OVARIAN MORPHOLOGY OF PIKE (ESOX LUCIUS) FROM THE DANUBE RIVER, DURING THE OOGENESIS CYCLE Lucica Geru 1, Angela Trofimov 2, Flavia Ruxanda 3, V. Rus 3, I. Radu 4, R. Pop 3, V. Miclăuş 3 1 SANITARY-VETERINARY DIRECTORATE FOR FOOD SAFETY, BRĂILA; 2 INSTITUTE OF RESEARCH AND DEVELOPMENT FOR AQUATIC ECOLOGY, FISHERY AND AQUACULTURE, GALAŢI; 3 FACULTY OF VETERINARY MEDICINE, UNIVERSITY OF AGRICULTURAL SCIENCES AND VETERINARY MEDICINE, CLUJ-NAPOCA; 4 SANITARY-VETERINARY DIRECTORATE FOR FOOD SAFETY, GALAŢI Summary In order to follow the evolution of the ovary during the oogenesis cycle in the Danubian pike, captures were made at the beginning of May, end of August, November and April. The macroscopical investigations have shown a slow development of the ovary from May until August, after which a more rapid one takes place, until it occupies the whole abdominal cavity and significantly distorts it starting from November. After spawning in April, the ovary gradually regains its former size from the previous year (May). The microscopical investigation has shown the presence of a heterogeneous population of small oocytes in May and starting from August two different types of oocytes could be observed: large, relatively homogeneous and small heterogeneous. Only large oocytes are engaged in the oogenesis cycle, fact showing that the pike ovary is the group synchronous type. There were no pathological aspects, thus demonstrating that the Danubian water is at such quality standards that allow the course of oogenesis in pike. Key words: pike, oogenesis cycle, ovarian morphology. Introduction Gametogenesis in pike is cyclic, and the time between two spawnings is termed as sexual cycle. A reproductive cycle in pike lasts for about one year. In teleosts, the ovary is a hollow paired organ suspended by the mesovarium (Khanna, 2004; Shukla, 2010). The ovarian cavity continues with the oviduct and a big part of it is lined by an epithelium, similar to the one in the oviduct. The rest of the ovarian cavity is compartmentalised by folds of the germinal epithelium, called ovigerous lamellae, which project in the ovarian lumen. This lamellae contain nests of oogonia, oocytes and follicles at various stages of development and differentiation (Jalabert, 2005). Oogonia keep on proliferating in adult female thus renewing the stock of young oocytes and follicles (Tokarz, 1978). The ovarian cavity is considered to be a 307 drlucirina@yahoo.com stockroom in which ovulated eggs are kept temporarily until they are spawned (Hoar et al., 1983). According to the pattern of oocyte development, three ovarian types have been classified in teleosts: synchronous, group synchronous and asynchronous. In the synchron type, all the oocytes are at the same development stage and it is found in teleosts which spawn once and then die. In the group synchronous there are at least two types of oocyte populations in different stages of development, a synchronous one with large oocytes and a heterogeneous one with smaller oocytes. They spawn once a year. The asynchronous ovary contains oocytes at all stages of development and they spawn many times during the breeding season (Hoar et al., 1983). The group synchronous type is the most common ovary in teleosts (Kunz, 2004). According to other authors (Craig, 1996), in the speciality literature
there is scarce information on pike oogenesis. In this context we considered the investigation on the ovary of pikes, captured in the Danubis throughout a year, as being pertinent. Material and methods In order to follow the macro- and microscopic changes of the ovary throughout a sexual cycle in Danubian pike, we had conducted investigations on the captures from the sector situated between km 76, the dischargement of Prut into the Danube and km 197, Gropeni locality, in May 2010 April 2011. The biological material was represented by captures made with gillnets in the following periods: beginning of May; end of August; November and April. After opening the abdominal cavity, the ovaries were examined, photographs were taken and fragments were harvested for histological investigation. The specimen were then chemically fixed in formalin 10% for 7 days. After dehydration with ethanol and clarification with n-butanol, the specimens were embedded in paraffin wax. Paraffin sections, 5 micrometers thick, were stained with Tricrom Goldner method and examined in the