23Cryopreservation of ovarian tissue has been studied for female germline preservation of 24 farm animals and endangered mammalian species. However, there are relatively few 25 reports on cryopreservation of fish ovarian tissue and especially using vitrification 26 approach. Previous studies of our group has shown that the use of a metal container for 27 the cryopreservation of bovine ovarian fragments results in good primordial and 28 primary follicle morphological integrity after vitrification. The aim of this study was to 29 assess the viability and in vitro development of zebrafish follicles after vitrification of 30 fragmented or whole ovaries using the same metal container. In Experiment 1, we tested 31 the follicular viability of five developmental stages following vitrification in four 32 vitrification solutions using fluorescein diacetate and propidium iodide fluorescent 33 probes. These results showed that the highest viability rates were obtained with 34 immature follicles (Stage I) and VS1 (1.5 M methanol + 4.5 M propylene glycol). In 35 Experiment 2, we used VS1 to vitrify different types of ovarian tissue (fragments or 36 whole ovaries) in two different carriers (plastic cryotube or metal container). In this 37 experiment, Stage I follicle survival was assessed following vitrification by vital 38 staining after 24 h in vitro culture. Follicular morphology was analyzed by light 39 microscopy after vitrification. Data showed that the immature follicles morphology was 40 well preserved after cryopreservation. Follicular survival rate was higher (P<0.05) in 41 vitrified fragments, when compared to whole ovaries. There were no significant 42 differences in follicular survival and growth when the two vitrification devices were 43 compared. 44
This study aimed to assess the effects of carp pituitary extract (CPE), follicle stimulating hormone (FSH) and luteinizing hormone (LH) on zebrafish oocyte maturation and the ability of these mature oocytes to be fertilized and developed until hatching. Stage III follicles were matured in eight treatments: five concentrations of CPE (16, 32, 48, 64 and 80 μg/mL), one of FSH (0.5 μg/mL), one of LH (0.5 μg/mL), or one combination of FSH (0.5 μg/mL) and LH (0.5 μg/mL). Maturation rates in CPE treatments were 12.8% (16 μg/mL), 24.8% (32 μg/mL), 27.0% (48 μg/mL), 22.7% (64 μg/mL) and 9.6% (80 μg/mL); in FSH was 15.7% (0.5 μg/mL), in LH was 31.8% (0.5 μg/mL) and in FSH (0.5 μg/mL) combined with LH (0.5 μg/mL) it was 50.4%. In vitro fertilization was performed in all treatments; however, only the treatment combining FSH and LH resulted in fertilized oocytes. After maturation using FSH combined with LH, the cleavage rate was 33.3% and hatching rate of live larvae was 20.0%. These results showed that FSH combined with LH was effective in IVM of zebrafish oocyte.
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