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International Journal of Morphology

On-line version ISSN 0717-9502

Int. J. Morphol. vol.21 no.3 Temuco  2003

http://dx.doi.org/10.4067/S0717-95022003000300005 

Int. J. Morphol., 21(3):211-219, 2003.

HISTOLOGY OF THE JAW DEFORMATION IN 
SALMON OF SOUTHERN CHILE (Salmo salar)

HISTOLOGÍA DE LA DEFORMACIÓN MANDIBULAR 
EN SALMONES DEL SUR DE CHILE (Salmo salar)
*Felipe Venegas; *Enrique Montiel; **Pablo Forno & *Mariana Rojas


VENEGAS, F.; MONTIEL, E.; FORNO, P. & ROJAS, M. Histology of the jaw deformation in salmon of southern Chile (Salmo salar). Int. J. Morphol., 21(3):211-219, 2003.

In adult salmon of the sea centres in southern Chile, a jaw deformation (JD) has been identified. It affects the dental and hyomandibular bones, which bend ventrally up to 90° of their normal position. The deformation affects also the dental articular bone. This pathology is related to weight loss and increased mortality of the salmons.

It was empirically postulated that a probable cause for this anomaly was food from vegetal origin in the diet of the fishes (which are carnivores) Therefore, the present work aims at comparing the biostructure of jaw bone of salmons fed either with vegetal (soja and gluten) formulation or animal formulation, mostly fish powder.

Fifty specimens were analyzed from Puerto Montt, 35 having JD and 15 normal control. Samples were obtained in June, July and September 1999. (group 1) and March, Sept and October, 2000 (group 2). Group 1 was fed mostly with vegetal flour and group 2 with fish flour. Each group was subdivided in two subgroups, one of healthy animals and the other of fishes with JD. Jaw and articular bones were fixed in 10% formaldehyde and 1% glutaraldehyde and processed for histology (hematoxylin.eosin, Alcian blue, Masson trichrome) histochemistry (Syrius red and von Kossa) and scanning electron microscopy (SEM).

The mandibular bone of group 1 with JD presented large amounts of osteoid tissue compared with its control. Collagen I disminishes and its architecture and composition changes, collagen III increases. No significant difference was found in calcium content between normal and JD fishes. SEM shows that the dental bone close to the joint in the fishes with JD displayed a disorganized structure and no trabecular formation, compared to controls, In group 2, these pathological findings were less evident, both macro- and microscopically.

Results suggest that JD is of multifactorial origin; the primary cause can be a genetic or congenital alteration of the Jaw cartilage. There should be susceptibility for presentation of the defect in this group of fishes, but its expression is triggered by deficit of phosphorous of animal origin in food, so that this pathology is not seen in fishes with adequate nutrition.

KEY WORDS: 1. Jaw; 2. Jaw deformation; 3. Histology; 4. Salmon; 5. Salmo salar.


INTRODUCTION

In adult salmons of sea centres in southern Chile a deformation of the lower jaw termed "jaw deformation" (JD) has been identified and related to decrease in weight and increased mortality up to 2.5% (INTESAL report, 1999).

JD is described in the literature as a deformity of adult Atlantic salmons, affecting the bones where teeth are inserted, such as the dental and hyomandibular bone, which bend ventrally at angles ranging from 45 to 90° from their normal position. A displasia of the jaw cartilage (Meckel) is evident.

In some fishes, displacement and lateral rotation of the articular bone lateral results in the -bone being pushed and the operculum is laterally deviated (Bruno et al., 1996).

Fishes with macroscopic deformation, feed and swimm normally, but the average weight of this group is lower than the same healthy fishes in the same cage, thus reducing their commercial value (Bruno et al). The malformation has been related to a variety of factors such as temperatura of the water from fertilization up to beginning of feeding period to speed up development (Lega et al., 1992; Baeversford et al., 1998a y b; Storset, 1998), problems during male sexual maturation (Eckhard & Hall, 2002), genetic factors (Miller et al., 2000) and heritage (Bruno et al.), environmental factors such as UV radiations (Bullock, 1982; Bruno et al.), evolutive factors and heterochronic factors in reference to the displacement of the timing of appearance and development in ontogenesis of an organ in regard to others (Alekseyev & Power, 1995; Ferguson et al., 1995, Richardson et al., 1997), chemical products, such as residues of malachite green (Bills et al., 1979; Bailey et al., 1989; Allen, 1991), use of antibiotic such as oxytetracicline in the reproductors (Toften et al., 1996; Baeverfjord et al., 1998).