optic microscope. Results and discussions Females captured at the beginning of May had elongated and weakly developed ovaries. They were translucent and the colour varied from grey-greenish to yellowish (Fig. a). The ovarian cavity and the ovigerous lamellae can be observed microscopically. They occupy most part of the ovary, and have a heterogeneous population of small oocytes (Fig. f). Females captured at the end of August had more developed ovaries that the preceding stage, with a double or larger diameter. They had a more yellowish colour and a more obvious vascularisation (Fig. b). Microscopically, a significant increase in the oocyte volume is observed and also complex substances that begin to 308 develop and accumulate in the cytoplasm. Oocytes are arranged in nests separated by fine connective trabeculae. Each nest contains two types of oocytes, large and small, the ratio between these cells differs from one nest to another (Fig. g). Vacuoles appear in large oocytes cytoplasm. Zona pellucida is now distinguishable, fine and continuous between the oocyte membrane and the follicular cells. Nucleoli can be seen inside the nucleus and they have a tendency to become located around the periphery of the nuclear membrane. In females captured in November a markedly increase in the ovaries volume is noticed, thus they occupy the entire abdominal cavity, significantly distorting it (Fig. c). The vascularisation is very clear in the ovarian wall, so that vessels, even the finest branches, can be easily appreciated macroscopically. The spawn reach an extent that allow their assessment with the naked eye. The two populations of oocytes can be observed microscopically, with the large ones increasing a lot in dimensions since August, while the small ones have nearly the same dimensions (Fig. h). The large oocytes display both diameter and shape polymorphism. The shape may vary from spherical to slightly folded or even displays polymorph folds. The cytoplasm is gradually occupied by yolk granules, in different maturation stages, excepting one perinuclear zone that appears to be occupied by an amorphous material (Fig. i). In females captured at the beginning of April the ovary and spawn reach the maximum volume and the colour changes to yellow-brown (Fig. d). Spawn can be discharged from the ovaries by simply massaging or inclining the body, which shows that the female is ready for spawning if the environmental conditions are auspicious. In females captured after the spawning, a significant decrease in the ovary dimension and a pink pale colour is observed. The mature spawn which was not
discharged during the spawning can be seen through the translucent capsule (Fig. e). Ruptured follicles, connective trabeculae and a relatively large number of young oocytes is observed microscopically (Fig. j), as well as a small number of mature spawn undischarged, which will be degraded. Gradually, the ovary changes colour and aspect, coming back to its features in stage II. The investigation we led showed the oogenesis stages in the Danubian pike. The females captured in May were in stage II of oogenesis and had thin fine ovaries that developped slowly until August (beginning of stage III), then the development rate amplifies. The ovaries come to occupy the entire abdominal cavity and significantly distort it until November (stage IV) and still develop until April (stage V). Spawning takes place around mid April and a significant increase in ovary size is observed. Microscopically, the stage II ovary contains a heterogeneous population of small oocytes in the ovigerous lamellae. The ovarian cavity is elongated and bordered by ovigerous lamellae on 2/3 of its surface and the rest of it by an epithelium similar to the one in the oviduct. The cells of this epithelium secrete a fluid, whose role is in assuring the spawn survival and its transport through the oviduct for spawning (Taati et al., 2010; Kabir et al., 2011). accompanied by the accumulation of larger quantities of yolk in the cytoplasm, therefore the oocytes increase in volume and at some point zona pellucida becomes distinguishable, thin and continuous, between the oocyte membrane and follicular cells. The synthesis and maturation processes of the yolk components finalizes in stage V of the oogenesis, when the completely formed spawn awaits the optimum moment for ovulation. Spawn is rapidly discharged, but