie, Shackelford et al . 1995).

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

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

The invention is founded upon the following novel observations:

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

2. That the number of SO fibres per cluster is

increased in the Duroc pig relative to the "White" pig;

3. That m calpain is preferentially localised in the SO fibres of pigs. Therefore pigs with more SO fibres (eg Duroc) have more m calpain in the muscle as a whole. Thus the amount of m calpain is increased per unit muscle in the Duroc pig relative to the "White" pig;

4. That the amount of μ calpain per fibre is increased in the Duroc pig relative to the "White" pig;

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

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

In the pig populations tested we have found that typically slaughter weight pigs (eg 50-100kg carcase weight) of the Duroc breed have an SO fibre frequency (mean + standard deviation) of 15.6% (±2.1%), whereas non-Duroc pigs have a much lower SO fibre frequency, generally 10.8% (±3.2%) (see Table 5, Examples) . Our observations have led us to conclude that such animals having a muscle fibre composition with an SO fibre frequency of approximately 13% or higher can be

classified as having a muscle fibre composition of the type characteristic of the Duroc pig. Consequently, it is now possible to analyse a muscle of any particular animal on the basis of percentage frequency of SO fibres in order to determine whether or not that animal has a muscle fibre composition characteristic of the Duroc pig. Such measurements may be made directly (eg by counting the number of SO fibres in a sample or by determining the proportional area of SO fibres in a sample) or indirectly by measuring the percentage frequency or proportional area of other fibre types, eg FG and FOG fibres.

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

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

Table 2

Total number of Number of SO fibres/cluster fibres/cluster

0% Duroc 3.98 ±0.8 2.65 ± 0.5

100% Duroc 7.60 ±0.1** 3.66 ±0.3**

** P < 0.01 compared with 0% Duroc

0% Duroc = Large White, Landrace or Large White/Landrace crosses.

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

It is well documented that post mortem storage of animal carcases at below ambient temperature, but above freezing, results in an improvement in meat tenderness. This increase in tenderness is due to the enzymatic breakdown of myofibrillar proteins and there is evidence that calpains are responsible for 90% of the tenderisation that occurs during post mortem storage (Taylor et al 1994) . Calpains are intracellular, calcium activated/dependent thiol proteases present to some extent in most body tissues. However, their exact role in normal physiological conditions is still undefined. Several isoforms of calpain are known to occur in various body tissues of birds and animals. Two isoenzymes, μ calpain and m calpain, with different calcium requirements were originally isolated (Huston and Krebs 1968, Mellgren 1980). More recently tissue specific calpains have been isolated from skeletal muscle and stomach (Sorimachi et al 1989, Sorimachi et al 1993) . It is the actions of μ calpain and m calpain that are thought to be involved in post mortem tenderisation of meat. In animal carcasses μ calpain is most active during the first 15 hours post slaughter whereafter its activity declines rapidly whilst the activity of m calpain is much more persistent. The activity of both μ and m isoforms of calpain is regulated by a natural inhibitor, calpastatin, which is also ubiquitously distributed in all body tissues.

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

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

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

Table 3

Total number of Number of SO fibres/cluster fibres/cluster

0% Duroc 3.98 ±0.8 2.65 ± 0.5

50% Duroc 5.15 ±1.1* 3.32 ±0.9*

100% Duroc 7.60 ±0.1** ++ 3.66 ±0.3**

FI 5.16 ±0.5* 3.92 ± 0.5*

* P < 0.05

** P < 0.01 compared with 0% Duroc

++ P < 0.01 compared with 50% Duroc or FI

0% Duroc = Large White, Landrace or Large White/Landrace Crosses

50% Duroc = Duroc x (Large White x Landrace) FI = Duroc x Large White.

