Influence of Breeding Systems on pH and Histochemical Properties of Muscle Fibres in Porcine
M. semimembranosus
Mara Lucia Stecchini, a Francesco Mascarello c & Adalberto Falaschini b
"Istituto di Tecnologie Alimentari, vie Maranpori 97, Udine, Italy ~Istituto di Produzione Animale, vie S. Mauro 1, Pagnacco (UD), Italy
CIstituto di Anatomia degli Animali Domestici con Istologia ed Embriologia, Facoltfi di Medicina Veterinaria, vie Celaria 101 Milano, Italy
(Received 15 August 1989; revised version received 22 November 1989; accepted 13 December 1989)
A B S T R A C T
The purpose of this study was to investigate pH values, fibre type frequency and histological features of Semimembranosus muscle o f 15 pigs subjected to three breeding systems. The A group was raised in a large paddodk; the B group animals were raised in a collective pen until they reached a Hve weight o f 120 kg and then in the same paddock as the A group; the C group was raised in a collective pen. Breeding systems had no significant effect on p H values and fibre type frequency. Histopathological changes in muscle fibres and connective tissue were observed in 12 of the animals studied regardless o f the group. However, analysis o f the incidence of alterations showed a higher frequency of lesions in the B group, followed by the C group.
Except for one case of DFD meat, muscle alterations were not associated with an adverse p H. In addition, regenerating fibres, with strong reactivity for neonatal serum, were observed both in apparently healthy and in pathological muscle samples.
I N T R O D U C T I O N
Techno log ica l p o r k qual i ty p rob lems are of ten re la ted to pig stress- suscept ib i l i ty resul t ing f rom a genetic select ion a imed at p roduc ing leaner
280 Mara Lucia Stecchini, Francesco Mascarello, Adalberto Falaschini
and faster growing animals. Stress-susceptible pigs, generally identified by a positive reaction to the anaesthetic halothane, are responsible for much of the pale, soft, exudative (PSE) condition.
As reported by Sellier et al. (1988), technological pig meat quality can be attributed to three main factors: (1) the rate ofpH fall, mainly (but not only) linked to halothane sensitivity; (2) the extent of pH drop, which primarily depends on the muscle glycolytic potential at the time of slaughter; (3) the composition and structure of the muscles, which is influenced by factors such as metabolic type, age and breed.
As far as the structure of muscles is concerned, it is generally assumed that muscle fibre number is relatively fixed at birth. However, the fibre type composition of porcine muscle is related to a number of genetic and environmental factors such as age, sex, breed and exercise. It was suggested that practices which favour transformation of eR (type IIA) fibres to ~W (type IIB) fibres could result in significant increases in animal muscularity (Ashmore et al., 1972).
Earlier investigations have shown that the occurrence of the PSE condition is related to an increase in the proportion of intermediate myofibres (type IIA) (Cooper et al., 1969) or white (type IIB) muscle fibres (Dildey et al., 1970) or both (Swattand & Cassens, 1973). Sosnicki (1987a) reported in White Zlotnicka pigs a positive correlation between meat pi l l (45 min after slaughter) and the presence of/~R (type I) fibres. On the other hand, Heffron et al. (1982) found, in Landrace pigs, no significant difference in the proportions of the three fibre types in normal and low-quality meat. Furthermore, meat quality was reported to decrease due to morphological lesions found in PSE muscle (Sosnicki, 1987b). Thus, this study was undertaken to investigate the effect of three breeding systems on pH and fibre muscle properties of genetically PSE-prone croSs-bred pigs.
MATERIAL AND METHODS
Fifteen genetically PSE-prone cross-bred (BL x LW and P x LW) pigs (females and castrated males), detected on the basis of the creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) tests at about 40 kg of live weight, were raised in a collective pen (18 m 2) on a commercial feed mixture. When their weight was approximately 80 kg, the pigs were divided into three groups and fattened as follows: the first group (A) in a large paddock (1500m2); the second (B) in a collective pen (18m 2) until a live weight of 120 kg was reached and then in the same paddock as the A group; the third (C) in a collective pen (18 m2). The pigs had a live weight of about 150 kg when they were transported to the slaughterhouse and killed after a 24 h lairage. During lairage, pigs received water but not food. Forty-five
Influence of breeding systems on pork muscle fibre 281
minutes, on average, after slaughter, samples of M. semimembranosus (SM) (the superficial portion of the ischiatic origin) were taken from the right side of the carcasses. Samples for histochemical analysis were rapidly frozen in isopentane cooled by liquid nitrogen and stored in a freezer at - 80°C. Parts (2g) of the SM samples were homogenized in 18ml of 0-005M sodium iodoacetate solution and the pH 1 (45 min after slaughter) of homogenate was measured by a glass electrode. The pH 2 was recorded 24 h post mortem on a SM fresh-cut homogenate.