The lack of balance of minerals in the diet and some environmental factors such as high mineral or CO2 content of the water (Bullock et al., 1981) phosphorous deficiency (Asgard & Shearer, 1997; Baeverfjord & Asgard, 1998; Sugiura et al., 2000), changes in its bioavailability (Nordrum et al., 1997), lack or excess of vitamins (Hilton et al., 1978; Bullock et al., 1982; Johnston et al., 1989; Naggar et al., 1991; Waagbo et al., 1993; Thompson et al., 1994; Bruno et al.) and parasites, may be cause of JD (Paperna, 1975; Bruno et al.).

In the 90´in Norway, a pathology affecting mostly the vertebral column and occasionally the jaw, was observed. It was thought to be due to high temperature during the ova stage or the use of large amounts of oxytetracicline. It is not a genetic defect, nor is due to fracture or trauma, neither was it related to nutritional factors or vaccines (Baeverfjord et al., 1998 a y b). Analysis of the diet has been done (Baeverfjord & Asgard, 1998). Atlantic smolt salmons and adults were fed a phosphorous deficient diet both provoking bone pathology with scoliosis of the vertebral column and the ribs, with reduced growth. When adequate phosphorous levels were provided for 9 weeks, body mass was normalizad but bone lessions did not improve.

The aim of this work was the histological characterization of the JD and comparison of the collagen, ossification and calcification of the jaw in salmons fed with formulations where vegetal flour replaces fish powder.

MATERIAL AND METHOD

Fifty specimens were analyzed from Puerto Montt, 35 having JD and 15 normal controls. Samples were obtained in June, July and September 1999. (group 1) and March, September and October, 2000 (group 2). Group 1 was fed mostly with vegetal flour and group 2 with fish flour. Each group was subdivided in two subgroups, one of healthy

animals and the other of fishes with JD. Jaw and articular bones were fixed in 10% formaldehyde and 2% glutaraldehyde and processed for histology (hematoxylin.eosin, Alcian blue, Masson trichrome) histochemistry (Syrius red and Von Kossa) and scanning electron microscopy (SEM).

All fishes captured were anesthesized, sacrificed and processed for different techniques. In two fishes of each subgroup, dissection eliminating soft tissues was done. Eight fishes of each subgroup were fixed in 10% neutral formaldehide. In 42, salmons serial histological sections of the head were obtained. Right sides were decalcified and left sides were not decalcified. The following techniques were used. a) Standard histological techniques b) Histochemical techniques Syrius red Junqueira (Junqueira et al., 1979) to detect collagen types I and III, Von Kossa method to detect calcium (Leiva et al., 1984).

The calcified samples were digitalizad in a NIKON OPTHIPHOTâ microscope with a video camera COHUâ scientific, adapted to analyze a scale of grays and then computer-analyzed using the Software OSIRISâ 3,5 Medical Imaging Software, University Hospital of Geneva, available in Internet.

SEM techniques; jaw pieces fixed in 2% glutaraldehyde were processed for critical point drying in CO2 and metallized to be observed in a Zeiss SEM model.

RESULTS

Figs 1A y 1B are schemes of the normal topography of the facial bones and the changes found in fishes with JD.


Fig. 1a).Normal facial bones lay out.
0001b)Lay out of bone in JD

In group 1, two types of JD were easily seen (Fig. 2 a y 2b). The dental bone bends ventrally in angles varying from 45 to 90° in respect to its normal position, a finding not related to age or size of the fishes. In 90% of them, JD was bilateral. Only in 10% was unilateral. Facial deformity was not related to alterations of the vertebral column. In all simples with JD, the articular and square bone changed their position, protruding towards the gingival mucosa, where hemorraghic areas, with increased number of melanomacrophages, loss of teeth, loosely inserted, and some of them fractured, were observed


Fig 2. Lateral (A) and rostral (B) view of an adult salmon ventral bending of the jaw is seen, affecting both dental bones. Bilateral (p) prominences are formed by lateral deviations of the articular and square bones. Rostrally. The oral mucosa (o), tongue (l) and part of the branchial arches (ab) with hemo-rraghic areas, are shown. Laterally part of the branchial tissue can be seen due to shortenning of the operculum.

In group 1 a second type of JD characterized by a bulky head, with exophtalmy, open mouth and a protruding low jaw was seen. In the gular area a bilateral increase in volume was seen.