Furthermore we have shown that the SO fibre frequency in animals containing only 25% 'Duroc' genes shows a variation among individuals consistent with a single gene controlling SO frequency. This observation is consistent with the inheritance of a dominant gene being inherited in a normal Mendelian manner. The results for fibre frequency in our studies can be seen in Table 5, Examples. The SO frequency seen in our 'White' pigs is similar to that found in Swedish Yorkshire ('Large White') at 8% (Karlsson, Essen-Gustavsson et al . 1994).

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

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

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

a. obtaining a tissue sample from said animal and subjecting said sample to genetic analysis to determine whether 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:

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 μ calpain present per unit muscle.

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

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

a) obtaining a tissue sample from said animal,

extracting genetic information therefrom and analysing said genetic information to determine whether the genotype of said animal includes said allele(s); and/or

b) obtaining a muscle sample from said animal, and analysing said sample by histochemical or immunochemical techniques to determine whether said sample exhibits phenotypic traits indicative of said allele(s) (for example the phenotypic traits set out above under paragraphs i to vi).

The results of the assay will indicate whether or not the animal tested has genetic information from the Duroc breed, specifically at least one copy of the (dominant) allele(s) determining Duroc muscle fibre type. This information can be used in selecting animals for breeding and/or for slaughter and use for provision of meat.

With reference to the genetic analysis referred to above (see paragraphs a) a number of different techniques may be used to give a genetic "fingerprint" of the test animal. This "fingerprint" can then be compared to known standards (eg typical "Duroc" and "non-Duroc" standards) . Whilst the present invention is not limited to any particular technique, mention may be made of techniques such as RAPD, AFLP, RFLP, SSCP and other mini-satellite or micro-satellite techniques or hybridisation techniques. Sequencing of genetic information can also be useful, when the test sequence can be compared to a known standard sequence. Thus, using techniques such as RFLP (restriction fragment length polymorphism), AFLP (amplified fragment length polymorphism), and RAPD (random amplification of

polymorphic DNA) it would be possible to identify suitable Duroc genotype marker or markers that constitute a genetic fingerprint and which associate with the improved eating quality of pork derived from this breed of pig.

RFLPs are detected in restriction enzyme digested genomic DNA which has been size fractioned by electrophoresis, Southern blotted to a membrane support then hybridised to a labelled probe. The probe used in RFLP analysis is usually a single gene locus of interest to the researcher. PCR (polymerase chain reaction) based techniques can also be used to detect single locus RFLP, eliminating the need for Southern blotting and hybridisation. In this instance the amplified DNA is digested with a restriction endonuclease prior to gel electrophoresis. Polymorphisms are evident as differences in the resulting DNA fragment sizes.

An alternative technique is AFLP. AFLP is based on the PCR amplification of genomic DNA after digestion with restriction enzyme(s) and ligation of oligonucleotide adapters. The technique is facilitated by the use of PCR primers that span a region of the oligonucleotide adapters and extend into the DNA restriction fragment. Only those fragments of DNA in which PCR primer extensions have a perfect match are amplified and the resultant mixture of PCR products can be analysed by electrophoresis. This technique could be used to identify a marker of the Duroc factor associated with the improved eating quality of pork derived from this breed of pig. A description of AFLP is given by Vos et al, 1995.

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

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

With reference to histochemical and immunochemical analysis referred to above (see paragraphs b) any technique able to analyse the number of SO fibres per cluster or the frequency of SO fibres and/or the amount of m calpain per unit muscle and/or the amount of μ calpain per unit muscle may be used.

For example, where the number of SO fibres per cluster is to be determined the muscle sample may be prepared and stained so that the muscle fibres can be viewed (for example using a microscope) and counted. Details of a suitable protocol are given in Example 1. Similar techniques can be used for determining the frequency of the SO fibres in the sample.

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

mean of 2 SO fibres per cluster or more can be characterised as having a muscle fibre composition characteristic of the Duroc pig.

Immunochemical techniques may be useful to aid visualisation of the SO fibres, by exposing the muscle sample to labelled antibodies which bind preferentially to SO fibres, eg MHCs (myosin heavy chain slow isoform) of Novocastra Laboratories Limited UK.

