Note: Descriptions are shown in the official language in which they were submitted.
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1
1 ASSAY FOR DUROC MUSCLE FIBRE TYPE
2
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.
11
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
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1 common synonym is 'Yorkshire'.
2
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.
8
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).
16
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.
33
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
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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.
8
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.
17
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.
24
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.
32
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
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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.
34
Within Europe, especially the United Kingdom, the
36 'Duroc' is characterised as being of reasonable growth
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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).
6
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)).
14
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.
31
' 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
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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.
7
8Table 1
9 DUROC CONTENT
0% 50%
1 Daily live weight gain (g) 806 803
1
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
8
19Pork flavour*
2 In lean 3.88 3.96
0
2 In fat 3.87 4.06
1
2 Pork odour in fat* 3.58 3.73
2
23
24
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.
29
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
9
10 To date there is no clear explanation of what causes
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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.
7
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.
14
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"
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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.
6
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.
19
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.
31
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
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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).
6
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.
21
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.
26
27 The invention is founded upon the following novel
28 observations:
29
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;
34
2. That the number of SO fibres per cluster is
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1 increased in the Duroc pig relative to the "White"
2 Pi9
3
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;
14
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.
19
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.
26
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
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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.
13
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.
24
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
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1 0~ Duroc - Large White, Landrace or Large
2 White/Landrace crosses.
3
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.
8
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.
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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.
6
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.
12
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).
22
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*
0
27 100o Duroc 7.60 t0.1** ++ 3.66 .3**
0
28 F1 5.16 0.5* 3.92 0.5*
29
*
P
<
0.05
31 compared with Oo Duroc
**
P
<
0.01
32 compared with 50% Duroc or
++ F1
P
<
0.01
33
34 ge Landrace or Large
Oa White,
Duroc
=
Lar
Crosses
White/Landrace
36
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1 50~ Duroc - Duroc x (Large White x Landrace)
2
3 F1 - Duroc x Large White.
4
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).
17
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.
27
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.
33
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:
37
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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
6
7 b. obtaining a muscle sample from said animal and
8 determining by histochemical or immunochemical
9 analysis:
10
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.
23
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.
29
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:
34
a) obtaining a tissue sample from said animal,
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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).
12
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
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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.
6
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.
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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.
4
Genetic analysis is preferred for both determination of
6 meat quality and in respect of devising breeding
7 programs.
8
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.
23
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
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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.
4
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.
26
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.
34
The method may conducted in vitro or in vivo using a
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1 sample from a living animal or post mortem following
2 the death of the animal tested.
3
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.
12
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.
19
20 In one preferred embodiment of the invention the animal
21 is a pig, although other mammalian species are also
22 included.
23
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.
29
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.
34
The increased proportion of SO fibres may lead to
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1 improved meat quality, less pale and more tender
2 muscle.
3
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.
8
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.
14
Alternatively, other genes and/or controlling sequences
16 may be involved, especially the genes controlling the
17 calpain/calpastatin system.
18
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.
31
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
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1 of SO fibres present in the muscle which forms said
2 meat.
3
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.
7
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.
14
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.
19
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.
23
24 For example, the means of detecting a higher frequency
of SO fibres could be any of the following:
26
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
33
34 iii. analysing the ~ calpain activity.
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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).
6
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.
13
14 The invention will now be described with reference to
the following examples and figures in which:
16
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.
23
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
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1 Section reacted to demonstrate the presenceof m-
2 calpain in 8kg Large White pig. The clustered
fibres
3 are of SO type.
4
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.
13
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.
22
23 All figures are transverse sect ions throughlongissimus
24 dorsi muscle of pigs.
26
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1 Example 1
2
3 Variation in fibre type associated with increasing
4 Duroc genotype
5
6 Background and Introduction
7
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.
21
22 Materials and Methods
23
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.
34
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1 Slaughter
2
3 Pigs were slaughtered on reaching 85kg liveweight.
4 Transport, lairage and slaughter was carried out in
accordance with MLC's Blueprint for pork.
6
7 Carcase Handling and Chilling
8
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.
14
Sample Transport
16
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
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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.
12
13 The sections were allowed to air dry at ambient
14 temperature for 2 hours and then frozen overnight for
staining the following day.
16
17 Fibre typing
18
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.
31
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1
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,
4
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.
13
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
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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.
6
7 Results
8
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.
27
28 The experiment was repeated using 8kg pigs (live
29 weight); sample results are shown in Figs 2A and 2B.
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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
5
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.
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1 Examele 2
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
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1 Novocastra Laboratories Ltd, Newcastle, UK) are shown
2 in Figs 5A and 5B.
