S ke I e tal Malformations

THE ANATOMICAL RECORD 233~314-320 (1992)

in Hatchery Reared Rana perezi Tadpoles IRENE MARTfNEZ, RAFAEL ALVAREZ, INMACULADA HERRAEZ, AND

Departamento de Biologia Celular y Anatomia, Facultad de Biologia, Universidad de Ledn, 24071 Lebn, Spain

PAZHERRAEZ

ABSTRACT Skeletal malformations at metamorphosis in Rana perezi tad- poles reared in culture have been studied. Tadpoles were fed ten artificially com- pounded and fresh diets and kept at two different temperatures. Animals were maintained in fiberglass tanks. Bone damages are related to the nourishment with compound diets. Tadpoles fed such diets showed variable percentages of scoliosis, kyphosis, luxations, and subluxations in the hind limbs, aplasia in phalanges, and wide failures of ossification. The data suggest the origin of the lesions is a defi- ciencv in some compounded diets that could alter collagen metabolism during skeleial adjustments at metamorphosis.

Intensive culture of frogs is one of the most compli- cated forms of aquaculture. It has been developed in several countries such as the United States, Japan, Cuba, Mexico, and Brazil since the beginning of this century (Lima and Agostinho, 1984; Martinez et al., 1990). The most widespread cultured frog is Rana catesbeiana and its main markets are the food industry, and biomedical education and research. This activity recently began to be established in Europe; in the last few years, studies have been undertaken on the semi- intensive production of the Spanish common frog, Rana perezi, with a view to establishing the ideal conditions for its industrial production (Martinez et al., 1990).

Little information exists on skeletal deformities in natural groups of amphibians (Sanchiz and Perez, 1974); reference is made to the presence of supernu- merary limbs (Luis and Baez, 1987), the duplication of the fore and hind limbs, and also of the phalanges as a result of the presence of parasitic cysts in the joints (Sessions and Ruth, 1990), etc. Deformities in the axial skeleton, the vertebral column, and the notochord have been experimentally induced in tadpoles through the use of toxics during embryogenesis (Schultz et al., 1985; Riggin and Schultz, 1986; Scaddmg, 1990), through incubation in water with low levels of calcium ion (Marshall et al., 19801, or through diets with vita- min C deficiencies (Leibovitz et al., 1982). Malforma- tions of the limbs have also been attributed to acid stress and rate of growth (Cummins, 1987, 1989). There are, however, few references to malformations in amphibians reared in culture (Mohanty and Dash, 1986; Leibovitz et al., 1982), and the factor or factors that produce them are still under discussion.

More data exist on skeletal lesions in fish, because the high proportion in which they occur is a serious problem in aquaculture. The non-congenital causes that have been suggested as possible origins of the problem in natural populations are very diverse, such as high levels of toxins, pesticides, or heavy metals (Hirose and Kitsukawa, 1986; Hodson et al., 1980; Neilson et al., 1984; Bengtsson et al., 1988), problems in the gallbladder (Kitajima et al., 1981), or bacterial endotoxins (Norton et al., 1969). In animals reared in

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0 1992 Wiley-Liss, Inc.

culture, nutritional deficiencies (Lovell, 1975; Sato et al., 1978; Fujita, 1979; Hodson et al., 1980), high den- sities of incubation (Devauchelle, 1976) high levels of solar radiation (Barahona-Fernandes, 19781, etc., should be added to these factors, as they have been shown to affect the development of the axial skeleton.

In this study malformations found in Rana perezi tadpoles kept under controlled conditions are de- scribed.

MATERIALS AND METHODS After hatching under laboratory conditions larvae of

Rana perezi were reared with natural lighting in 20 litre fiberglass tanks with a water flux about 200 mli min and 100 specimens per tank. The pH of the water was between 7 and 8, the dissolved oxygen was greater than 7 mg/l, and the calcium content of the water was 37-38 mg/l. Ten different diets, whose contents are shown in Table 1, were tested in triplicate. Animals were fed ad libitum, the diets were replaced every sec- ond day, and food consumption was calculated weekly as percent of biomass. The experiment was carried out a t two different temperatures (19/22"C and 22125°C). The specimens which presented skeletal malforma- tions during metamorphosis were fixed in 10% formol, their deformities studied and classified, and several representative specimens of each type were dyed alcian blue-alizarin red, following the Hanken and Wassersug (1981) modified method, so that the skeleton could be observed.

The percentages of deformed specimens in each pro- cess, with respect to the number of survivors, were compared using the multiple range test Student-New- man-Keuls (P < 0.05).