With regard to determining the amount of m calpain or μ calpain immunochemical techniques may be used, for example an ELISA assay. Anti-m calpain, anti-μ calpain, (anti-calpastatin) and anti-myosin (heavy chain slow isoform) antibodies are available commercially. Examples include MAB3082 (anti-μ calpain antibody), MAB3084 (anti-calpastatin antibody), AB1625 (anti-m calpain antibody), all of Chemicon International, Inc (Temecula, CA 92590, USA) . These antibodies (and other similar antibodies) can be used as described in the manufacturer's instructions or according to known protocols. Reference is also made to the description of immunocytochemical locations of the calpain proteolytic system in porcine muscle described in Example 4.

In a further aspect, the present invention provides 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 as described above and segregating these animals found to have said allele or said composition from the other animals.

The method may conducted in vitro or in vivo using a

sample from a living animal or post mortem following the death of the animal tested.

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

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

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

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

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

The increased proportion of SO fibres may lead to

improved meat quality, less pale and more tender muscle.

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

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

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

According to another aspect of the present invention there is provided a method of enhancing tenderness and/or colour of the muscle of a mammalian animal, said method comprising influencing said animal or its parents to increase the proportion of SO fibres present in the muscles.

The present invention also provides a method of enhancing the eating quality of musculature in a mammalian animal, said method comprising enhancing the proportion of SO fibres in the skeletal muscle of said animal.

In a further aspect the present invention provides meat from a mammalian (non-human) animal, said meat having improved meat quality wherein said animal has been selected for or influenced to increase the proportion

of SO fibres present in the muscle which forms said meat.

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

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

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

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

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

i. analysing genes of the calpain/calpastatin system and comparing the results to a known standard; and/or

ii. analysing the m calpain activity (eg in muscle and/or in SO fibres); and/or

iii. analysing the μ calpain activity.

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

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

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

Figure 1A ATPase from 8kg Duroc pig showing clusters of SO fibres. Figure IB ATPase from 8kg Large White pig showing clusters of SO fibres.

Figure 2A ATPase from slaughter weight Duroc pig showing clusters of SO fibres Figure 2B ATPase from slaughter weight Large White pig showing clusters of SO fibres.

Figure 3A Section reacted to demonstrate the presence of m- calpain in 8kg Duroc pig. The clustered fibres are SO type. Figure 3B

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

Figure 4A Section reacted to demonstrate the presence of μ- calpain in 8kg Duroc pig. The overall brightness is compared with that in Fig 4B. Figure 4B Section reacted to demonstrate the presence of μ- calpain in 8kg Large White pig . The overall brightness is less than that in Fig 4A.

Figure 5A Section reacted to demonstrate the presence of myosin heavy chain slow isoform in the clusters in 8kg Duroc pig. Figure 5B Section reacted to demonstrate the presence of myosin heavy chain slow isoform in the clusters in 8kg Large White pig.

All figures are transverse sections through longissimus dorsi muscle of pigs.

Example 1

Variation in fibre type associated with increasing Duroc genotype

Background and Introduction

Evidence from various trials (for example MLC, 1992) indicates that Duroc genes enhance the eating quality of pork, in particular tenderness. Whilst Duroc cross pigs tend to be fatter with higher levels of intramuscular fat, it is not clear whether this is the cause of the enhanced eating quality. Durocs also have redder muscle with a higher concentration of the muscle pigment, haem. This indicates a higher oxidative capacity and therefore, fibre types were expected to differ from "WhiteW genotypes. It is possible that differences in fibre type may be related to eating quality differences between Duroc crosses and "White" pigs.

