The cryopreservation protocols are species‐specific owing to variable sperm sensitivity towards temperature reduction and contact with cryoprotectant solutions. However, little is known about spermatic pathologies, especially after the cryopreservation process. Thus, the objective was to evaluate the effect of cryopreservation on morphological changes in semen of jundiá (Rhamdia quelen). Sperm pool of five males, with >80% motility, as collected, diluted in a cryoprotectant solution and frozen in liquid nitrogen (−196°C). There was a reduction in the percentage of normal cells and sperm motility, accompanied by an increase in the percentage of sperm abnormalities after cryopreservation of R. quelen spermatozoa, indicating a substantial fragility of the spermatozoa towards the cryopreservation process. The most frequent types of morphological changes in the cryopreserved semen were macrocephaly, folded tail, strongly curled tail and distally curled tail. This is the first study to evaluate the spermatic morphology of R. quelen before and after cryopreservation, paving way for further investigations on morphological alterations and for a new classification of these changes in fish semen due to cryopreservation.

Purpose Cryopreservation of ovarian tissue is paramount for fertility preservation, with important clinical applications, especially for women suffering from an oncological condition. Several cryopreservation methodologies have been tried in search of better outcomes, especially in terms of primor-dial and primary follicles integrity post-cryopreservation. Vitrification has successfully been applied to ovarian tissue using different carriers for tissue exposure to the liquid nitrogen (LN2). Methods We developed an enclosed metal vessel, which has the advantage of a faster heat transfer, when in contact with LN2 avoiding at the same time, the direct contact with tissue. Additionally, we assessed the effect of different times and temperatures of transport between the collection of mouse ovaries and the beginning of cryopreservation, on follicular morphology after vitrification.Results Our results suggest that 37°C and R.T. help to maintain normal primordial and primary follicle morphology for up to 4 hrs after collection and beginning of vitrification in a metal container. Conclusion These data show that the metal container is an appropriate carrier for mouse ovary vitrification. The rate of morphologically normal primordial follicles up to 4 hrs.

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

The aim of the present study was to compare the efficiency of vitrification and slow freezing techniques for the cryopreservation of zebrafish ovarian tissue containing immature follicles. In Experiment 1, assessment of cell membrane integrity by trypan blue exclusion staining was used to select the best cryoprotectant solution for each cryopreservation method. Primary growth (PG) oocytes showed the best percentage of membrane integrity (63.5 ± 2.99%) when SF4 solution (2 M methanol + 0.1 M trehalose + 10% egg yolk solution) was employed. The vitrification solution, which presented the highest membrane integrity (V2; 1.5 M methanol + 5.5 M Me2SO + 0.5 M sucrose + 10% egg yolk solution) was selected for Experiment 2. Experiment 2 aimed to compare the vitrification and slow freezing techniques in the following parameters: morphology, oxidative stress, mitochondrial activity, and DNA damage. Frozen ovarian tissue showed higher ROS levels and lower mitochondrial activity than vitrified ovarian tissue. Ultrastructural observations of frozen PG oocytes showed rupture of the plasma membrane, loss of intracellular contents and a large number of damaged mitochondria, while vitrified PG oocytes had intact mitochondria and cell plasma membranes. We conclude that vitrification may be more effective than slow freezing for the cryopreservation of zebrafish ovarian tissue.

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The cryopreservation promotes cellular damage that could compromise sperm quality in terms of motility and fertility rates, which may be caused by oxidative stress. Thus, the aim of this study was to assess the effects of cysteine addition on post‐thaw sperm quality, DNA damage and indices of oxidative stress of the South American silver catfish (Rhamdia quelen) sperm, compared with the cryoprotectant solution without cysteine addition. Sperm collected from five males were cryopreserved in cryoprotectant solution (fructose 50 g/L, powdered milk 50 g/L and methanol 100 ml/L) containing different cysteine concentrations (0, 2.5, 5, 10 and 20 mM). After thawing, the following were measured: sperm motility, morphology, sperm viability, DNA damage, lipid peroxidation, concentration of carbonyl and sulfhydryl groups and the activity of SOD, CAT, GST and GPx enzymes. The lowest sperm motility was determined for semen cryopreserved with addition of 20 mM of cysteine. The control group had the lowest DNA damage and lipid peroxidation. The findings of this study show that cysteine addition had no positive effect on evaluated parameters. Therefore, the concentrations tested are not recommended for the supplementation of cryoprotectant solution for semen of R. quelen.

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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