Bakou et al. (Vol 3, N°3-4, 2005)

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Revue Africaine de Santé et de Productions Animales © 2005 E.I.S.M.V. de Dakar

A RTICLE ORIGINAL Histopathological alterations of bovine skeletal muscle observed at the slaughterhouse of Dakar (Senegal) S. BAKOU1*, Y. KANE2, B. MUSABYEMARIYA3, W. NDJOYI1, G. DAYO1, Mg. SEYDI3 1 2 3

EISMV - Service d’Anatomie-Histologie-Embryologie, BP 5077 Dakar-Fann, Sénégal EISMV - Service de Pathologie Médicale, Anatomie Pathologique et Clinique Ambulante, BP 5077 Dakar-Fann, Sénégal EISMV - Service d’Hygiène et Inspection des Denrées Animales et d’Origine Animales, BP 5077 Dakar-Fann, Sénégal

* Correspondance et tirés à part , e-mail : serakou2001@yahoo.fr

Abstract The aim of this study was to report the nature and incidence of muscular changes found upon histological examination at the time of slaughter in the slaughterhouse of Dakar (Senegal). Eighty cattle (Zebu and N’dama) coming from Senegal, Mali and Mauritania, and transported by trucks and train to the Slaughterhouse of Dakar (Senegal) were used in this study. Five muscles [Sternocephalicus (SC), Infraspinatus (IS), Pectoralis descendens (PD), Obturator (OB) et Diaphragma (DI)] were collected immediately after slaughter from each of the animals. Among these 400 samples processed for histology and examined in cross-sections, 10.5% exhibited isolated necrotic cells, 21.2% fibers splitting, and 60% hypercontracted fibers. Ten percent (10%) of samples showed a high incidence of hypercontracted fibers, 53.3% a medium incidence (less than de 10%) and 36.7% none. Hypercontraction was more frequent in DI muscles (P<0,05) (RASPA, 3 (3-4) : 164-168).

Keys – Words: Hypercontracted fibers - Skeletal muscle – Bovine - Meat quality - Senegal.

Résumé Les altérations histopathologiques du muscle squelettique des bovins observées aux abattoirs de Dakar (Sénégal) Le but de cette étude était de rapporter la nature et l’incidence des altérations histopathologiques des muscles de bovins observées aux abattoirs de Dakar (Sénégal). Quatre-vingts bovins (Zébu et N’dama) provenant du Sénégal, du Mali et de la Mauritanie et acheminés par camions aux abattoirs de Dakar (Sénégal) ont été utilisés dans cette étude. Quelques minutes après l’abattage des animaux cinq muscles [sternocephalicus (SC), infraspinatus (IS), pectoralis descendens (PD), obturator internus (OI) et diaphragma (DI)] ont été prélevés, traités par les techniques histologiques et examinés. Sur les 400 échantillons musculaires examinés, 10,5% ont présenté des fibres nécrosées, 21,2% de fibres clivées et 60% de fibres hypercontractées. Dix pour cent (10%) des échantillons examinés ont présenté une forte incidence en fibres hypercontractées, 53,3% une incidence moyenne et 36,7% n’ont présenté aucune lésion. Le diaphragme est le muscle le plus atteint (71,4%, p<0,05).

Mots-clés : Fibres hypercontractées - Muscle squelettique - Bovin - Qualité de la viande - Sénégal.

Introduction The perception of meat quality depends on three criteria: tenderness, color and shelf life ability. Tenderness seems to be the most important sensory attribute of meat, affecting consumer acceptance of beef [14], [27]. Nevertheless, in developing countries like Senegal, the safety of food is the main concern for the average consumer. For instance, when he buys meat, he wants to be tasteful. But most of all, he requires that the product does not present any risk at an unacceptable level for his health, and for his life, that may be related to the presence of dangerous agents (micro-organisms, toxins, various contaminants)[19].