Materials and Methods

Animals

Samples from 0, 25% and 50% Duroc animals were sourced from an MLC University of Newcastle-upon-Tyne trial designed to examine the influence of lean tissue growth rate on the eating quality of pork. These were taken from pigs fed ad libi tum from weaning to slaughter. 100% Duroc animals were sourced separately from a commercial abattoir and no control over rearing or slaughter was exercised for these.

Slaughter

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

Carcase Handling and Chilling

Carcases were chilled according to normal plant practice. Carcases were transported to MLC at Winterhill following overnight chilling. Loin samples were frozen after a five day ageing period.

Sample Transport

The samples were transported from Milton Keynes by air in insulated boxes and maintained in the frozen state. The chops were then stored at -70°C.

Histochemistry Blocking and sectioning chops

Before the initiation of this study a novel method was developed which allowed the retrospective examination of blast frozen meat. This method is simple, and relies on slowly thawing the chops overnight at +4°C. Subsequently, approximately 1 cm ² blocks were cut from the centre of the longissimus dorsi muscle. Care was taken to ensure that the same area was sampled from each of the chops. These cubes of muscle were orientated for transverse sectioning, mounted on a piece of cork with optimal cutting temperature compound (OCT) , covered with more OCT and with unperfumed talcum powder and frozen in liquid nitrogen with constant

agitation. Twelve blocks were taken from each chop and once frozen, were stored in aluminium tins submerged in liquid nitrogen. Throughout the period of the study the blocks were maintained in the liquid phase of the nitrogen dewar to limit any freeze drying. The tins were removed from the liquid nitrogen storage and placed in the cryostat at -20°C 2 hours before sectioning. Serial transverse sections were cut at lOμm using a Frigocut 2800 cryostat with motor driven cutting stroke to reduce variation in section thickness.

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

Fibre typing

The characterisation of fibre typing adopted in this study is based upon the reaction of individual fibres to a minimum of three stains. The stains used were chosen to demonstrate the activities of Ca ^z+ activated myofibrillar adenosine triphosphatase (ATPase) , nicotinamide adenine dinucleotide diaphorase (NADH) , and ct-glycerophosphate dehydrogenase (GPOX) , which then allowed the characterisation of the fibres based on their contractile and metabolic activities as follows and as illustrated in Table 4; ATPase - contractile activity (fast or slow twitch); NADH - oxidative activity; GPOX - glycolytic activity.

Table 4 The histochemical basis of characterisation of muscle fibre types in pig meat.

FIBRE TYPE STAIN

ATPASE NADH GPOX

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

FG +++ + +++

SO + +++ +

Quantification of fibre type and size,

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

The ATPase stained sections were examined under a light microscope fitted with a Sony video camera, the output of which was applied to the image handling software of the Torch computer. The use of the ATPase stain generates an image in which three fibre types can be distinguished based on their grey levels. Fibre type was confirmed through examination of printed images of the NADH and GPOX stains to give information on the metabolic character of each fibre. The three fibre types were analysed separately, and thresholding was altered to detect all fibres of the same type. Where adjacent fibres were thresholded and detected as a single unit, manual editing operations were undertaken to separate the fibres through the use of a

superimposed 'live' camera image to visualise the sarcolemmal membranes accurately. The data for size, frequency and percentage area was computed for each animal. Approximately 1600 fibres were analysed for each pig.

Results

The results were clear and showed that possession of 50% or more Duroc genes was associated with a significant increase in both the frequency and percentage area of SO fibres, and a significant reduction in the frequency of FG fibres; there was also a tendency towards a reduction in FG percentage area (Table 5). In addition, pigs possessing 25% Duroc genes showed an increased SO frequency over 0% pigs, with a mean frequency value lying half way between the value for 50% and 0% pigs (Table 5) . Close inspection of the individual values showed that half the animals had SO frequencies similar to those seen the 50 and 100% Duroc animals (mean frequency 14.8 (±2.2 (sd), n= 6), while the remaining animals had SO frequencies which resembled those seen in the 0% pigs (mean frequency 10.9 (±1.3 (sd), n=6)).