3
4 ExamQle 3
6 RAPD Assay
7
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
11
12 50~ G+C con tent
13 GTGCAATGAG AAATCGGAGC
14 CAATGCGTCT GTCCATAGCA
AGGATACGTG TACATCAGCG
16 TCCCTTTAGC CATAGCGGTT
17 CGGATAACGT CTACTAGGGT
18 AGGTTCTAGC AGTGAATGCG
19 TCCGACGTAT ACGATTCCTG
GGAAGACAAC TTTACGGTGG
21 AGAAGCGATG ATGGTGTAGC
22 CCATTTACGC AATCACACCC
23
24 60~ G+C content
CGCAGTACTC GAGTCTGTCG GAGTGTCTGC
26 GTCCTACTCG CGAACTCGTC CACATAGCGC
27 CTACTCAGGC GGAACCCATG CGAAGCGATC
28 GTCCTTAGCG CGCTATCTGC CCCTCATCAC
29 GTCCTCAACG GCAGTATGCG CCTGTTAGCC
CTACTACCGC GGCGATATGG GCAGCTCATG
31 GAGTCACTCG CCCTTACTGG CGCTTGCTAG
32 GTCCTCAGTG GTCGACAACG GAACCTACGG
33 CGTCGTTACC CCTGATGACC CTAGCTGAGC
34 GCAGACTGAG GACCGACACG GAGCAGGCTG
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1 70~ G+C content
2 CATCCCGAAC TCCCTGTGCC
3 CAGGGTCGAC GCTCTCCGTG
4 ACGGTGCCTG GAGACGTCCC
CGCATTCCGC GTATGCCGCG
6 GAGATCCGCG GCACCGAACG
7 GGACTCCACG CCGGCGTATC
8 ATCTCCCGGG AGCCTGACGC
9 CTGTACCCCC GCTCTGGCAG
TGCAGCACCG CGCACTCGTC
11 CAGACACGGC CTGTCCGGTC
12
13 80~ G+C con tent
14 GCACCCGACG GCAGCAGCCG ACGCGCCAGG
CGCCCAAGCC CGACGCGTGC ACTCGGCCCC
16 CCATGGCGCC ACCCGTCCCC GGCCCCATCG
17 CGCCCGATCC GCAGCTCCGG CGCGAGGTGC
18 ACCCCAGCCG CGAGACGGGC CGATCCGCGC
19 GCACGGAGGG ACCGCCTCCC CCCGACTGCC
GCACGCCGGA GCAGGTCGCG GGCAAGCGGG
21 CGATGAGCCC CGCCCTCAGC CGCACCGCAC
22 CGCTGTTACC GCACGGTGGG ACGGCGGCTC
23 CTAGGTCTGC CGCCCTGGTC CGCGCTACGC
24
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
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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
16
17 RAPD Assay Development
18
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
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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.
9
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.
23
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.
2B
29 CGGAATTCCG AGGGGAGCCG GGCCTTCAGG CCCGACTGCC
31 CGCCACGAGC CGCCCGATCC GCAGCTCATG GCACCCGACG
32
33 CCTGTTAGCC TACACTAGCG GAACCTACGG CAATGCGTCT
34
GCGGTACCCG CGGAATTCCG GGAAGCTTCG GGTCGACGCG
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1 GCCCCATGCG.
2
3 Example 4
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).
17
18 Materials and methods
19
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.
29
Results
31
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
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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.
19
Discussion
21
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
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1 suggesting a specialised role for each of these
2 proteins in skeletal muscle.
3
4 Example 5
Breeding Program
6
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).
23
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
33
34 At least 12 samples were taken per animal and the SO
cluster sizes pooled.
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1 References
2 Barnoy et al. (1996) Biochem Biophys Res Com 220: 933-
3 938.
4
Barton-Gade, P. (1989). The effect of breed on meat
6 auality characteristics in pigs. 35th International
7 congress of meat science and technology., Kulmbach.
8
9 Bejerholm A. C. (1984). Experience in taste testing
fork at the Danish Meat Research Institute Proceedings
11 of the 30th European Meeting of Meat Research workers.
12
13 Brustis et al. (1994) Eur. J. Cell Biol. 64: 320-327.
14
Calkins, D.R., T. R. Dutson, et al. {1981).
16 "Relationship of fibre type composition to marbling and
17 tenderness of bovine muscle." Journal of Food Science
18 46: 708.
19
Carpenter C. E., O. D. Rice, et al. (1996). "Histology
21 and composition of muscle from normal and callipyge
22 lambs." Journal of Animal Science 74: 388-393.
23
24 Glutton-Brock, J. (1981). Domesticated animals from
early times. London, Heinemann/British Museum.
26
27 Cockett, N. E., S P Jackson, et al (1994). "Chromosomal
28 localisation of the callipygegene in sheep (Ovis Aries)
29 using bovine DNA markers. "Pro. Nat. Acad. Sci. USA 91:
3019-3023.
31
32 Elamrani et al. (1995) Biology of the cell 85: 177-183.
33
34 Gandemer, G. and C. Legault (1990). Contribution du
Genotype et du system d'elevage a la
CA 02267347 1999-03-30
WO 98/15837 PCT/GB97/02741
1 production dun porch labelisable. Porc Macrazine. 222:
2 31-37.
3
4 Garlick et al. (1989) Am. J. Physiol. 2S7: E828-832.
5
6 Handel, S. E. and N. C. Stickland (1987). "The growth
7 and differentiation of porcine skeletal muscle
8 fibretypes and the influence of birthweight.~ Journal
9 of Anatomv 152. 107-119.
