Experimental procedures
Tissue samples and transportation
Sixteen ovaries from goats (Capra hircus) aged 9–13 months were obtained at a local slaughterhouse in Yogyakarta, Indonesia, during the period of September to November 2018. After the goats were slaughtered, ovaries were transported to the Laboratory of Physiology at the Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada within 1 h using a sterile transport box which was filled with ice to maintain a stable temperature between 5 and 8 oC. Transport medium was composed of 10% M199, serum-substituted supplement (10% SSS), 2% penicillin-streptomycin (10,000 U/mL) (Thermo Fisher Scientific) and 1% fungizone (Thermo Fisher Scientific).
Preparation of the ovarian tissue
After ovarian tissue collection (n = 16 tissue samples), the ovarian medulla was dissected using small scissors (Orthotech, Palermo, Italia) to form an ovarian cortical strip. Ovarian cortical strips (1–2 mm thickness) were prepared and cut into a small size of 5 × 5 mm. The ovarian strips were divided equally into four groups: the fresh group (Fr), fresh-transplanted group (FrTr), vitrification group (Vi), and vitrification-transplanted group (ViTr) (Fig. 1).
Ovarian tissue vitrification-thawing and transplantation
We developed the vitrification method based on modifications of the method described by Suzuki et al. in 2015 [19]. Ovarian cortical strips were washed in M199 medium (Thermo Fisher Scientific, MA, USA) supplemented with 20% SSS. Ovarian cortical strips were equilibrated in equilibrium solution (ES) I that consisted of M199 medium, 10% ethylene glycol (EG, Wako Oure Chemical Industries, Tokyo, Japan) and 20% SSS for 5 min. The second equilibration was done in ES II that consisted of M199 medium, 20% EG, 20% SSS for 5 min. Next, ovarian cortical strips were transferred into the vitrification solution which consisted of M199 medium, 35% EG, polyvinylpyrrolidone (Sigma-Aldrich, St. Louis, MO, USA) for 15 min. Then, ovarian cortical strips were loaded into the cryodevice (Ova Cryo device type M, Kitazato, Tokyo, Japan) and stored in liquid nitrogen (− 196 oC) until they were used. Ovarian cortical strips were successfully vitrified if the ovarian cortex strips appeared transparent [19].
For thawing, ovarian strips were transferred into the thawing solution that consisted of M199, 20% SSS, and 0.8 mol/l sucrose for 1 min on a warming plate set at 37 oC. Next, ovarian strips were incubated in diluting solution that consisted of 20% SSS, 0.4 ml/l sucrose for 3 min, followed by incubation in the washing solution that consisted of M199 and 20% SSS for 5 min and repeated twice. Then, the ovarian cortical strip was transplanted for 5 days in CAM of 5-day-old fertilized eggs which were incubated at 37 oC and 44% humidity. After 5 days transplanted, the ovarian cortical strips were harvested and loaded into paraformaldehyde for the next step.
Histological observation of follicle density
Ovarian strips from the four groups were fixed in 10% formalin, embedded in paraffin wax, serially sectioned in 4 μm thickness, stained with hematoxylin and eosin (HE) and observed under light microscope with × 100 and × 400 magnification. Follicles were classified into two groups: primordial follicles which were surrounded by a single layer of flattened granulosa cells and growing follicles which have the appearance of one or more cuboidal granulosa cells surrounding them, which included primary, secondary, preantral, and antral follicles. Ovarian cortex strips were evaluated for their primordial follicle density which was defined as the number of healthy primordial follicles per total follicles from a random section from four ovarian strips in each group [20].
DNA fragmentation assay
The DNA fragmentation was assessed using an In Situ Cell Death Detection Kit (Roche, Germany). Paraffin sections were deparaffinized, rehydrated, and permeabilized with 20 μm proteinase K in 10 mM Tris pH 7.4 (Qiagen, Netherlands). The washed slides were incubated with TdT-mediated dUTP-biotin nick-end labeling (TUNEL (Roche, Germany)) reagents according to the manufacturer’s instructions and counterstained with methyl green. DNA fragmentation of primordial follicles was demonstrated by the presence of positive staining in ≥ 49% of granulosa cells [19]. DNA fragmentation of follicles was defined as the number of primordial follicles with positive TUNEL staining per total follicles from a random section from four ovarian strips in each group.
Statistical analysis
Statistical analysis was performed using SPSS 25.0 software (IBM Corp., Chicago). Follicular density and DNA fragmentation in the four groups were compared by one-way analysis of variance (ANOVA). Various characteristics were summarized by mean and standard deviation (SD) within groups. The level of statistical significance was set at p < 0.05 with 95% confidence interval (CI).