Sample results are shown in Figs 1A and IB.

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

1 Table 5. The fibre type distributions in muscle

2 from pigs containing different proportions of Duroc

3 genes .

FOG FG so

AREA ZFREQ ZAREA AREA ZFREQ ZAREA AREA ZFREQ ZAREA

OZ 2079 28.5 26.9 2352 60.6 64.5 1740 10.8 8.5

Duroc (401) (2.1) (2.8) (478) (3.6) (3.8) (243) (3.2) (2.2)

25Z 2137 32.2 31.5 2338 54.8 58.4 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 56.2 61.6 1675 15.6 10.3

Duroc (507) (1.8) (2.8) (438) (3.6) (3.5) (195) (3.6) (1.9)

100Z 2006 32.8 27.4 2867 51.5 61.7 1628 15.6 10.8

Duroc (440) (1.8) (1.8) (508) (3.2) (1.7) (103) (2.1) (1.4)

4 Discussion

5

6 Pigs containing Duroc genes have more SO fibres . The

7 results show clearly that animals with 50% or more

8 Duroc genotype have a significantly increased number of

9 SO fibres and decreased numbers of FG fibres compared 0 to 0% genotypes. The observations of the SO frequencies 1 in the 25% Duroc suggests independent segregation of 2 genes and supports the concept that the Duroc factor is 3 a heritable trait inherited in a normal Mendelian 4 manner. 5 6 Consequently, the present data provide a basis that 7 there is/are inheritable muscle specific Duroc gene(s) 8 which confer properties beneficial to the eating 9 quality of Duroc pig meat.

Example 2

Antibodies raised against μ calpain, m calpain and calpastatin are now commercially available (MAB3082, AB1625 and MAB3084 respectively, all of Chemicon International Inc, Temecula, CA USA) . Data from the supplier of these antibodies show that these antibodies bind to the calpain proteins of a number of different species and there was no reason to believe that they would not bind to the porcine calpain and calpastatin epitopes as at the amino acid sequence level the polypeptide products of the calpain and calpastatin genes are highly conserved from species to species. Using standard immunocytochemical techniques, transverse sections of Longissimus dorsi muscle from Large White new-born and 10kg pigs were prepared and developed using the panel of antibodies described below. For Duroc animals transverse sections of Longissimus dorsi muscles from new-born and 10kg pigs were also prepared as were transverse sections of semimembranous and biceps femorus muscles from 10kg animals. In addition, longitudinal sections were cut from a block of semimembranosus muscle. The antibody to m calpain was a rabbit polyclonal which was developed and visualised with an anti rabbit FITC conjugate. Both anti calpastatin and anti μ calpain were murine monoclonal antibodies that were developed using an appropriate anti mouse conjugate. For some of these samples serial sections were cut and stained using both standard histochemical and immuno- cytochemical techniques. Pooled information from these serial sections were then subjected to analysis and interpretation. Sample results are shown in Figs 3A, 3B, 4A and 4B. Alternative staining using antibodies to myosin heavy chain slow isoform (eg NCL-MHCs of

Novocastra Laboratories Ltd, Newcastle, UK) are shown in Figs 5A and 5B.

Example 3

RAPD Assay

120 primers were purchased from Genosys Biotechnologies Inc. One hundred primers had a G+C content of 50, 60, 70 and 80% and had the sequences as follows:

50% G+C content GTGCAATGAG AAATCGGAGC

CAATGCGTCT GTCCATAGCA AGGATACGTG TACATCAGCG TCCCTTTAGC CATAGCGGTT CGGATAACGT CTACTAGGGT AGGTTCTAGC AGTGAATGCG TCCGACGTAT ACGATTCCTG GGAAGACAAC TTTACGGTGG

