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RP2E INRA Université de Lorraine

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Histological study of embryogenesis and classification of developmental impairments in the Eurasian perch, Perca fluviatilis

5th International Workshop on the Biology of Fish Gametes, 07-11 septembre, Ancone, Italie

Alix, M., Chardard, D., Feizbakhsh, O., Ledore, Y., Schaerlinger, B., Fontaine, P.

2015

INTRODUCTION

The Eurasian perch is an important candidate to promote diversification of the freshwater fish species in Europe. Indeed, its delicate flesh is appreciated and has a high economical value. Since several years the control of perch reproduction through photo-thermo periodic variations has been unlocked (Abdulfatah et al., 2011). Now, a new stock point appears to be the gametes quality and developmental impairments (mortality and/or abnormalities occurrence) during embryogenesis. However, this question is difficult to discern because criteria used to define quality are not always well characterized. In addition, the normal development of the perch is still poorly known. A first description of its embryogenesis remained elusive (Chevey, 1925). We thus choose (i) to better characterize the normal development of P. fluviatilis through video-microscopy and histological studies and (ii) to use this developmental table to define most common deformities met in perch development and mortality stages. When possible, those data further allow us to preset spawns ‘quality and then do a classification of the spawns.

 

MATERIALS AND METHODS

To ensure a large diversity of spawn quality experiments have been conducted independantly on fish with diverse origins: (i) domesticated fish from Geneva Lake (Lucas Perches (Hampont, France)) induced in our indoor facilities thanks to Abdulfatah et al., 2011 and (ii) wild perch caught in two ponds in Lorraine (GAEC Piscicole du Saulnois and D. Nippert pond) one month before the spawning season and kept in our outdoors facilities. Artificial reproductions were performed according to Zarski et al. (2012), in order to determine the beginning of development. After fertilization and until the first oral feeding, embryos were incubated in our indoor hatchery at 13°C ± 0.5°C. Embryogenesis of 35 spawn was studied by time lapse-video microscopy, using a light upright optical microscope (Nikon Eclipse Ni-U). Embryos were sampled once a day and fixed in Bouin’s solution to later perform histological studies and analyze organs ontogeny. In addition, the survival rate was checked daily for 5 days (300 randomly choosen egg/spawn) to determine mortality stages and hatching rates. Abnormal embryos were retrieved and fixed in 4% formaldehyde to perform microscopic studies or in Bouin’s solution for histological experiments.

 

RESULTS

In Eurasian perch, embryogenesis, from ova’s activation to the first oral feeding lasts for 15 days at 13°C (optimal temperature according to Saat and Veersalu, 1996). It can be divided into 5 main periods: zygote, cell cleavage, gastrulation, organogenesis and free embryo periods from hatching to the first food intake..Some of these periods can further be subdivided. The cell cleavage begins 2.5 hours after fertilization and lasts for 24 hours as the gastrulation. So the third day post-fertilization (dpf) the first tissue differentiation begins with the definition of antero-posterior axis, the notochord and the optic vesicle apparitions before the tailbud closure. We choose to focus a particular attention on the ontogeny of digestive and visual functions because they play an important role in the first oral feeding and larval survival. The optic capsule first appears 3 dpf and, the various layers of the eyes develop in parallel. At 7dpf, the first choroid melanocytes appear and give their color to the eyes. From 8 dpf, the eyes are histologically mature. The digestive system first develops from 4 dpf with the apparition of intestine anlage. The first peristaltic movements take place at 10 dpf and all embryos first feed 15 after the fertilization. First hatching occur from 6 dpf and last for 5 days in average within a spawn, whatever the origin of the breeders. The last embryo to hatch was observed14.5 dpf while the first food intake took place 15 dpf in every tested spawn. It means the free embryo period can elapse from 9 to 1/2 days during which the embryo fast.

Once the normal development described, it was interesting to make an overview of the developmental defects that could be met during perch development. We focused on the characterization of deformities occurrence and the identification of key steps that could lead to a massive lethality. Concerning the later one, three main groups of spawn were found with different survival rates upon the time. The first category groups spawn with hatching rates over than 90%, the second group spawn had high mortality during early embryogenesis and low hatching rate (60 % and 14 %, respectively) and finally the third one corresponds to embryos that never hatch and die during early embryogenesis. An accurate study of these lethalities showed two stock point 24h and 48h after the fertilization suggesting that the cell cleavage to gastrulation and gastrulation to organogenesis transitions are key steps for reproduction success. In addition, the study of the deformities occurrences showed that, in our conditions, the deformities rates range from 0 to 20 %. In the meantime, 6 main categories of deformities have been characterized according the abnormal organ (e.g. skeletal, yolk, jaw, eyes or heart). A statistical analysis of these data allows to make a classification of spawn according to the lethality stage and the type of abnormalities.

 

DISCUSSION AND CONCLUSIONS

Pierre Chevey first (1925) investigated Perca fluviatilis development. Most of our observations seem similar to these his study and we complete with histological observations. Among the crucial point is the definition of the end of the embryonic development. While some believe that this is hatching, other use the first food intake (Peñaz, 2001). In our study, we clearly observed that the onset the first food intake always takes place 15 dpf while hatching can last over 5 days. In these conditions this last step better define the end of embryogenesis. Our study of the developmental impairments showed that (i) the end of the cell cleavage and the gastrulation both constitute crucial stages the defects of which could lead to death and (ii) some organs seem particularly touched by potential deformities. These data constitute a solid basis to investigate further the reasons of the developmental defects in perch and their links with the gametes quality.

 

AKNOWLEDGEMENTS

This work was supported by Research Ministry (PhD fellowship of M.A.), INRA Departement PHASE, Lorraine region and the COST Action FA1205 AQUAGAMETE.

 

REFERENCES

Abdulfatah, A.; Fontaine, P.; Kestemont, P.; Gardeur, JN.; Marie, M., 2011: Effect of the photothermal kinetics and amplitude of photoperiod decrease on the induction of the reproduction cycle in female Eurasian perch Perca fluviatilis. Aquaculture 322-323, 169-176.

Chevey, P. 1925: Recherches sur la perche et le bar. Etude embryogénique, systématique et biogéographique des percidés européens. In: Bulletin biologique de la France et de la Belgique. Paris

Saat, T.; Veersatu, A., 1996: The rate of early development in perch Perca fluviatilis L. and ruffe Gymnocephalus cernuus (L.) at different temperatures. Annales Zoologici Dennici 33, 693-698.

Peñaz M., 2001: A general framework of fish ontogeny: a review of the ongoing debate. Folia Zoologica 50 (4): 241-256.

Zarski, D.; Horvath, A.; Kotrik, L.; Targonska, K.; Palinska, K.; Krejszeff, S.; Bokor, Z.; Urbanyi, B.; Kucharczyk, D., 2012: Effect of different activating solutions on the fertilization ability of Eurasian perch, Perca fluviatilis L., eggs. Journal of Applied Ichthyology 28, 967-972.

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