RESULTS The malformations observed were divided into four

groups:

Received July 22, 1991; accepted December 24, 1991.

315 SKELETAL MALFORMATIONS I N TADPOLES

TABLE 1. Proximate composition of the diets1

Carbo- Moisture Protein Lipid hydrate Ash

P1 Compounded Different flours, sugar, milk, and egg yolk (Lima and 39.32 39.96 9.71 36.61 13.72 Diets Composition (%I (%) (%I (%I (%I

Agostinho, 1984) PlVH Mixed P2 Compounded

P2VH Mixed P3 Compounded

P3VH Mixed V Fresh H Fresh VH Fresh L Fresh

P1 six days a week and VH on the seventh Shrimp and fish meal, yeast, bran, soy protein, whey, 50.84 40.30 11.12 34.03 14.55

oil, gelatin, vitamins, linolenic acid, oxytetracycline, sulfamerazine, and agar as a binder (Culley et al., 1978)

and agar as a binder

P2 six days a week and VH on the seventh Commercial animal feed for trout (Ipes Iberica, S.A.) 64.21 48.73 10.98 28.57 11.72

P3 six days a week and VH on the seventh Boiled lettuce, boiled carrot, and agar 94.25 13.27 0 77.59 9.14 Boiled calf liver and agar 83.58 70.04 12.99 11.22 5.75 Boiled lettuce, carrot, calf liver, and agar 89.26 55.31 9.87 27.88 6.94 Boiled lettuce, carrot, and calf liver (w5thout agar) 85.53 43.40 8.22 39.66 8.72

'Moisture content expressed as percentage of fresh diet; protein, lipid, carbohydrate as percentage of dry weight or diet (analysis according to AOAC, 1990).

1. Malformations of the tail. 2. Malformations of the vertebral column. 3. Malformations of the hind limbs. 4. Multiple malformations.

The total percentages of malformed animals which appeared in each treatment, the distribution within the various types and the time in weeks they take to complete metamorphosis are shown in Table 2.

Malformations of the Tail These comprise the highest percentage of the de-

formed specimens. They consist of different degrees of S-shaped lateral curvatures. These curvatures affect the notochord, which in some cases appears dilated, following an irregular path (Fig. 1). In the most acute cases this type of malformation leads to a lateral dis- placement if the iliacus and the urostile (Fig. 2), which, nevertheless, disappears when the tail finally re- gresses during metamorphosis, and the specimens show a completely normal skeleton, apart from a slight deviation of the urostile, which apparently does not affect their capacity of movement.

Malformations of the Vertebral Column The greatest percentage of this type of alteration is

made up of the lateral deviation of single or double- curved scoliosis. They are produced at different levels of the backbone, with angles of up 60" and generally provoke the tilting of the pelvic girdle. This is accom- panied by a rotation of the vertebrae and shortening of the lateral apophyses on the convex side (Fig. 3). De- fects in the vertebral body do not usually occur, al- though at times there is a slight wedging of the verte- brae. Some specimens show dark brown, irregularly shaped marks, apparently located between the verte- bral body and the neural tube, above the latter, the nature of which has not been identified (Fig. 4). Defects in the closure of the back vertebral arch have also been observed (Fig. 5). A small number of specimens showed dorsoventral deviations, of the kyphosis type.

Limb Deformities The observation of specimens in vivo shows that

these consist of stiffness in one or both hind limbs, which may affect one or more joints. The dying of the skeleton shows complete luxations (Fig. 6) or dysplasia of subluxations of the affected joints (Fig. 6, 7).

Multiple Malformations These consist of the presence in the same specimen of

deformities in the legs and in the column. These ani- mals generally suffer a high degree of deformity and in them can be seen generalized problems in bone forma- tion (Fig. 71, seen in the shortening, widening, and arching of the diaphysis of the long bones, the appear- ance of cup-shaped epiphysis with undulating edges, and the presence in the diaphysis of clearly delimited, oval-shaped non-ossified areas, similar to the fibrous cortical defects in higher vertebrates (Fig. 8). In some cases these failures in ossification are accompanied by the loss of phalanges in one or more fingers (Fig. 9).

The percentage of affected animals is significantly different (P < 0.05) in animals fed fresh or compounded diets. Only two of the specimens fed on fresh diets show slight malformations of the tail, which do no affect the formation of the skeleton, but the presence of lesions is constant when the diet is compounded. Of the three compounded diets tested, P2 is that which produces the least number of deformities; with P3 a significant in- crease in the percentage of deformed animals is pro- duced (P < 0.05). The use of fresh food once a week with the P3 (PSVH) compounded diet significantly reduces the number of lesions, which drops to the levels ob- served with P1 and PlVH at both tested temperatures iP < 0.05).