11 Hong et al. (1995) Biochem Biophys Acta 1267: 45-54.
12
13 Huston and Krebs (1968) Biochemistry 7: 2116.
14
Iwamoto, H., H. Awaida, et al. (1983). "Breed
16 differences in the distribution of muscle fibre types
17 in the fasciculi of porcine psoas major muscle."
18 Japanese Journal of Zootechnical Science 54(6):
I9 392-400.
21 Jeffreys, Wilson and Thein (1985) Nature 316: 76.
22
23 Karlsson A. B Essen-Gustavsson, et al. (1994). "Muscle
24 glycogen depletion pattern in halothane gene-free pigs
at slaughter and its relation to meat quality." Meat
26 Science 38: 91-101.
27
28 Koohmaraie, M., S. D. Shackelford, et al. (1995). "A
29 muscle hypertrophy condition in lamb (callipyge):
Characterisation of effects on muscle growth and meat
31 quality." Journal of Animal Science 73: 3596.
32
33 MacLennan, D. H. and P. J. 0'Brien (1992). Diagnosis
34 for porcine malignant hyperthermia. Patent Cooperation
treatv, The University of Toronto Innovations
CA 02267347 1999-03-30
WO 98/15837 PCT/GB97/02?41
41
1 Foundation.
2
3 Maltin, C.A., K. D. Sinclair, P.D. Wariss, C.H. Grant,
4 A.D. Porter, M.I. Delday and C.C. Warkup. In press
Animal Science.
6
7 Martel J. F. Minveille, et al. (1988). "Effect of
8 crossbreeding and sex on carcase composition, cooking
9 properties and sensory characteristics of pork."
Journal of Animal Science 66: 41-46.
11
12 Mason, I. L. (1988). World Dictionary of Livestock
13 Breeds. Wallingford, Oxon, UK, C.A.B International
14
Mellgren (1980) FEBS Letts 109: 129.
16
17 Mellgren (1991) J. Biol. Chem. 266: 13920-13924.
18
19 MLC (1992}. Stotfold pig development unit - Seconds
trial results., Meat and Livestock Commission Milton
21 Keynes.
22
23 MLC (1996). Pia Yearbook. Milton Keynes, Meat and
24 Livestock Commission.
26 Ockerman, H. W., D. Jaworek, et al (1984). "Castration
27 and sire effects on carcase traits and meat
28 palatability and muscle fibre characteristics in Angus
29 cattle." Journal of Animal Science 59: 981.
31 Peter, J. B., R. J. Barnard, V.R. Edgerton, C.A>
32 Gillespie and K.E. Stempel (1972) Biochemistry 11:
33 2627 - 2633.
34
Purchas, R. W., W C Smith, et al. (1990). "A comparison
CA 02267347 1999-03-30
WO 98I15837 PCT/GB97/02741
42
1 of the Duroc, Hampshire, Landrace and Large White as
2 terminal sire breeds of crossbred pigs slaughtered at
3 85kg live weight 2. Meat quality." New Zealand Journal
4 of Agricultural Research 33: 97-l04.
6 Ruusunen, M. (1993). The fibre-type composition and
7 capillary density in M L. Dorsi of different pig
8 cross-breed. Pork Quality: Genetic and metabolic
9 factors E. Puolanne, D. I. Demeyer, with, M. Ruusunen
and S. Ellis. Wallingford, CAB International: 301.
11
12 Savart et al. (199S) FEBS Letters 359: 60-64.
13
14 Seideman, S. C and J. D Crouse (1986). "The effects of
sex condition, genotype and diet on bovine muscle fibre
I6 characteristics " Meat Science 17: 55.
17
18 Seideman, S. C and L. K. Theer (1986) "Relationships of
19 instrumental textural properties and muscle fibre types
to the sensory properties of beef." Journal of Food
21 Quality 9: 251.
22
23 Skorjanc, D, A. Salehar. et al (1994) "Breed
24 differences in morphometric characteristics of muscle
fibre types of pigs longissimus dorsi and
26 semimembranosus muscle." Zbornik biotehniske fakultete
27 universe Liublj_ani Kmetiistvo 64: 63-70.
28
29 Sorimachi et al., (1993) J. Biol. Chem. 268: 10593.
31 Sorimachi et al., (1989) J. Biol. Chem. 264: 20106.
32
33 Szentkuti, L. and R. G Cassens (1978) "Die verteilung
34 Dr fasertypen I, II A and II B im M Longissimus dorsi
and M. semitendinosus von schweinen verschiedenen
CA 02267347 1999-03-30
WO 98/15837 PCT/GB97/02741
43
1 alters" Deutsche Tierarztliche Wochenschrift 8S(1):
2 23-27.
3
4 Taylor et al., J. Anim. Sci. 73: 1351.
6 Uhrin, V., L. Kuliskova. et al. (1986) "Histochemical
7 analysis of some muscles in the final hybrids of pigs "
8 Zivocisna Vyroba 31(12):1065-1074.
9
Vos, P., et al, (1995) "AFLP: a new technique for DNA
11 fingerprinting" Nucleic Acids Research 23:4407-4414.
12