AGAAGCGATG ATGGTGTAGC CCATTTACGC AATCACACCC

60% G+C content

CGCAGTACTC GAGTCTGTCG GAGTGTCTGC

GTCCTACTCG CGAACTCGTC CACATAGCGC

CTACTCAGGC GGAACCCATG CGAAGCGATC

GTCCTTAGCG CGCTATCTGC CCCTCATCAC

GTCCTCAACG GCAGTATGCG CCTGTTAGCC

CTACTACCGC GGCGATATGG GCAGCTCATG

GAGTCACTCG CCCTTACTGG CGCTTGCTAG

GTCCTCAGTG GTCGACAACG GAACCTACGG

CGTCGTTACC CCTGATGACC CTAGCTGAGC

GCAGACTGAG GACCGACACG GAGCAGGCTG

70% G+C content CATCCCGAAC TCCCTGTGCC CAGGGTCGAC GCTCTCCGTG ACGGTGCCTG GAGACGTCCC CGCATTCCGC GTATGCCGCG GAGATCCGCG GCACCGAACG GGACTCCACG CCGGCGTATC ATCTCCCGGG AGCCTGACGC CTGTACCCCC GCTCTGGCAG TGCAGCACCG CGCACTCGTC CAGACACGGC CTGTCCGGTC

80% G+C content GCACCCGACG GCAGCAGCCG ACGCGCCAGG CGCCCAAGCC CGACGCGTGC ACTCGGCCCC CCATGGCGCC ACCCGTCCCC GGCCCCATCG CGCCCGATCC GCAGCTCCGG CGCGAGGTGC ACCCCAGCCG CGAGACGGGC CGATCCGCGC GCACGGAGGG ACCGCCTCCC CCCGACTGCC GCACGCCGGA GCAGGTCGCG GGCAAGCGGG CGATGAGCCC CGCCCTCAGC CGCACCGCAC CGCTGTTACC GCACGGTGGG ACGGCGGCTC CTAGGTCTGC CGCCCTGGTC CGCGCTACGC

A further twenty primers were purchased into which a restriction endonuclease site had been incorporated. This facilitates cloning of any amplified products after digestion with the appropriate restriction endonuclease into a suitably cut cloning vector.

CGGGATCCGC BamHl GGCTGCAGCG Pstl GCGGTACCCG Kpnl CCCTCGAGGC Xhol CCAGATCTGC Bgll

CCAAGCTTGC Hindlll

GCATCGATCG Clal

GGCTGACGCG Sail

GCGTTAACGC EcoRl

CGAATTCGGC EcoRl

CGGATCCCGC EcoRl

CGCGATGCGC Sphl

GGGATCCGCC BamHl

GCCAAGCTTC Hindlll

CGATCGATGC Clal

GCGAGCTCTG Sacl

GGAAGCTTCG Hindlll

CGTCTAGAGC Xbal

CCCTGCAGGC Pstl

CGCGAGCTCG Sacl

RAPD Assay Development

Oligonucleotide primers were dissolved in sterile distilled water at a concentration of 25 pico moles per microlitre and used to optimise the PCR based RAPD reaction. To make the RAPD test as sensitive and reproducible as possible nine different buffer regimes were assessed with a panel of the primers chosen at random. In these buffers the pH, salt concentration, and buffer component were varied. Reactions performed in a 50/tl volume containing 20mM Tris HCI pH 8.75, lOmM KC1, lOmM (NH ₃ ) ₂ SO _ή , 0.75mM MgCl ₂ , 0.75mM MgSO _ή , 0.1%Triton, 0.01%Tween, 0.001%gelatin, 200 mM dNTPS, (IX PCR assay buffer) using 25pmoles of appropriate oligonucleotide primer, 200ng of template DNA and 2.5 units of Taq DNA polymerase were found to be consistently better than any of the others tried in the assay. Amplifications were performed using an APPLIGENE/ONCOR crocodile III microprocessor controlled

incubation system programmed for 25 or 30 cycles of 94°C for 45 sec, 37°C for 3 min, 72°C for 3 min. The products of these reactions were analysed by electrophoresis in gels containing 2.5% agarose in IX TAE buffer. Using this technique 120 primers of known arbitrary sequence have been assessed and 12 which show differences when DNA templates isolated from pure bred Duroc or Large White pigs were examined.