In group a second type of JD was seen, characterized also by bulky head, bilateral exophtalmy, open mouth and a protruding low jaw. In the gular region a bilateral increase in volume, was seen.


Fig. 3. Jaw deformation, bilateral exophtalmos, open mouth, lower jaw protrusion, increased volum of the gular region.

In group 2, JD was mild and difficult to contrast to control fishes.

Dissection of fishes with JD revealed that the dental and articular bone in adult salmon have a ventral bending that starts from the medial region of the dental bone and is very marked towards the symphysis, as if this area was mechanically driven towards the gular area (Fig. 4a). In some cases, signs of fractures and bone repair was seen. In the normal samples, the dental bone has a smaller surface and is not bent (Fig. 4b). The joint is smaller and does not show fibrosis. In medial view, part of ossified jaw cartilage is seen running over the gular plate. In fishes with JD of group 2, the bending was very mild (Fig. 4 C).


Fig. 4. Dental salmon bone (Salmo salar).
a) Group 1, grossly affected.
b) Control.
c) Group 2, slightly affected.
 

The articular joint of fishes with JD was soft to section compared to the other facial bones. In most cases, dense connective tissue with great increase of capillary formation was seen. With Junqueira´s Syrium red technique, a large proportion of collagen I fibers and very few type III (Fig. 6 A) are seen. In the JD specimens, a small quantity of collagen I fibers, was seen. They are found normally in the extracellular matrix of fish bone with more type III fibers, arranged in a loose network. Therefore, quality and quantity of collagen fibers is affected (Fig. 6 B).


Fig. 6. Salmon jaw a) normal bone trabecules (TO) showing type I collagen fibers, densely arranged, yelow to red. b) in JD, collagen fibers loosely arranged, do not show the same amount of collagen I as in bone tissue, Collagen III in green (Junqueira Syrius red). Polarized light microscopy, 400X.

Histological analysis of facial bones dental, articular and square of smolt and adult fishes with JD of groups 1 and 2, osteogenic cells, osteoblasts and morphologically normal osteoblasts in normal number were observed. These cells make up the endostium that surrounds the bone trabecules. However, in group 1 with JD osteoid tissue (with non mineralized matrix) was seen. It was very thick, located between the osteoblasts and the bone trabecules (Fig. 5 A). Some areas presenting changes in the staining of the extracellular matrix, characteristic of bone, were observed. Hyaline cartilage was identified inside the trabecules. In normal adult fishes no osteoid tissue was found or only a thin layer could be seen. In the fishes with JD of group 2, there was a thin layer of osteoid tissue (Fig. 5b).


Fig. 5a. Bone Trabecules of dental bone, Group 2. Osteoid tissue (arrow) Bone trabecule (T). Masson´s trichromic. 400X. Fig. 5b. Medium zone of the bone trabecules of dental bone, Group 2. There was a very thick layer of osteoid tissue. Masson´s trichromic. 400X.

Von Kossa technique, which reveals calcium localization as black or blue spots in tissues, shows little differences between normal and deformed fishes in most facial bones. In the fishes of group 1, low levels of calcium in the bones of both normal and macroscopically deformed fishes were found. In salmon of group 2 with JD intensity of the staining denotes higher calcium content in all cases (Fig. 4a). Some areas are seen, however, with less calcium in the dental and articular bone which are brown stained (Fig. 7a y 7b).


Fig. 7. A) Spongy bone tissue of dental bone in a normal salmon homogeneously black. B) Id of a JD salmon hetereogenously brown stained Von Kossa, 200X.

Using SEM, in control animals of both groups, well organized bone trabecules are seen. In the fishes with JD, the trabecules are not observed but there is increased connective tissue (Fig. 8).


Fig 8. SEM of dental bone a) bone trabecules of a control salmon b) severely distorted trabecules of affected specimen, group 1. 2000X.

In Table I, the morphological traits of each of the subgroups are summarized.

Table I Morphoogical traits of salmon subgroups.


Group I Salmons without JD Salmons with JD
Vegetal protein diet Horizontal dental bone. Two types of JD
No osteoid tissue is seen. Macroscopically rotated dental bone.
Collagen I predominant. Great thickness of osteoid tissue.
Low collagen III proportion. Increased proportion of collagen III.
Reduced Ca in bones as shown by Von Kossa. Reduced Ca in bone as shown by von Kossa.
     
