Histology and hormonal effect of Ghrelin on ovary of Benni (Barbus sharpeyi) in order to study sexual maturity, zygote and larval generation

Benni (Barbus sharpeyi) is valuable fish that Khuzastan fisheries office propagated it artificially in Susangerd Fish Propagation Center every year. Pituitary gland is used for this aim but female fish lost their fertilization power after 2-3 years, so in present research, new hormone, that is called Ghrelin. The aims of this research are histology, hormonal, zygote and larval generation studies and comparing the results with each other. Ghrelin is a multifunctional peptidyl hormone which increases GTH-II in fish, amphibian, and birds and mammalian so its effect on Benni sexual maturation was studied. Human Ghrelin (hGRL) was obtained from ANASPEC, Canada, with 28 amino acids. In the present study, three levels of ghrelin including 0 (sham treatments), 0.10 (treatment 1) and 0.15 μg/g (treatment 2) body wt and one level of pituitary gland 4000 μg/g (pituitary treatment) with two replications were used. 56 specimens were injected intraperitonealy and their ghrelin level was evaluated immediately after injection and after 24 h. Control fish(n=16) were just injected by physiological saline. For hormonal studies sham and experimental fish(n=40) were anesthetized with MS-222 at a concentration of 250 mg l-1, and blood samples were collected and kept at 4ْC, then spun to collect serum. Serum samples were stores at -20ْC until the RIA for CTH-II. For histology studies immediately after injection a piece of ovary was collected from control fish (Sham zero) after being anesthetized. The sampled ovaries were fixed in Buin solution and embedded in paraffin, and stained to Sections of 5–6 μm using haematoxylin and eosin. The ovarian samples were performed with a compound microscope. Histology and micrometry studies had done. The mature oocytes had given from mature fish, then weighted and the working fecundity were counted. The mature oocytes fertilized, the eggs were incubated and the percentage of fertilization was calculated. After 72h the eggs hatched and the percentage of hatch was counted. The percentage of hindrance was calculated after 6 days. Hormonal results indicate that ghrelin and pituitary increase significantly the GTH-II level in comparison to sham. Macroscopic observations (before taking ovary) showed that ovaries with green colored have couple oval structure located in the abdominal cavity. Microscopic studies of dissected ovaries indicated simultaneous growth of 127 oocytes with 6 stages. The type of the ovary is asynchronous. The results indicated that both of the ghrelin treatment increased the percentage of mature follicles followed by decrease of immature follicles. There were significant differences (P<0.05) between the number of mature and immature follicles. Average diameter of follicle in both of the ghrelin treatment was significantly (P<0.05) declined in the stages of the vitellogenesis when the result compared to the other treatment. Just treatment 1 and pituitary treatment can give mature oocytes. The fecundity of pituitary treatment significantly increase in comparision to ghrelin treatment (P<0.05). In food-restricted fish where endogenous ghrelin levels are known to be increased, a chronic administration of ghrelin induces overt negative effect in releasing mature oocytes. The percentage of fertilization was significantly increase (P<0.05) in ghrelin t. in comparison to pituitary t. and the percentage of hatch was significantly increase (P<0.05) in pituitary t. in comparison to ghrelin t. There was no significant difference (P>0.05) in terms of percentage of hindrance between treatments. In conclusion, the present study demonstrated that ghrelin has positive effect on the level of GTH-II, oocyte maturation, ovarian vitellogenesis and the number of mature follicles of Barbus sharpeyi ovary. Increasing of the mature follicles number reduces their average diameter, indicating stimulating effect of ghrelin in sexual maturation of Barbus sharpeyi.The ghrelin and pituitary treatment have equal chance in the post-stage of spawning.

Study on biological characteristics, sexual maturation and reproduction of Liza abu in the water of Khozestan Province

As the most of the fish resources are known and exploited, protecting their generation is of the greatest importance. Aquaculture is one of the efficient procedures in protecting and reviving fish resources and knowing about the reproductive cycle and gonads development has an important role in approaching this aim. Liza abu belongs to the family Mugilidae that according to its resistance to the environmental condition and its fast growth , can be introduced as a fish with economical value. As there is no scientific data on the reproductive biology of this species , study on the reproductive biology and gonad development is considered as the aim of this research . For this purpose , 360 samples of this species were investigated during the period from February 2007 to January 2008 in Khozestan Province . After studing morphological and histological characteristics of gonad specimen , they were prepared through histological method. Samples were prepared through usual histological method and studied under light microscope. According to the results, the maturity stages of male and female Liza abu were separated to six different successive stages. In ovaries , these stages were as follow : In stage І, the oocytes were small , this stage was observed from July to October . In stage ІІ, considerable growth was observed in the oocytes . This stage was observed from October to January . In stage III, due to vitellogenesis, the maximum growth was observed and three layers of theca, granullosa and follicle cells were visible. This stage was observed during January and February . In stage IV, migration of germinal vesicle was observed and due to hydration of the oocytes , their diameter was increased. The ovaries were yellowish and in maximum size and ovules could be easily observed with naked-eye . This stage was observed in February and March . In stage V, spawning occured. This stage was observed in April . In stage VI, ovaries consisted of immature and atretic oocytes and also empty follicles. This stage was observed in May and June. In testes , these stages were as follow : In stage I , the testes were small in size and contained the spermatogonia which were the only cellular components.This stage was observed in August and September . In stage II (maturing virgin ) , the spermatogonia and the primary spermatocytes were visible. This stage was observed in October . In stage III (developing), intensive spermatogenesis was occured and the primary and the secondary spermatocytes were the most visible cells during this stage .This stage was observed from November to January. In stage IV(developed), cells of all stages of spermatogenesis could be seen but the secondary spermatocytes and spermatids were in large number. This stage was observed from January to March. In stage V , the testes were filled with sperms. This stage was observed in March and April .In stage VI, residual spermatozoa and the spermatogonia were visible in the testes. This stage was observed from May to August. According to cyclic changes in GSI, sexual maturation in breeding begins in January and spawning occurs in April. The ova diameter ranged from 30.75 μ in stage I to 472.19 μ in stage IV. In this study , the sex ratio was 1:2.7, and male and female percentage were 27.02% and 72.98% respectively. This means that females predominate males. In this study absolute fecundity was calculated and changing between 30805.44 to 431247.3 was observed and absolute fecundity was calculated 111275.3 in average.