Thus, the quality of the meat is guaranteed by monitoring livestock health, and ante and post mortem inspection of animals. However, apart from the average consumer, in big cities like Abidjan, there is a new category of consumers concerned by the organoleptic quality of the beef meat as well.

Technological, visual and organoleptic attributes of meat are influenced by genetic factors (species, sex, breed strain and genotype) and environmental factors (rearing, preslaughter handling and post slaughter management) [11].

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In numerous studies the significant influence of preslaughter handling on meat quality characteristics have been noted [16], [17]. Hypercontrated fibers (giant fibers) are known as a result of the stress of slaughtering [4], [22] and are incriminated in the default of technological and sensory qualities of meat. This type of muscle modification was observed in bovines [20], pigs [1], [9], [10], [12], [13], [21], turkeys [5], [15], [18], [22], [26], chickens [7] and Grasscutters [2]. The hypercontracted fibers are usually oval or round in shape and have a larger cross section than other the normal muscle fibers in most of the time. They can be found grouped or isolated and are generally located at the periphery of the muscle bundle. These muscle fibers have myofibrils excessively contracted with abnormally short sarcomeres [3].

These alterations of the muscle fibers are not identifiable by the current methods used during meat inspection in the slaughterhouses of Africa in general and Senegal in particular. The outcome is a misappreciation of this type of alteration. Therefore, the purpose of the present study

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HF

was to report the nature and incidence of muscular changes found upon histological examination at the time of slaughter in the slaughterhouse of Dakar (Senegal).

Materials and Methods 1. ANIMALS

HF

One hundred 3 to 10-years-old cattle from different origins (Senegal, Mali, Mauritania) were used in this study. Most animals have move to summer pastures (transhumance) before being brought by trucks and trains to the slaughterhouse of Dakar (Senegal). Upon their arrival, they are put in lairage rest.

2. SLAUGHTER PROCEDURE

Cattle were slaughtered at the Dakar slaughterhouse according to the Muslim (Halal) way by severing the throat and major blood vessels in the neck. After slaughtering, animals are dressed.

3. MUSCLE SAMPLING

Five muscles were sampled: Sternocephalicus (SC) on the neck, Infraspinatus (IS) on the hindlimb, Pectoralis descendens (PD) on the breast, Obturator externus (OE) on the forelimb and the Diaphragma (DI). The choice of the muscles used was motivated by the fact that there are located in different areas of the animal body and are usually removed in this type of study.

4. HISTOPATHOLOGICAL EXAMINATION

Transverse sections (about 2 cm thick), taken from the muscle midbelly were collected immediately after dressing (between 15 and 30 minutes after bleeding), fixed by immersion in a neutral buffered formalin solution, dehydrated and embedded in paraffin according to standard histological methods. Four (4) mm thick transverse sections were stained by Hematoxylin-Eosin-Saffron (HES) method and examined under light microscope to evaluate changes in muscle morphology. The same area of muscle was examined in every case.

SF Figure 2: Transverse section of a Obturatorius externus muscle (HES, x 168).

Cluster of hypercontracted fibers (HF) and splitting fiber (SF)

1. FIBER SPLITTING Fiber splitting was characterized by partial or total division of myofibers. With a variable size, they are retracted in their basal membrane.

Their cytoplasm is smooth and have a basophilic tinctorial affinity. Their nuclei are small, compact and rather located at the periphery. In the latter case, splitting fibers were composed of a number of small cells surrounded by the same basal membrane (Figure 3).

Among the samples, 21.2% exhibited splitting cells (Table1), and only 1 or 2 split fibers were noted among 400 fibers.

5. HISTOPATHOMORPHOMETRICAL ANALYSIS

Quantification of fibers splitting, necrotic cells and hypercontracted fibers was performed at magnification of X 160 in 5 microscopic fields from each sample, i.e., among 400 fibers.

6. STATISTICAL ANALYSIS

Statistical analysis was performed using ANOVA followed by Fischer PLSD.

