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Vitrification and ultra-rapid freezing, which are more commonly used for oocytes and embryos, have recently been applied to spermatozoa in an attempt to make semen cryopreservation in field conditions easier compared to conventional freezing. It is well-known that in case of unexpected death of rare and wild animals, preserving epididymal spermatozoa from isolated testicles represents a great chance of salvaging male germplasm for future use in assisted reproductive technologies. The aim of this study was to evaluate the morphofunctional integrity of cat epididymal spermatozoa ultra-rapid frozen in pellets or straws with two different extenders [E1 (Tris buffer with 20% egg yolk and 0.25 M sucrose) or E2 (Ham's F10 with 1% bovine serum albumin and 0.4 M sucrose)] and to test whether spermatozoa preserved by the best combination were able to fertilize oocytes and produce embryos

The in vitro development of vitrified oocytes (VOs) is still suboptimal (Mandawala et al., 2016) and the traditional two-dimensional (2D) culture systems might not be adequate to fully exploit VOs potential. The use of three-dimensional (3D) follicle-like structures, i.e. a combination of granulosa cells (GCs) and semipermeable 3D matrices, could mimic the physiological microenvironment and enhance VOs maturation and embryo development. The aim of this study was to assess the steroidogenic ability (estradiol and progesterone secretion) of GCs encapsulated in 3D barium alginate microcapsules (follicle-like structure) compared to GCs cultured in a 2D monolayer and the maturation outcomes of VOs cultured in these systems. After purification (Simsek & Arikan, 2015), cat GCs retrieved from isolated ovaries were in vitro cultured for 6 days in 3D microcapsules (Vigo et al., 2005) or in 2D monolayers. On days 2 and 6, conditioned medium was collected and hormonal determination by enzyme-linked fluorescent assay was performed. On the same days, 3D and 2D cultured GCs were used as artificial milieu for in vitro maturation of VOs obtained by Cryotop protocol. Nuclear maturation was assessed by bis-benzimide staining. Steroidogenesis was observed in 3D follicle-like structures as well as in 2D monolayers; hormonal concentration increased over time and on day 6 it significantly differed between systems (p=0.02). Vitrified oocytes resumed meiosis in presence of GCs cultured for 2 days (3D: 45.5%; 2D: 56.7%), while GCs cultured for 6 days significantly hindered VOs meiosis progression in monolayers (21.7%, p=0.007), but supported high proportions of full maturation in follicle-like structures (26.7%, p=0.07). Granulosa cells in 3D microcapsules maintained their physiological features and these follicle-like structures were able to restore VOs developmental abilities. However, further advancements in VOs culture methods would optimize the use of these valuable resources., International Journal of Health, Animal Science and Food Safety, Vol 5, No 1s (2018)