The survey of sexual maturity in Mugil cephalus so measuring sex hormones and histologic studies

In this study the process of female gray mullet brooders was carried out by using histological study and masurment of sex steroids. Results of histological studies showed that oocyte of gray mullet brooders in Gomishan Rearing Center conditions of develop to the end of yolk globule stage. The results were observed with oocyte in chromatin nucleolar stage (first stage) with means of diameter of 20 p m, in August, perinucleolar stage (second stage) in September with mean diameter of 87 p m, yolk vesicle stage (third stage) in October with mean diameter 200 p m and yolk granules stage (forth stage) from October to November with average diameter of 180 — 650 p m. For the reason of stopping oocyte develop at the end of fourth stage, hormonal induction to final oocyte maturation and ovulation was used. For this purpose, carp pituitary , HCG and LRH-A2 with different combinations were used in two stages, second injection was used 24 hours after first injection. 15 females brooders were divided in 5 groups, different hormonal combinations were injected to four groups and to fifth group as control, only saline, was injected. The process of female brooder rippening in hormonal induction was studied via masurment of sex steroids including 17 a - hydroxy progestrone, estradio1-17)6 and testosterone. Blood samples were collected from caudal vein during first injection, 24, 30 and 48 hours after the first injection. At the same time, for distinguishing histological changes the sample has been attained from the gonads Sex stroid fluctuation patterns in different brooder groups that injected hormon were similar, however hormonal composition had similar effects. All brooder that their oocyte in the beginning of hormonal injection were At the end of fourth stage with oocyte diameter average of 600 p m received to final maturation and ovulation. The brooder that its oocytes were At the begining or mid-fourth stage did not show ovulation but hormonal induction caused oocyte develop at the beginning of fifth stage. Study of 17-hydroxy progestrone fluctuation showed that the maximum level of this steroid (0.347 ng/ml) measured 30 hours after the first injection and was significantly higher (p< 0.05) than those of control group. So, 17-hydroxy progestrone is probably precursor of maturation inducing steroid (MIS). However the maximum level of that observed was coincident with germinal vesicle breakdown, oil droplets coalescence and dissolution of yolk granuls The maximum levels of esteradiol— 17/0 and testosterone (3.778 and 16.801ng/ml,respectively) in spawned brooders,were observed 24 hours after the first injection. levels of those steroids were significantly higher (p<0.05) than control group. Maximum level of sex steroids in the brooders that did not spawn to the end of treatment was observed with more delay than those in spawned brooders. Therefor maximum level of 17a-hydroxy progestrone (0.264 ng/ml) in those brooders observed in fourth sampling time and the maximum levels of estradio1-17a and testosterone (2.944 and 18.993 ng/ml, respectivly)observed in third sampling time that was significantly higher (p<0.05) than those of control group. For the study of stress effect on brooders during the hormonal induction, level of cortisol was measured in every sampling time. level of cortisol had high fluctuation that showed handling level and stress effect on brooders. However maximum level of cortisol in majority of brooders was dominant in third sampling time that was coincident with final maturation.

Evaluation on propagation of common carp (Cyprinus carpio Lin.) with hormonal and natural stimuli

The success of breeding of common carp (Cyprinus carpio) using hormonal inducement and environmental stimuli was evaluated considering different sex ratios, and natural and artificial substrates. A total of 18 females (weighing 250 to 2200g) divided into 6 treatments were investigated. A successful spawning was observed in all the treatment groups, only. 66.66% female responded successfully to LHRH-A combined with dompheridone and 83.33% female in natural stimuli. Females induced with LHRH-A and dompheridone found prompt ovulation than that of natural stimulation. A significant variation (F=7.45, P<0.05) was found among the different treatment groups. The number of eggs released appear to depend on body weight (t=15.72, P<0.05), sex ratio (t=7.96, P<0.05) and percentage of ovulated females (t=5.34, P<0.05). Although environmentally stimulated females released more eggs than injected female (t=5.18, P<0.05) but their survival rate was similar (t=1.77, P<0.05). Comparison between the two approaches under the conditions of AIT hatchery shown that both are suitable for spawning induction in common carp. However, environmental stimulation is advantageous because of the less labor and lower cost required for ovulation.

Triploidy induction in Heterobranchus longifilis (Family: Clariidae) by cold shock

Triploid was induced in African Catfish (Heterobranchus longifilis) by cold shocking activated eggs at 5 degree C for forty minutes starting 3-4 minutes after fertilization. Triploidy was confirmed from mitotic chromosomes prepared from embryo which showed 100% triploidy in the cold shocking treatment and 100% diploidy in the control treatment

Induction of mitotic and meiotic gynogenesis and production of genetic clones in rohu, Labeo rohita Ham.

