The bovine ovary contains a large number of follicles at the different developmental stages. The objectives of this study were to establish the optimal vitrification condition for bovine secondary and primordial follicles, and to examine the developmental ability of vitrified follicles by xenotransplantation to SCID (severe combined immune deficiency) mice. In the first experiment, secondary follicles 150–200 �m in diameter were dissected individually from the ovarian cortex. The follicles were exposed to equilibration solution containing 7.5% ethylene glycol (EG), 7.5% DMSO, and 20% fetal calf serum (FCS) in modified (HEPES-buffered) TCM-199 medium (m199) at room temperature (RT) for 15 min. They were randomly allocated to 6 experimental groups, treated with the vitrification solution containing 15% EG, 15% DMSO, 20% FCS, and 0 or 0.5 M sucrose (Suc) in m199 at RT for 1 or 30 min or at 4�C for 30 min, and then put on the Cryotop (Kitazato Supply, Tokyo, Japan) and directly plunged into liquid nitrogen (LN2). For warming, vitrified follicles with 0.5 M Suc were put in m199 supplemented with 1 M Suc and 20% FCS at 38.5�C, and then transferred to m199 supplemented with 0.5 M Suc and 20% FCS at RT for 5 min. Vitrified follicles without Suc were warmed in Suc-free medium in the same manner. After washing, they were fixed and examined histologically. In the secondary follicles vitrified in 0 M and 0.5 M Suc-containing solutions after treatment at RT for 1 min, 59% (10/17) and 91% (10/11) of oocytes showed normal morphology, respectively. In the follicles vitrified after 30 min treatment, 63–100% of the oocytes showed abnormal cytoplasm and/or pyknotic nuclei, regardless of temperature and Suc contents. In the second experiment, tissues containing 40 to 50 primordial follicles (1 mm � 1 mm � 0.5 mm) were dissected from the ovarian cortex. Sixteen tissues were allocated to 4 groups, equilibrated at RT for 5 or 15 min in the equilibration solution, treated with the vitrification solution containing 0 or 0.5 M Suc at RT for 1 min, and then put on the Cryotop and plunged into LN2. After warming and washing in a similar manner, they were examined histologically. After treatment with the equilibration solution for 5 min followed by Suc-free vitrification solution, 82 � 5% (mean � SEM of 4 tissues) of primordial follicles showed normal morphology, and the rate was significantly higher than for other groups (P < 0.05, Student's t-test). In the third experiment, secondary follicles and primordial follicles vitrified by each best method were transplanted under the kidney capsule of SCID mice for 1 and 6 months, respectively, and examined histologically. After transplantation of 40 secondary follicles into 4 mice, about 40% of the follicles developed to the antral stage. After transplantation of 8 tissues containing primordial follicles into 3 mice, 2 or more follicles developed to the antral stage in each mouse. These results indicate that bovine secondary and primordial follicles can be vitrified without losing their developmental capacity to the antral stage.
Quantification of multi-oocyte follicles in ovaries of bitches