Frozen SM samples were serially cut (10/am) on a cryostat and sections for the analysis of morphological aspects were submitted to haematoxylin- eosin (HE) and periodic-acid2 Schiff(PAS) staining. For fibre typing, sections were stained for myosin adenosine triphosphatase (m-ATPase) activity after alkaline and acid preincubation (Brooke & Kaiser, 1970) according to the A method described in Snow et al. (1982). The oxidative and glycolytic activity of the fibres was assessed by staining the section for succinic-dehydrogenase (SDH) and ~-glycerophosphate dehydrogenase (~- GPDH) activities (Lojda et aL~ 1976). Based on m-ATPase activity after preincubation at pH4-55, fibre type frequency was evaluated by photomicrographs of serial sections taken in different muscle areas. From different areas of each sample at least 2000 fibres were identified. In addition, sections were taken for immuno- histochemical staining by the indirect immunoperoxidase technique. An antiserum to myosin heavy chain (extracted from bovine embryo) specific for neonatal myosin (anti-neonatal serum) was used. Fibres which reacted positively with this antibody were counted using a template of 22-74 mm 2 in area which was placed on each slide.
The results of pH and fibre type frequency were subjected to statistical evaluation with the Kruscall-Wallis test for variance analysis.
RESULTS
pH measurements
In Table 1 average pH values for the three pig groups are reported. Breeding systems had no significant effect on pH measurements. None of the pigs had a pH 1 < 5.8 (PSE condition). However, one sample of the B group had a pH 2 higher than 6-20 (a case of dark, firm, dry (DFD) meat).
Fibre types and frequency
The fibre types found in the samples examined were classified on the m- ATPase activity as type I, IIA and IIB. Type I fibres showed a strong acid- stable and alkali-labile myosin ATPase activity, a strong reaction for SDH
282 Mara Lucia Stecchini, Francesco Mascarello, Adalberto Falaschini
TABLE 1 Mean Values and Standard Deviations for pH~ and pH 2 Values in M.
semimembranosus of the three (A, B, C) Pig Groups
Group No. of pigs pH 1 pH 2
.~ SD .~ SD
A 5 6-44 0.10 5.82 0-21 B 5 6-31 0-15 5-85 0-28 C 5 6-28 0-22 5-84 0.23
and a negative reaction for e - G P D H activities. Type IIA fibres exhibited an acid-labile and alkali-stable myosin ATPase reaction, moderate to strong S D H activity and a strong to moderate e - G P D H reaction. Type l iB myofibres showed a moderately acid-labile and moderately alkali-stable m- ATPase, a weak S D H and strong ~ - G P D H activities.
Fibre type frequency, evaluated on the basis o f m-ATPase activity after incubation at pH 4-55 (type I dark, type l iB intermediate and type IIA negative), is illustrated in Table 2. No significant difference in the fibre types between the three different management systems was found.
Morphological and histopathological findings
O f the animals studied, 12, equally distributed in the three groups, exhibited histopathological changes at varying degrees in muscle fibres and connective tissue.
Altered muscle fibres exhibited central nuclei, focal necrosis (Fig. 1) and a t rophy with phagocytosis. Giant fibres, observed in muscles from stress- susceptible pigs (Cooper et al., 1969; Dutson et aL, 1978), could be distinguished in six samples regardless of the group. Very small fibres were
TABLE 2 Proportions and Standard Deviations of the Three Muscle Fibre Types (I, IIA, IIB) in M.
semimembranosus of the Three Pig Groups (A, B, C)
Group No. of pigs % type I % O'pe IIA % type IIB
SD .~ SD .~ SD
A 5 18-54 8"91 11.76 4-31 69-74 12.97 B 5 17-80 10"15 9"68 6-05 72.54 14.90 C 5 11"42 5"51 8-48 4.78 80" 12 9.26
Influence of breeding systems on pork muscle fibre 283
Fig. 1. M. semimembranosus. HE x 75. Central nuclei and focal necrosis.
distributed randomly in the fibre bundles. In the necrotic areas proliferation of connective tissue was observed. Giant fibres showed an SDH and :~- G P D H irregular reaction characterized by a thickening of the precipitate network. Moth-eaten fibres of all fibre types were observed. Analysis of alteration incidence showed a higher frequency of lesions in the B group, followed by the C group.
Immunohistochemical findings
Immunohistochemical reaction for the presence of neonatal heavy-chain myosin revealed a strong reaction in fibres of small (10-20#m) and very small (5 F~m) diameters. In some fibres o f medium diameter, the reaction also was positive (Fig. 2). It should be noted tha t the intensity of reaction decreased as the diameter increased.
