Vitrification with Cryotop and DAP213 of ex situ and in situ feline cumulus-oocyte complexes

Introduction and aim. Banking of cryopreserved female gametes may ensure the future survival of high genetic value individuals. Among cryopreservation methods, vitrification brought promising results. We previously demonstrated that feline oocytes retrieved from ovarian tissue (in situ oocytes) vitrified with DAP 213 (2 M DMSO, 1 M Acetamide, 3 M Propylene Glycol) maintain the capability of resuming meiosis (1). However, the efficacy of different vitrification procedures is still controversial. Recently, Cryotop method, that consists in the vitrification of gametes in less than 0.1 microliter medium droplet placed on the surface of a fine polypropylene strip attached to a plastic handle, has been developed (2). The aim of this study was to evaluate Cryotop and DAP213 for the vitrification of ex situ (isolated) and in situ (included in the ovarian tissue) feline cumulus-oocyte complexes (COCs). Material and methods. Domestic cat (Felis catus) ovaries were collected after routine ovariohysterectomy. Of each pair of ovaries, one was sliced to release COCs and the ovarian cortex of the other ovary was sectioned into small fragments (1.5-2 mm3). Grade I COCs and fragments were vitrified with Cryotop (2) and DAP 213 (3). Fresh COCs were used as control group. After warming, vitrified ex situ (isolated) and in situ (retrieved from vitrified ovarian fragments) COCs were matured in vitro for 48 h in Kreb’s Ringer Bicarbonate as previously described (1). Oocyte viability, cumulus cells presence and viability were evaluated immediately after warming and after IVM with FDA-PI (fluorescein diacetate-propidium iodide; Sigma) staining. Meiotic configurations were assessed with Hoechst 33342 (Sigma) staining after IVM. Nuclear stages ranging from germinal vesicle breakdown to Metaphase II were considered to have resumed meiosis. Data were analyzed by Chi-square test. Results. Ex situ oocytes vitrified with Cryotop showed rates of viability after warming (97.1%, 33/34) and after IVM (84.6%, 33/39) higher (P<0.05) than those vitrified with DAP 213 (72.2%, 26/36 and 54.8%, 23/42) and similar (P>0.05) to that of fresh oocytes (97.1%, 34/35 and 94.2%, 49/52). No differences were observed in viability rates of in situ oocytes vitrified with the two methods. Presence and viability of cumulus cells layers were remarkably decreased in all the groups after warming (between 5.6% and 20.6%; P<0.00001), and after culture (between 0% and 7.9%; P<0.00001) compared to fresh oocytes (77.1% and 62.3%). After IVM, ex situ oocytes vitrified with Cryotop achieved rates of meiosis resumption similar (P>0.05) to fresh oocytes (67.5%, 27/40) and higher (P<0.001) than oocytes vitrified with DAP 213 (53.8%, 21/39 vs 15%, 6/40). However, DAP 213 applied to in situ oocytes resulted in higher rates of meiosis resumption than Cryotop (28%, 14/50 vs 4.9%, 2/41; P<0.01), even though a significant difference was observed with fresh oocytes (P<0.00005). Conclusions. Cryotop is a reliable method for the vitrification of isolated (ex situ) feline COCs. Vitrification with DAP213 of in situ COCs confirms the results previously obtained (1) and it can be considered an option for the vitrification of feline ovarian tissue.