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

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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.