Rex Rabbit Somatic Cell Nuclear Transfer with In Vitro-Matured Oocytes

Area of Science:

• Reproductive Biology
• Developmental Biology
• Animal Biotechnology

Background:

• Somatic cell nuclear transfer (SCNT) is crucial for animal cloning but requires a substantial supply of mature oocytes.
• In vitro maturation (IVM) offers a potential solution for oocyte sourcing in SCNT applications.
• Optimizing IVM and SCNT protocols is essential for improving efficiency and success rates in species like the Rex rabbit.

Purpose of the Study:

• To investigate and optimize the in vitro maturation (IVM) of Rex rabbit oocytes for somatic cell nuclear transfer (SCNT).
• To determine the relationship between follicle diameter and oocyte meiotic ability.
• To refine SCNT procedures, including denucleation, activation, and donor cell selection for Rex rabbits.

Main Methods:

• Oocytes were isolated from Rex rabbit ovaries obtained from abattoirs and categorized by follicle diameter.
• Meiotic ability was assessed after in vitro culture (IVC) for 18 hours.
• SCNT procedures were optimized, involving denucleation, activation with ionomycin and 6-dimethylaminopurine (6-DMAP), and donor cell synchronization.
• Fetal fibroblast cells (FFCs) and cumulus cells (CCs) were compared against skin fibroblast cells (SFCs) as nuclear donors.
• Optimal electrofusion parameters were determined.

Main Results:

• Oocytes from follicles >0.7 mm acquired maturation ability, with those >1 mm showing greater developmental potential.
• Oocytes from <0.5 mm follicles failed to resume meiosis, remaining arrested at the germinal vesicle (GV) stage.
• The highest activation rates were achieved using 2.5 μM ionomycin followed by 2 mM 6-DMAP and 5 μg/mL cycloheximide (CHX).
• Fetal fibroblast cells (35.6%) and cumulus cells (38.0%) yielded higher blastocyst rates compared to skin fibroblast cells (19.7%).
• Optimal fusion parameters involved a 2DC interval, 1.6 kV/cm voltage, and 40 μsec duration.

Conclusions:

• The study successfully optimized in vitro maturation of Rex rabbit oocytes, establishing a reliable source for SCNT.
• Follicle diameter is a critical determinant of oocyte meiotic competence and developmental potential.
• Optimized SCNT protocols using FFCs or CCs significantly enhance blastocyst formation rates in Rex rabbits.