Group II Horizontal dental bone. Slight rotation of dental bone
Animal protein diet No osteoid tissue is seen. Reduced osteoid tissue
Predominant collagen I. Less proportion of collagen III. Increased collagen III
Heavy Ca deposit as shown by von Kossa. Heavy Ca deposit as shown by von Kossa.

DISCUSSION

The present work established that the JD affecting the anterior region of the dental and specially the articular bone of smolt and adult salmon does not compromise other facial bones (lacrimal, maxilar and premaxilar, which are normal).The supportive connective tissue of the lower jaw of the affected fishes is reduced. This description, in general, agrees with that Bruno et al. 10% of the fishes with JD present unilateral malformation, a fact not reported by other authors. Moreover, this malformation was not associated with alterations of the vertebral column, such as scoliosis or lordosis, in agreement with the work of Baeverfjord & Asgard, 1998 who observed scoliosis and lordosis but not JD in fishes.

In salmons of group 1 the joint of the articular square bones is altered, with increased neoformed capillaries and fibrous tissue plus numerous melanomacrophages. This area is more fragile than other joints. According to Baeverfjord et al. (1998) this may be a sign of phosphorous defficiency.

Works done in Norway, where smolt and adult salmons are fed with a phosphorous defficient diet, scoliosis of the vertebral column and reduced rate of growth was observed (Asgard & Shearer, 1997; Baeverfjord & Asgard, 1998) However, the salmons in our work did not show column alterations associated to JD. Group 2, fed with a diet of animal origin probable is subjected to changes in absorption and his availability and other minerals, explaining why salmons in group 1 but not group 2, fed with vegetal diet were affected.

In the bone trabecules of the jaw thick osteoid tissue was found, similar to the description for racchitism or osteomalacia in mammals (Carlyle et al.; 1983; Cotran et al.; 1996). In normal bones, osteoid tissue is not detectable, since it is quickly calcified alter being formed by the osteoblasts. In cases of Ca or P differiency, osteoid tissue accumulates, since mineralization proceeds very slowly (Young & Heath, 2000). In this case, however, there is not cronic kidney disease since the deformation is localizaded, not generalized.

Parathyroid hormone release is stimulated by low Ca serum levels, promoting bone reabsorption by osteoclasts. If there are low Ca levels or parathyroid hyperfunction, the endostium of the trabecules should show osteoid tissue and numerous osteoclasts. This markers were not found in fishes with JD, implying that the parathyroid gland is not affected. In any case, the changes affected only the jaw and not other bones.

Histological analysis of the dental and articulates bones show changes in collagen fibers synthesis and their arrangement. In salmons with JD type I and II collagen fibers decreased and III (forming fine fibers), increased specimens with vitamin C deficiency have ð chains in the tropocollagen molecules unable to form estable loops to form fibrils. This change affects first the tissues with a hight collagen turn-over, such as periodontal and gingival tissue. (Gartner & Hiatt, 1997) If the deficit is prolonged, there will be changes in the vertebral column, decreased hematopoyetic tissue in the kidney shortening of the operculum and cell degeneration and vacuolation in the branchial lamelae (Bruno et al.). Salmons with JD show hemorraghic areas in the oral mucosa fracture and loosening of teeth and altered architecture of the collagen fibers; all these changes may be due to vitamin C deficiency indirectly related to altered mineralization of osteoid tissue.

Von Kossa technique was used to analyze Ca in the affected bones. Results showed that calcium content is similar in normal and deformed fishes, Moreover, the phitates ingested by the fish may have and inhibitory effect on Zn absorption (Ruz et al., 1996).

Group I samples analyzed by von Kossa, showed a low intensity of staining in all of them. Differences may be in bio availability of Ca rather than its amount. In group II, staining was greater both in healthy and in JD fishes

Food of animal origen is rich in proteins and in minerals (Ca, P, Fe, and Zn) for a fish that is carnivore, Food of vegetal origin has P as phitates and is not well absorbed. In addition phitates form insoluble complexes with Ca, so that is bioavailability decreases. Phitates also inhibit absorption of other minerals, such as Mg, Fe and Zn (Ruz et al.).

From a long time it has been advocated that protein supply (as fish flour) should be lowered in the diet for salmonids, to be replaced by energetic food, such as carbohydrates (cereals) and lipids. Formulations for replacement may hinder contamination by nitrites (NH3), which is also important to prevent diseases and reduce environmental pollution (Kinkelin et al., 1991).

