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  • G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
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  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/156—Polymorphic or mutational markers

Definitions

• the present invention relates to animals, especially pigs, having improved meat quality.
• 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.
• 'Landrace' breed 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).
• 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.
• 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.
• PSE Pale Soft Exudative meat
• 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.
• 'Duroc' 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.
• 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) .
• 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.
• 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 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.
• the present invention is concerned with determining, by immunochemical, histochemical or genetic analysis, whether a particular individual animal has desirable muscle characteristics.
• 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;
• 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.
• 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.
• 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.
• Duroc Large White, Landrace or Large White/Landrace Crosses 50%
• Duroc Duroc x (Large White x Landrace)
• FI Duroc x Large White.
• 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.
• 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
• 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:
• 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.
• 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.
• RFLP restriction fragment length polymorphism
• AFLP amplified fragment length polymorphism
• RAPD random amplification of polymorphic DNA
• 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.
• the amplified DNA is digested with a restriction endonuclease prior to gel electrophoresis. Polymorphisms are evident as differences in the resulting DNA fragment sizes.
• 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.
• 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 ⁇ calpain per unit muscle may be used.
• 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.
• 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.
• MHCs myosin heavy chain slow isoform
• m calpain or ⁇ calpain immunochemical techniques may be used, for example an ELISA assay.
• Anti-m calpain, anti- ⁇ calpain, ( anti-calpastatin) and anti-myosin (heavy chain slow isoform) antibodies are available commercially. Examples include MAB3082 (anti- ⁇ 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.
• 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.
• 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.
• the animal is a pig, although other mammalian species are also included.
• the present invention provides a mammalian animal having increased proportions of SO fibres in its musculature.
• the animal will be the progeny of animal(s) selected for breeding by the method given above.
• the increased proportion of SO fibres may lead to improved meat quality, less pale and more tender muscle.
• 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.
• genes and/or controlling sequences may be involved, especially the genes controlling the calpain/calpastatin system.
• a method of enhancing tenderness and/or colour of the muscle of a mammalian animal 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.
• 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 .
• 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.
• 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.
• the means of detecting a higher frequency of SO fibres could be any of the following:
• the present invention provides the use of anti-m calpain antibodies or anti- ⁇ 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 .
• 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 ⁇ - calpain in 8kg Duroc pig. The overall brightness is compared with that in Fig 4B.
• Figure 4B Section reacted to demonstrate the presence of ⁇ - 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.
• 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.
• Duroc 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.
• 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.
• 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.
• 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 lO ⁇ m 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.
• 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.
• 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.
• Antibodies raised against ⁇ 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.
• 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.
• 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.
• 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 ⁇ calpain were murine monoclonal antibodies that were developed using an appropriate anti mouse conjugate.
• 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:
• 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.
• 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.
• 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.
• 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 ⁇ calpain which was localized around the sarcolemma with a variable level of cytoplasmic staining.
• Ruusunen M. (1993). The fibre-type composition and capillary density in M L. Dorsi of different pig cross-breed. Pork Quality: Genetic and metabolic factors E. Puolanne, D. I. Demeyer, with, M. Ruusunen and S. Ellis. Wallingford, CAB International: 301.

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