Results Each sample exhibited several well-structured muscle bundles. Each myofiber had an ovoid shape and was generally removed from its basal membrane by histological treatment. The myofibers showed pink cytoplasm and 2 or 3 blue nuclei located at the periphery, just beneath the plasma membrane (figure 1). These normal myofibers are associated in some bundles to altered myofibers: splitting fibers, necrotic fibers and hypercontracted fibers (figure 2).

Figure 3: Transverse section of a Infraspinatus muscle (HES, x 320).

A splitting fiber.

Table 1: Distribution of histopathological alterations among muscle samples Histopathological alterations Hypercontracted fibers Fiber splitting Cell necrosis

2. NECROTIC FIBERS

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Figure 1: Transverse section of a Infraspinatus muscle (HES, x 84).

Features of a normal fibers.

Total count

% of samples

240

60

82

42

21.2

10.5

Necrotic muscle cells showed weakly stained â&#x20AC;&#x153;liquefiedâ&#x20AC;? or hyaline cytoplasm with picnotic nuclei or were invaded secondarily by phagocytic cells. Necrotic cells were isolated and scattered throughout the muscle.

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Only 10.5% (Table1) of all the samples exhibited necrotic cells. Moreover, on some samples of IS muscle, we notice the presence of a chronic inflammatory reaction associated with inflammatory cell infiltrate mainly macrophages and esoniophils, neovascularization and fibroplasia, (figure 4).

These areas contain in some sites hemorrhagic center.

These changes are generally associated to muscular regenerative features with the formation of myotubes (figure 5).

Figure 6: Transverse section of a Pectoralis descendens muscle (HES, x 336).

A hypercontracted fiber was swollen and rounded in shape

Table 2 : Hypercontracted fibers: distribution of samples

fp

Number of hypercontracted fibers per field

Total count

% of samples

0

147

60

More than 10

40

1 - 10

Figure 4: Transverse section of a Infraspinatus muscle (HES, x 84).

Necrotic fibers invaded by mononuclear cells (arrows) associated with fibroplasia (fp).

60

Number of samples

50

IS

21.2

10.5

PD

OE

DI

40 30 20 10 0

Figure 5: Transverse section of a Infraspinatus muscle (HES, x 84).

Feature of myoblastic regeneration with numerous myotubes (arrows)

SC

213

0 1 to 10 > 10 Number of hypercontracted fiber per field

Figure 7: Hypercontracted fibers: frequency of distribution of the 3 categories [0, (1 to 10), (> 10)] per muscle

3. HYPERCONTRACTED FIBERS

4. ASSOCIATION OF LESIONS

Hypercontracted fibers also known as « giant fibers » were swollen and rounded in shape, and their cytoplasm was homogeneous in appearance (Figure 6). They were grouped together by 2 or 3 most often and located at the periphery of the muscle fascicles. Their number varied greatly from slide to slide, with a mean (± SD) of 3.8 ± 5.5 per field. They were more than 10 hypercontracted fibers in 10% of samples, and most samples showed a medium incidence (Table 2).

The distribution of samples per muscle is shown in Figure 7. The number of giant fibers varies from 60% in the OB muscle to 71% in the DI muscle. No significant differences were noted between the examined muscles (ANOVA, Fischer PLSD P = 0.03).

Necrotic features, splitting fibers and more than 10 hypercontracted cells were never observed simultaneously in samples. No association was found between necrosis and hypercontracted fibers (2 factors ANOVA).

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Discussion The experiment was conducted in a way to minimize as much as possible, the variation between cattle. Thus only one slaughterhouse was used, the time of sampling after death was as constant as we could achieve (less than 10 min of variation). The main sources of uncontrolled variation were the origin (genetic) and the technical

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specifications of rearing. Our results showed the presence of hypercontracted fibers in different muscles of the cattle after slaughter, as being the most significant and frequent alterations and are in concordance with earlier reports in turkeys [5], [15], [18], [22], [26], Pigs [1], [9], [10], [12], [21], chickens [7], Grasscutters [2] and cattle [20].