JD is limited only to the jaw, In some laboratory rodents it has been observed that corticoids induce retrognathia due to inhibition of the growth of Meckel´s cartilage (Ten Cate, 1994). In addition, diazo-oxo-norleucine (inhibitor of glucosaminoglycans synthesis), reduces the length of the cartilage and delays growth of the jaw (Diewert & Pratt, 1979). These results reveal the importante of Meckel´s cartilage for growth of the jaw (Rojas et al., 1996; Gilbet, 1997). In humans, it is believed that Meckel´s cartilage determines, as cartilage center of ossification the shape of the jaw during development (Ten Cate, 1994). A fact that could be easily extrapolated to salmons.

The fact that only some of the salmon fed same diet, are affected, suggests that the JD is multifactorial. It can be triggered by nutritional deficit, mainly when animal P is replaced by vegetal P (phitates). However, it is probably that the disease will occur when other alterations, genetic or congenital, such as defects of the jaw cartilage, are present. 


VENEGAS, F.; MONTIEL, E.; FORNO, P. & ROJAS, M. Histología de la deformación mandibular en salmones del Sur de Chile (Salmo salar). Int. J. Morphol., 21(3):211-219, 2003.

RESUMEN: In the South of Chile aquacultures, it has been identified in mature salmon a deformation in the jaw inferior denomEn salmones adultos de pisciculturas del Sur de Chile se ha identificado una deformación en la mandíbula inferior denominada "deformación mandibular" (DM) la cuál afecta al hueso dentario, que se curva ventralmente hasta 90° de su posición normal, también afecta a los huesos hiomandibular y articular. Esta patología se relaciona con la pérdida de peso y un aumento de la mortalidad de los salmones. Se ha postulado empíricamente que una causa probable de esta anomalía sería el origen vegetal de la dieta de los peces (los cuáles son carnívoros). El propósito de este trabajo fue comparar la bioestructura del hueso dentario de salmones alimentados con una formula de reemplazo vegetal (soya y gluten) con los salmones alimentados con harina de pescado.

Se analizaron 55 especímenes provenientes de Puerto Montt, 35 con DM y 15 controles sanos. Las muestras del grupo 1, fueron obtenidas en Junio, Julio y Septiembre 1999, y las del grupo 2 en Marzo, Septiembre y Octubre del año 2000. El grupo 1 fue alimentado principalmente con reemplazo vegetal y el grupo 2 con harina de pescado. Cada subgrupo fue dividido en dos subgrupos, uno de animales sanos y el otro de peces con DM. La mandíbula y huesos articulares fueronm fijados en formalina al 10% y glutaraldehido al 1%, procesados para histología (Hematoxilina-eosina-azul de Alcian, tricromico de Masson, histoquímica (rojo sirius de Junqueira y Von Kossa) y microscopía electrónica de barrido (SEM).

El hueso mandibular del grupo I con DM presentó grandes cantidades de tejido osteoide comparado con su control sano. El colágeno I disminuye y cambia su composición y arquitectura, mientras que el colageno III aumenta. No se encontraron diferencias significativas en contenido de Ca entre peces normales y con DM. En los salmones con DM la microscopía electrónica de barrido muestra que el hueso dentario próximo a su articulación presenta una formación desorganizada no trabecular, comparada con el control. En el grupo 2, las deformaciones macroscópicas y microscópicas fueron menos evidentes.

Estos resultados sugieren que la DM tiene un origen multifactorial; La causa primaria puede ser genética o una alteración congénita del cartílago dentario. Esto representaría una mayor susceptibilidad en este grupo de peces, pero su expresión es gatillada por una situación de stress que podría ser un deficit de fósforo de origen animal en el alimento. Esta patología no se observa en peces con nutrición adecuada.

PALABRAS CLAVE: 1. Mandíbula,; 2. Deformación mandibular; 3. Histología; 4. Salmón; 5. Salmo salar. 


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Dirección para correspondencia
Prof. Dra. Mariana Rojas Rauco
PLaboratorio de Embriología Comparada
PPrograma de Morfología - ICBM

Facultad de Medicina
PUniversidad de Chile
PIndependencia 1027
PSantiago - CHILE

Email : mrojas@machi.med.uchile.cl
00000fvenegas@machi.med.uchile.cl

Recibido : 14-01-2003
Aceptado: 26-06-2003 


* Programa de Morfología, ICBM, Facultad de Medicina, Universidad de Chile
** Ewos Chile

Este estudio fue financiado parcialmente por EWOS-CHILE.

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