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

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

CGGAATTCCG AGGGGAGCCG GGCCTTCAGG CCCGACTGCC

CGCCACGAGC CGCCCGATCC GCAGCTCATG GCACCCGACG

CCTGTTAGCC TACACTAGCG GAACCTACGG CAATGCGTCT

GCGGTACCCG CGGAATTCCG GGAAGCTTCG GGTCGACGCG

GCCCCATGCG.

Example 4

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

Materials and methods

Longissimus dorsi muscles were removed rapidly from 10kg pigs, orientated for either longitudinal or transverse sectioning and frozen in liquid nitrogen. Standard immunocytochemical techniques were employed using commercially available antibodies raised against μ calpain, m calpain and calpastatin (Chemicon International Inc, Temecula, CA 92590) . Localization of μ and m calpain was carried out in sections serial to those used for localization of calpastatin.

Results

Calpastatin was localized around, but not in, the nucleus. In addition to this perinuclear localization, in transverse sections a granular dispersion of stained material scattered throughout the cytoplasm was

observed but with no fibre type specific distribution. Longitudinal sections showed that calpastatin immunoreactivity was also present down the inner surface of the sarcolemma, and in association with some component of myofibril ultrastructure. There was no fibre specific distribution of μ calpain which was localized around the sarcolemma with a variable level of cytoplasmic staining. Examination of longitudinal sections however, showed that μ calpain was localized in a regular banded pattern, indicating a highly ordered localization of this protease in muscle cytoplasm, and suggesting some association with contractile proteins in the A or I bands. In contrast to both calpastatin and μ calpain, m calpain localization did appear to be associated with specific fibre types. Immunoreactivity for m calpain was primarily associated with slow twitch fibres; much less intense staining being noted in all other fibre types.

Discussion

Mellgren 1991, proposed a role for the calpains in the turnover of nuclear proteins, hence a perinuclear site for calpastatin might be predicted. However, this apparent compartmentation raises some questions as to its role in the inhibition of the calpains at the sarcolemma and in the cytoplasm. The presence of μ calpain at the sarcolemma is consistent with its role in the regulation of the activity of enzymes and cytoskeletal protein degradation. The fibre type localization of m calpain in slow fibres may relate to the higher rate of protein turnover in these fibres types (Garlick et al 1989) . Overall, the results suggest that the calpains and calpastatin have an ordered localisation in porcine skeletal muscle

suggesting a specialised role for each of these proteins in skeletal muscle.

Example 5 Breeding Program

Method Large White and Duroc pedigree pigs were used in a breeding programme to produce an FI population which were 50% Duroc and 50% Large White. Specifically two crosses were set up. In the first cross Duroc boars were mated to Large White sows and in the other Duroc sows were served by Large White boars. The resulting FI populations showed the Duroc muscle phenotype indicating a dominant Duroc gene or gene cluster. Sows from the FI population were then crossed with Large White boars to generate an F2 backcross population. Classical Mendelian genetics suggests that in such crosses there should be a 50% 50% segregation of any given genetic trait. In this FI backcross population there is such a segregation of muscle phenotype into Duroc and Large White types (SO fibres per cluster).

Results The number of SO fibres per cluster (mean + SD) in the F2 population (8kg live weight) fell into two distinct groups as follows (see Figs 5A and 5B wherein the SO fibres are stained using the myosin heavy chain slow isoform antibody NCL-MHCs):

Group A (n=3; non-Duroc phenotype): 1.6 ± 0.1 Group B (n=5; Duroc phenotype): 2.5 + 0.2

At least 12 samples were taken per animal and the SO cluster sizes pooled.

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