Histological characteristics of hypercontracted fibers in cattle are identical to that described in the literature, i.e. in a cross section, they often have an increased diameter and are rounded; which earned them the appelation “ballonised fibers” or “giant fibers” in reference to the observations of DUTSON et al. [9].

Our results also reveal that 60% of the samples show at least one hypercontracted fiber. In an equivalent study on the muscles of hindlimb in turkey, CHEREL et al. [5] report 76.3%. It is generally accepted that the hypercontracted fibers repressent less than 2% of the total number of muscle fibers. In our study this proportion varies, according to the muscles studied, and goes from about 1% in PM, ST and OB muscles to 3.5% in DI muscle.

The DI muscle was probably more sensitive to hypercontraction than the other muscles due to its particular physiological function (most inspiratory muscle).

The impact of genetic background on the occurence of the hypercontracted fiber has been studied in pigs [10], [21] and turkeys [18], [26]. All authors agree that the giant fiber syndrome is an intrinsic susceptibity to muscle fibers enlargement in some animals and is not necessarily associated with breeding. For instance, FAZARINC et al.

[10] concluded that the giant fiber syndrome depends mostly upon the rate and intensity of early post-mortem glycolysis, which results in acidity of muscle tissue.

Moreover, REMIGNON et al. [18] reported that hypercontracted fibers were found almost equally in three lines of turkeys. In the light of our results, we suggest possible relations between the frequency of hypercontracted fibers and the stress of slaughtering (transport and bleeding). Indeed in the slaughterhouse of Abidjan, the bleeding is done without electrical stunning, as conditions of slaughter which are likely to reveal this type of lesion in the muscles of the domestic animals in general and the bovines in particular. More studies are necessary to fully understand this assumption.

The status of hypercontracted fibers is more controversial.

CARPENTER and KARPATI [3] noted that fibers became hypercontracted during biopsy. SOSNICKI et al. [22] demonstrated that the histoenzymological patterns of giant cells were atypical, exhibiting a strong positive succinic dehydrogenase reaction, a positive ATPase reaction and a diffuse reaction for alkaline and acid phosphatases. They concluded that hypercontracted fibers were not artifacts but constituted real changes associated with developing muscle pathology.

CULLEN and FURTHORPE [6] reported that hypercontracted fibers in Duchenne muscular dystrophy represent an early stage of fiber necrosis. SOSNICKI et al.

[22] reported an association of hypercontracted fibers and necrotic fibers with an adipose tissue infiltration in turkey muscle. VELLEMAN et al. [26] also reported that the hypercontracted muscle fibers were observed in muscle areas undergoing fiber degeneration and suggested that hypercontraction of muscle fibers is a step in the process of muscle necrosis. In the present study, no evidence of association between hypercontracted fibers and necrotic fibers was noted. Our results are consistent with the conclusion of the work of CHEREL et al. [5]; they concluded that hypercontracted fibers in turkey muscle are

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not indicative of an early stage in the necrotic process.

The relationships between fibers splitting and hypercontracted fibers are not clear. Splitting fibers are found in muscular dystrophies as well as in neurogenic diseases than in normal circumstances at the myotendinous junction [8].

The number of hypercontracted fibers varies considerably between samples, but the significance of this remains unclear. However, the influence of the hypercontracted fibers on the organoleptic quality of the meat seems obvious. In turkey skeletal muscle, SOSCNICKI and WILSON [25] concluded that hypercontracted fibers represent a condition of muscle damage and causes a toughening of meat. In the pig, giant cells are often found in association with pale, soft, exudative meat [1], [9].

Further studies could enable to better determine the consequences of hypercontracted fibers on the physical and organoleptic qualities of the cattle meat slaughtered in our countries and thus contribute to the improvement quality of meat delivered to the consumers.

Acknowledgment We are grateful to Doudou DIAGNE for technical assistance.

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