Skeletal malformations are more numerous at high temperatures, with a special, significant increase in batches fed on P3 (P < 0.05).

The increase in the number of affected specimens fed on P3 and P3VH is mainly due to an increase in lesions in the limbs and in multiple deformities.

All the diets were well accepted and seem to be pal-

316 I. MARTfNEZ ET AL.

TABLE 2. Percentage of deformed tadpoles (x% Def.), percentage of animals with deformities in tail (%D.T.), column (%D.C.), limbs (%D.L.), and multiple malformations

(%.D.M.), and average time in weeks to reach metamorphic climax (W) in each treatment

Diets x % Def. % D.T. % D.C. % D.L. % D.M. W 19122°C

PlVH 2,06 16,6 66,6 0 16,6 12

P2 2,12 33 0 33 33 11 P3VH 879

P1 5,53 25 18,7 31,2 25 11,6 P2VH 1,02 33 0 66 0 11,3

23,l 7,69 69,2 0 11,3 33 0 33 33 11,3 P3 10,7

V 0 0 0 0 0 20,6 H 0 0 0 0 0 12,3 VH 0,6 1 0 0 0 16,6 L 0 0 0 0 0 21

PlVH 9,5 62,9 11,l 22,2 3,6 7,3 P1 8,62 56 28 11,l 3,7 723 P2VH 1,06 66 33 0 0 793 P2 0,34 1 0 0 0 636 P3VH 15,7 21,l 5,26 52,6 15,8 8 P3 44,2 38,7 3,22 3 5 3 22,5 7,6 V 0 0 0 0 0 20,3 H 6 1 0 0 0 9,6 VH 0 0 0 0 0 936 L 0 0 0 0 0 9.3

22125°C

atable to the species. Food consumption is bigger a t high temperatures and increases gradually during de- velopment. The percentage of food consumption per week with respect to the biomass does not show big differences within the various diets, except for P3 and P3VH, with a higher consumption. Thus, in the treat- ment a t low temperatures their values (maximums of 20.1% and 25.9%, respectively) are even four times big- ger than those of the other diets (maximums between 2.24% and 6.93%).

The increase in temperature substantially dimin- ishes the period of metamorphosis. The specimens kept at 19122°C show slower development with V, VH, and L diets than those fed on compounded diets and liver (Pl, P2, P3, and H). At high temperatures prepared diets result in a shorter time to metamorphic climax, P2 specially promotes development, but there are slight differences between P3 or liver nourishment.

DISCUSSION Given these results, the clear relation that exists be-

tween diet and the appearance of the bone damage would seem to indicate that the origin of these lesions may be in the deficiencies in the formulation of P1 compounded diet and, in particular of P3. The decrease in deformities that occurs when P3 compounded diet is complemented with fresh food supports the hypothesis of the metabolic origin of the problem. Food consump- tion shows that there are no nutritional deficiences be- cause of low palatability in any diet.

The types of lesions observed may correspond to dis- orders in the connective tissue. Likewise, the defects in the tail and the backbone are probably produced in the same way as the lordoses described by Schultz et al. (1985) and Riggin and Schultz (1986). They observed that the use of lathyrogenic drugs causes an incorrect formation of the connective notochordal sheath by pre-

venting the cross-linking of the collagen fibers. The laxity of this notochordal sheath would produce the curvature of the tail and the malformation of the no- tochord, as well as rotations and displacements of the vertebral column (which is formed according to the axis marked by the notochord). Treatments with lathyro- genic drugs during embryogenesis (Schultz et al., 1985; Riggin and Schultz, 1986) fundamentally cause lor- doses but not scoliosis, from the first stages of develop- ment, probably because the great laxity of the noto- chordal layer causes its curvature a t the point of greatest tension, just a t the beginning of the tail, below the viscera. In the specimens studied here the damage appeared in more advanced states, basically in the tail and as scoliosis, probably as a result of the S-shaped movement when swimming, which causes lateral ten- sion rather than dorsoventral. Either because the dam- age is not so serious, or because it appears late, or for other reasons, the lordoses described by other authors are not produced. Failures in the formation of collagen fibers have also been related to skeletal malformations in fish (Sato et al., 1978).

