G ivf medium

Following IVM, the two-cell embryo development
potential rate of MII oocytes was evaluated by IVF.
Male mice spermatozoa were obtained from the cauda
epididymis that capacitated for 1 hour at 37°C. MII
oocytes were incubated with sperms in the GIVF medium
(Vitrolife, Sweden) for 4 hours. For the removal of
additional spermatozoa, oocytes were washed and then
cultured in a G1-plus droplet (Vitrolife, Sweden; 5-6
oocytes in one drop) and in 37°C and 5% CO₂in air for
3 days. The embryo development rate was checked and
evaluated everyday by using a stereomicroscope.

Spermatozoa were collected postmortem from the right cauda epididymidis and ductus deferens and expressed into 0.5-ml G-IVF medium (Vitrolife, Gothenberg, Sweden) and incubated for at least 10 min in 6% (v/v) CO₂ and 5% (v/v) O₂ at 37°C.35

A total of 308 mouse oocytes classed as resistant immature following in vitro culture were randomly allocated to control (n = 113) and treatment groups (n = 195). This study conducted at Animal Science Laboratory of IMERI, Human Reproductive, Infertility, and Family Planning Research Center of IMERI, and Electrophysiology Imaging of Terpadu Laboratory, Faculty of Medicine, University of Indonesia. Immature oocyte after in vitro culture was included in this study, whereas exclusion criteria were mature and degenerated oocytes.
Immature mouse oocytes were obtained by priming 8–12-week-old DDY hybrid female mice with 7.5 IU of a pregnant mare's serum gonadotropin (Intervet, The Netherlands) (intraperitoneal). A total of 44‒48-h later ovarian dissection was performed to collect immature oocytes using a 26.5G sterilized needle in G-MOPS medium (Vitrolife, Sweden). Subsequently, maturation cultures were divided into two groups based on cumulus status (intact or without cumulus cells) [Figure 1] in G-IVF medium (Vitrolife, Sweden) for 14‒16 h at 37°C and 5% CO₂. Oocytes in both germinal vesicle (GV) and MI stages were randomly allocated to activation treatment and control groups by an embryologist [Figure 2].

After 16 h, COCs ovulated from follicles were retrieved. COCs were digested by hyaluronidase (Sigma) in about 2 min at 37°C. Oocytes with first polar bodies and homogeneous cytoplasm were considered normal mature oocytes. Mature oocytes that are at the meiosis II (MII) stage are known as MII oocytes. As illustrated in Fig. 1, mature oocytes were counted and transferred to a drop of G-IVF medium covered (Vitrolife, Sweden) with paraffin liquid (Sigma) in a 35-mm dish, which was incubated for about 4 h with 5% CO₂ in the air and 100% humidity at 37°C. Mature spermatozoa were collected from the cauda epididymis of ICR mice over 8 wk of age [18 (link)]. Spermatozoa were added at a final concentration of 2 × 10⁶/mL to a drop of G-IVF medium containing the MII oocyte and incubated for 20 h at 37°C. Spermatozoa were removed by pipetting, and the eggs were cultured for 72 h in the G-1 medium (Vitrolife).
In the positive control group, female mice were injected intraperitoneally with 10 IU of pregnant mare serum gonadotropin (PMSG; ShuSheng), followed by 10 IU hCG (ShuSheng) at 48 h post-PMSG. Ovulated COCs were retrieved from the oviduct at 16 h post-hCG [19 (link)].