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.

Biological traits, and egg and fecal pellet production of Calanus finmarchicus and Calanus glacialis in situ and during experiments

The effects of temperature and food was examined for Calanus finmarchicus and C. glacialis during 3 phases of the phytoplankton spring bloom in Disko Bay, western Greenland. The 2 species were collected during pre-bloom, bloom, and post-bloom and exposed to temperatures from 0 to 10°C, combined with deficient or excess food. Fecal pellet and egg production were measured as indices for grazing and secondary production, respectively. Furthermore, changes in body carbon, nitrogen, and lipid content were measured. C. glacialis sampled before the bloom and incubated with excess food exhibited high specific egg production at temperatures between 0 and 2.5°C. Higher temperatures did not increase egg production considerably, whereas egg production for C. finmarchicus more than tripled between 2.5 and 5°C. Starved C. glacialis produced eggs at all temperatures stimulated by increasing temperatures, whereas starved C. finmarchicus needed temperatures above 5°C to produce eggs fueled by their lipid stores. Few C. finmarchicus had mature gonads at the initiation of the pre-bloom and bloom experiment, and egg production of C. finmarchicus therefore only increased as the ratio of individuals with mature gonads increased. During the bloom, both C. glacialis and C. finmarchicus used the high food availability for egg production, while refueling or exhausting their lipid stores, respectively. Finally, during the post-bloom experiment, production was low by C. finmarchicus, whereas C. glacialis had terminated production. Our results suggest that a future warmer ocean will reduce the advantage of early spawning by C. glacialis and that C. finmarchicus will become increasingly prevalent.

Characteristics of eggs and female adults of Calanus finmarchicus, C. glacialis and C. hyperboreus from Disko Bay, western Greenland

This is the first high temporal-resolution study in Disko Bay covering population dynamics, grazing, reproduction, and biochemical composition of 3 dominating copepod species (Calanus finmarchicus, C. glacialis and C. hyperboreus) from late winter to midsummer in 2008. C. finmarchicus and C. glacialis ascended to the surface layer at the onset of the spring phytoplankton bloom, followed by C. hyperboreus 2 wk later. C. finmarchicus spawning occurred during the bloom and postbloom period, partially fueled by wax esters. C. glacialis commenced spawning before the bloom, yet it was greatly stimulated when food became available. However, feeding and reproduction was terminated after the main bloom despite the presence of food. In terms of feeding, this was also the strategy for C. hyperboreus. Between pre-bloom and post-bloom, C. finmarchicus showed an increase in carbon, nitrogen, and phospholipid content but a decrease in total lipid content. This was likely the result of protein synthesis, oocyte maturation, and spawning fueled by wax esters and by feeding. C. glacialis showed a similar pattern, although with an increasing total lipid content from pre-bloom to post-bloom, and an increasing wax ester and decreasing phospholipid content after reproduction was terminated. C. hyperboreus showed greatly increased content of carbon, nitrogen, and all lipid classes between the pre- and post-bloom periods. Hence, C. finmarchicus commenced feeding and spawning at the onset of the bloom and continued throughout the remaining study period. Both C. glacialis and C. hyperboreus females refueled their storage lipids (wax esters) during the bloom and post-bloom period, suggesting that they may spawn in an additional year.

Mesozooplankton biomass data from Disko Bay, West Greenland, 2008

The study site was located in the Disko Bay off Qeqertarsuaq, western Greenland. Due to land-connected sea ice coverage during winter, 2 sampling sites were combined. At the first site in winter (21 February to 23 March 2008), sampling was conducted through a hole in the ice at ca. 65 to 160 m depth approximately 0.5 nautical mile (n mile) south of Qeqertarsuaq (69° 14' N, 53° 29' W). In spring and summer (9 April to 18 July), sampling was done at a monitoring station 1 n mile south from Qeqertarsuaq (69° 14' N, 53° 23' W) at 300 m depth. Sampling was carried out between 10:00 and 17:00 h. During sampling from the ice, mesozooplankton was collected using a modified WP-2 net (45 µm) equipped with a closing mechanism (Hydrobios). Samples were collected in 3 depth strata (0-50, 50-100, and 100-150 m). During ship-based sampling, mesozooplankton was collected with a multinet (50 µm) equipped with a flow meter (Multinet, Hydrobios type midi), and 2 additional depth strata (150-200m and 200-250 m) were included. In addition to the seasonal study one diurnal investigation with sampling every 6 h was conducted from 29 April at 12:00 h to 30 April 30 at 12:00 h. Samples were immediately preserved in buffered formalin (5% final concentration) for later analyses. Biomass values of the different copepod species were calculated based on measurements of prosome length, and length/weight relationships. Two regressions for Calanus spp. were established for biomass calculations: one applicable prior to and during the phytoplankton bloom until 4 May, and another from 9 May onwards.

Mesozooplankton size data from Disko Bay, West Greenland, 2008

The study site was located in the Disko Bay off Qeqertarsuaq, western Greenland. Due to land-connected sea ice coverage during winter, 2 sampling sites were combined. At the first site in winter (21 February to 23 March 2008), sampling was conducted through a hole in the ice at ca. 65 to 160 m depth approximately 0.5 nautical mile (n mile) south of Qeqertarsuaq (69° 14' N, 53° 29' W). In spring and summer (9 April to 18 July), sampling was done at a monitoring station 1 n mile south from Qeqertarsuaq (69° 14' N, 53° 23' W) at 300 m depth. Sampling was carried out between 10:00 and 17:00 h. During sampling from the ice, mesozooplankton was collected using a modified WP-2 net (45 µm) equipped with a closing mechanism (Hydrobios). Samples were collected in 3 depth strata (0-50, 50-100, and 100-150 m). During ship-based sampling, mesozooplankton was collected with a multinet (50 µm) equipped with a flow meter (Multinet, Hydrobios type midi), and 2 additional depth strata (150-200m and 200-250 m) were included. In addition to the seasonal study one diurnal investigation with sampling every 6 h was conducted from 29 April at 12:00 h to 30 April 30 at 12:00 h. Samples were immediately preserved in buffered formalin (5% final concentration) for later analyses. Biomass values of the different copepod species were calculated based on measurements of prosome length, and length/weight relationships. Two regressions for Calanus spp. were established for biomass calculations: one applicable prior to and during the phytoplankton bloom until 4 May, and another from 9 May onwards.