Studies were undertaken to produce genetic clones derived from all homozygous mitotic gynogenetic individuals in rohu, Labeo rohita Ham. ln view of this, attempts were made to interfere with the normal functioning of the spindle apparatus during the first mitotic cell division of developing eggs using heat shocks, there by leading to the induction of mitotic gynogenetic diploids in the F1 generation. Afterwards, viable mitotic gynogenetic alevins were reared and a selected mature female fish was used to obtain ovulated eggs which were fertilized later with UV-irradiated milt. Milt was diluted with Cortland’s solution and the sperm concentration was maintained at 10⁸/ml. The UV-irradiation was carried out for 2 minutes at the intensity of 200 to 250 µW/cm² at 28± 1°C. The optimal heat shock of 40°C for 2 minutes applied at 25 to 30 minutes a.f. was used to induce mitotic gynogenesis in first (F1) generation and at 3 to 5 minutes a.f. to induce meiotic gynogenesis in the second (F2) generation. The results obtained are presented and the light they shed on the timing of the mitotic and meiotic cell division in this species is discussed.

Comparison of effectiveness of heat and cold shocks applied in the induction of gynogenesis in Clarias gariepinus (Burchell)

An experiment was conducted to optimize the procedure of gynogenesis in African catfish, Clarias gariepinus by suppressing meiotic and mitotic cell divisions in fertilized eggs. Gynogensis was conducted by fertilizing normal eggs with UV-irradiated sperm followed by either heat or cold shocking Irradiation of spermatozoa was given for a duration of 1 min and the eggs were fertilized in vitro. Cold shock at a temperature of 3± 1°C for a duration of 30 and 60 min and heat shock at a temperature of 39± 1°C for a duration of 1 and 2 min was applied to induce diploidy. Higher percentage of hatching (68.66) was observed for meiotic gynogens at a shock temperature of 39± 1°C for a duration of 1 min, 5 min after fertilization (af). Higher percentage of mitotic gynogenetic induction (15.33) was observed at a temperature shock of 39± 1°C for a duration of 1 min, 30 min af.

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 macroscopic, microscopic and hormonal changes in gonads of the crayfish Astacus leptodactylus in Aras Dam

This study was carried out to seasonal determination of some morphological characteristics, Seasonal fecundity, Seasonal fluctuations of vertebrate-type steroids and seasonal analysis of gonadal histology in both female and male sexes of freshwater crayfish (Astacus leptodactylus Eschscholtz 1823) in the area of Aras dam Lake. Crayfish were collected respectively in June, August, November (2011) and January (2012). The average length and weight of male crayfish was higher than that of females. %GSI of females fluctuated within an extended range (between 0.6 and 13.5% from June to January). Both of synchronous and asynchronous ovaries were seen in August sampled ovaries; however asynchronous form was higher than another. The annual reproductive cycle of male A. leptodactylus was surveyed by study on the seasonal changes of the external appearance of the testes and vasa deferentia, fluctuations in the gonadosomatic index (GSI%) and the histological analysis of the male reproductive system. Based on the histological differentiation of testis, spermatogenisis devided to 5 separated stages. The findings suggested asynchronous testis in the species A.leptodactylus. The presence of primary spermatophore layer may help keeping spermatozoa alive while the secondary spermatophore layer may produces spermatophore or synthesize of acellular material which forms spermatophre. Pleopodal fecundity was 37.3%lower than ovarian fecundity observed. The significantly higher number of eggs attached to 3rd and 4th pairs of pleopods. The egg number and gonadosomatic index increased with female size while egg weight and egg diameter didn’t increase with female size. Hemolymph levels of 17β-estradiol and progesterone followed a similar fluctuation pattern with % GSI in females, while testosterone didn’t follow the mentioned pattern. The testis of November sampled crayfish presented significantly higher gonadosomatic (%GSI) index (P < 0.05).The most observed gonadosomaticindices were 13.5%(forfemales) and 1.21% (for males, in autumn. Althogh the lowest GSI was (0.50%) formales in spring and (0.26%0 for spent females in January. Testosterone which followed a similar pattern with %GSI in males increased remarkably in November. 17β-estradiol increased strictly in January. The strictly enhancement of the three estroid hormones in January in both male and female sexes could bedue totheir stimulating role in in spermatophre and egg lying in the mating season (In January). Most of the ovaries followed the asynchoronous growth pattern. Also the testes presented asynchoronous growth pattern in autumn.