Fibres with neonatal myosin were present in 13 of the samples examined. The number of such fibres counted from each sample within an area of 22-74 mm 2 (where about 1600-1700 fibres were present) is reported in Fig. 3.
284 Mara Lucia Stecchini, Francesco Mascarello, Adalberto Falaschini
Fig. 2. M. semirnembranosus. Immunoperoxidase, anti-neonatal serum x 100. Small and medium diameter fibres showing a strong reactivity for neonatal serum.
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Fig. 3. Bars represent the total number of fibres with neonatal myosin within an area of 22.74 ram-" of M. semimembranosus of the three pig groups (al-5: group A; bl-5: group B;
cl-5: group C).
Influence of breeding systems on pork muscle fibre 285
DISCUSSION
Pig production in Italy is unique in that the animals are slaughtered at a higher weight than in many other European countries (Dazzi et al., 1987). Furthermore, all of the pig carcass, except for the loin, is used for processing and special interest is focused on the quality of ham (Russo et aL, 1987). It is widely accepted that both the rate and the extent ofglycolysis are important in the determination of pork quality. Furthermore, a relationship between these events and pH values is well proven. It follows that pH values recorded post-mortem at the end of glycolysis are considered suitable indicators of meat quality (Hofmann, 1988). In terms of pH values, all the samples examined except for one belong to a normal quality category.
The expression of different fibre types in skeletal muscle is under neuronal and hormonal control. The variable expression of myosin heavy chain genes enables muscles to adapt their contractile properties to changing physiological environments. For example, endurance training can induce a transformation of type IIB fibres into type IIA and perhaps an increase in type I (Bandman, 1985). In our study, however, variation in management did not promote a difference in physical activity sufficient to modify fibre type proportions.
As far as the morphological and histochemical findings are concerned, evidence of a relationship between stress susceptibility and muscle alterations was recently reported by Bader (1987) and Sosnicki (1987b). According to Bader (1987), muscle fibres in stress-susceptible pigs show varying degrees of alteration related to the existence of myopathy in different stages. Acute lesions are characterized by the swelling and rounding of muscle fibres; subacute lesions take place as removal (myophagia), regeneration and repair. In this study, lesions could be attributed largely to the subacute or chronic mode of alteration as reported by Bader (1987). Furthermore, a higher incidence of fibre lesions in the B group could be attributed to the change in breeding system at 120 kg body weight. On the contrary, pigs raised in the large paddock showed a lower incidence of fibre lesions. It should be noted that in this investigation, muscle alterations, except for one case of DFD meat, were not associated with adverse values of pH. Furthermore, as described by Muir (1970) very small fibres were observed in the skeletal muscle from stress-susceptible pigs.
Muscle retains a regenerative potential due to the presence of satellite cells. These are small mononucleated cells that reside beneath the basement membrane of muscle fibres (Mauro, 1961). Satellite cells are capable of DNA synthesis and mitosis followed by fusion and myofibrillar protein synthesis (Allen et al., 1979).
During muscle development, a sequential transition in the myosin
286 Mara Lucia Stecchini, Francesco Mascarello, Adalberto Falaschini
isozymes takes place with the embryonic and neonatal myosin heavy chain being replaced by the adult isoforms. The myosin heavy chain isozymes are very homologous in structure but are distinct polypeptides, as shown by protein chemical and immunological techniques (Pierobon-Bormioli et al., 1980, 1981; Butler-Browne et al., 1988). The same sequential transition has been reported for regenerating fibres (Sartore et al., 1982).
In this study, the finding of very small diameter fibres associated with muscle lesions prompted the testing of samples with neonatal heavy chain myosin antiserum. Fibres with strong reactivity for neonatal serum were observed both in apparently healthy and in altered muscle samples. Whereas the detection of neonatal myosin in regenerative fibres of pathological muscle has already been described (Sartore et al., 1982), its discovery in apparently normal muscle samples is significant. Furthermore, the presence of focal damage and regeneration areas cannot explain the random distribution of this type of fibre. However, t he presence of some fibres containing neonatal heavy chain myosin in the adult pig could be explained by the recent work of Darr and Schultz (1987). These authors suggested that in rat muscle satellite cells were activated even in the absence of fibre necrosis and that, in exercised muscle, lesions not discernible by light microscopy could induce a release of mitogenic factors to increase proliferation of satellite cells.
A C K N O W L E D G E M ENTS
The authors thank Dr R. M. A. P. Ridge and Dr A. Rowlerson for the gift of the antibody to neonatal myosin and Mr P. Stortini for technical assistance. This work was partially supported by MPI 40%.
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