the few remaining in the ovaries enter relatively rapid in degenerative process and will be resorbed. Gradually, the ovary returns to its features in stage II. Besides the oogenesis stages, the investigation accentuates the fact that the Danubian water is not polluted in such a way that could significantly influence the oogenesis, at least qualitatively. This information is backed up by the fact that we did not identify anomalies, degenerative or alteration processes of the female sex cells of the pikes taken into the present study, regardless of the oogenesis stage in which the female was. The susceptibility of the female sex cells of pike to certain disturbing agents was highlighted, among others, by Belova and Emel yanova (2009) that investigated pikes and 4 other fish species from Teterev river and Kyev Reservoir, 20 years after the Chernobyl accident, claiming that the pike was the most sensitive. Oocyte growth is slow until the end of August, when a significat increase in the development rate of some oocytes is observed. Besides, there are small oocytes that do not take part in this oogenetic cycle. They are arranged in nests separated by fine connective trabeculae. Each nest contains both large and small oocytes, the ratio between them being different from one nest to another. The presence of two types of oocytes at once, suggests that the pike ovary is the group synchronous type, like in the majority of teleosts (Kunz, 2004). The beginning of the vitelogenesis stage is 309 Conclusions The ovarian cavity of the pike has an elongated shape and is bordered by ovigerous lamellae on 2/3 of its surface and the rest of it by an epithelium similar to the one in the oviduct. The oviduct continues with the ovarian cavity. Throughout the oogenesis cycle in pike there are two types of oocytes, one that participates in oogenesis and other that awaits, which makes the pike ovary the group synchronous type. There were no anomalies, degenerative or alteration processes of the
female sex cells of the pikes in the present study, regardless of the oogenesis stage in which the female was. The investigation we led highlights the fact that the Danubian water is at quality standards that ensure a normal oogenesis. References Belova, N.V.; Emel yanova, N.G.: Status of the Reproductive Sistem of Bony Fish from the Teterev River and the Kiev Resorvoir 20 Years after the Chernobyl Accident, Journal of Ichthyology, 49, 9, 793 802, 2009. Craig, J. F.: Pike: Biology and Exploitation, Published by Chapman and Hall Fish and Fisheries Series, Ed. Springer, Printed in Great Britain at the University Printing House, Cambridge, vol. 19, chap. 2, 25, 27, 28, 1996. Hoar, W.S.; Randall, D. J.; Donaldson E.M.: Fish Physiology, vol. IX, Reproduction Part A, Endocrine Tissues and Hormones, United Kingdom Edition published by Academic Press, INC. (London), 229-231, 238 245, 1983. Jalabert, B.: Particularities of reproduction and oogenesis in teleost fish compared to mammals, Reproduction Nutrition Development, 45, 261 279, 2005. Kabir, M.; Imanpoor, M. R.; Asghari, M.: The effects of different times of reproductive migration on biochemical compounds of ovarian fluid and on fertilization rate of Persian sturgeon (Acipenser persicus) brood stocks, AACL Bioflux, 4, 3, 351 360, 2011 Khanna, D.R.: Morphogenesis, Published by Discovery Publishing House, India, 88 89, 2004. Kunz, Y. W.: Developmental Biology of Teleost Fishes, vol. 28, In: Fish and Fisheries Series, Published by Spinger, Netherlands, 9, 2004. Shukla, J. P.: Fish and Fisheries, Second Edition, Ed. Rastogi Publications, Printed at Arihant Electric Press, Meerut, India, 184-188, 2010. Taati, M. M.; Jafaryan, H.; Mehrad, B.; Golpour A.: Changes of ovarian fluid compositions and sperm quality parameters in koi (ornamental Cyprinus carpio) during spawning season, ABAH Bioflux, 2, 2, 83 90, 2010. Tokarz, R.R.: Oogonial proliferation, oogenesis and folliculogenesis in nonmammalian vertebrates. In: Jones RE (Ed), The vertebrate ovary, Comparative Biology and Evolution, Ed. Plenum Press, New York, 145 179, 1978. 310
a f b g c h d i e j Fig. a-e Pike ovaries macroscopical aspects : a) stage II; b) stage III; c) stage IV; d) stage V; e) stage VI Fig. f-j Pike ovaries microscopical aspects (Tricrom Goldner) : f) stage II (ob.2); g) stage III (ob.2); h) stage IV (ob.2); i) yolk vesicles (stage IV,ob.40); j) stage VI (ob.2) 311