Marshall et al. (1980) also observed scoliosis and crooked limbs in Rana catesbeiana as a result of rear- ing in culture water with low calcium ion content; these decrease when the diet is supplemented with this element. However, the joint laxity observed in this study could also be explained by a problem in the for- mation of collagen which would affect the skeletal re- adjustment that takes place during metamorphosis, rather than by calcium ion deficiencies are suggested by Marshall et al. (1980) and Cummins (1989) since, as has been observed, the lesions in the limbs are lux- ations and subluxations, which can be due to the hy- poplasia of ligaments and tendons. Moreover, the cal- cium content in the water used here was considerably superior to the one tested by Marshall et al. (1980).

SKELETAL MALFORMATIONS IN TADPOLES 317

Fig. 1. Notochord with multiple curvatures showing an irregular

Fig. 2. Animal with a S-shaped tail (arrowheads) displaying the

the hip. b: Detail of the assymetric lateral apophysis (arrows) and irregular marks between the vertebrae and the neural tube (as- terisk).

Fig. 4. Several spots between the vertebral archs and the neural

thickness (arrowheads).

rotation of the iliacus and urostile (discontinuous arrows). tube (arrows).

Fig. 3. a: Double scoliosis with rotation of vertebrae, shortening of the lateral apophysis at the convex side (arrows), and tilting of

318 I. MARTINEZ ET AL.

Fig. 5. Defect in the closure of the vertebral arch (asterisk).

Fig. 6. Iliofemoral luxation (arrow) and femorotibial subluxation

Fig. 8. Arched, shortened, and enlarged fibula and tibiale showing non-ossified areas (arrowhead) and epiphysis displaying undulating edges (arrow).

(arrowhead). Fig. 9. a: Foot with only two fingers, one of them lacking two pha-

langes. b: Foot lacking two complete fingers with every bone arched. Fig, 7. Multiple malformations. All joints are dysplasic, the dia- physes are arched (asterisks), and the epiphysis cup-shaped (arrow- head).

Cummins (1987, 1989) also describes in vivo defor- food. The results obtained in the present study do not, mities in the limbs of Rana temporaria, which seem however, show any relation between the appearance of similar to those described here and which relate to the deformities and these parameters. Moreover, the growth rate and the intensity of the competition for method of feeding was the same in all cases, so that

SKELETAL MALFORMATIONS IN TADPOLES 319

there is no difference in the access to food with one or another diet. Although prepared diets promote the de- velopment with regard to the fresh diets, we deem this is not the cause for the systematic appearance of le- sions, because there is not a direct relation between the percentage of deformities and the speed of develop- ment. So, with P2 there are few affected tadpoles and the fastest development; besides, the time taken to complete metamorphosis is not statistically different in those specimens fed on P3 or on liver (HI, although there are great differences with regard to their mor- phogenetic effects.

It is known that the organogenesis of long bones in- volves the synthesis, remodeling, and degradation of collagenous matrix. The alterations of this matrix could cause the generalized defects in ossification, in the same way as some types of osteogenesis imperfecta described in higher vertebrates (Prockop et al., 1985; Minor et al., 1985). As Minor et al. (1985) indicate, the diseases of collagen must result from 1) a quantity de- crease in the amount of collagen per unit area of tissue, 2) qualitative changes in the organization of collagen in fibrils andlor fibers, or 3) a decrease in the formation of covalent cross-links between collagen molecules within fibrils. The regulation of all these processes in- volves the participation of a high number of enzymes. One of the critical points in collagenic metabolism is the hydroxylation of amino acids which allows the cross-link of collagen fibrils and requires vitamin C as a cofactor. Thus, Leibovitz et al. (19821, working with Rana catesbeiana tadpoles, report evidence of lesions in the backbone and in the tail, seen in X-rays, that seem to be similar to those described in this study, and that are also related to the administration of diets low in vitamin C. The administration of this vitamin in tad- pole feed is problematic, given the slow way in which these animals eat, since the vitamin breaks down after a long period in water (Leibovitz et al., 1982). This diminution can be seen to accelerate a t high tempera- tures, which would explain the increase in deformities a t 22125°C.

To sum up, although our data support the hypothesis that nutritional deficiencies obstruct the correct forma- tion of collagen, provoking skeletal malformations, fur- ther histophysiological studies are necessary to deter- mine exactly which of the steps in the processing of collagen is affected and which types of collagen have been modified, as well as the possible spreading of the alterations to other organs and tissues.

ACKNOWLEDGMENTS This reasearch was supported by a grant from

C.D.T.I. (Ministry of Industry and Energy) (89-0098) through RANASSAS, S.A. We wish to thank Dra. Con- cepci6n Dominguez for their help in analyzing food samples.

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