Timing factors affecting blastocyst development in equine somatic cell nuclear transfer

Area of Science:

• Reproductive Biology
• Animal Biotechnology
• Developmental Biology

Background:

• Nuclear transfer (NT) efficiency in equids is limited by the need for donor cell reprogramming within the ooplast cytoplasm.
• The developmental competence of the oocyte restricts the feasible duration of nucleus-cytoplasmic exposure prior to activation.

Purpose of the Study:

• To investigate the impact of nucleus-cytoplasmic exposure duration and in vitro maturation (IVM) timing on equine NT outcomes.
• To determine optimal conditions for enhancing blastocyst development and subsequent live foal production in equine NT.

Main Methods:

• Examined varying delays (2, 5, 8 hours) between cell reconstruction and activation following 24-hour IVM.
• Assessed blastocyst rates using different donor cell lines and IVM durations (20 vs. 24 hours).
• Investigated a modified protocol involving maturation of metaphase I oocytes for 3 hours before activation (20+3H treatment).

Main Results:

• Blastocyst rates increased with longer delays (5 or 8 hours) between reconstruction and activation, reaching up to 15% and 11% respectively.
• A 5-hour delay yielded 17-22% blastocysts with two cell lines.
• The 20+3H treatment combined with an 8-hour activation delay resulted in a 30% blastocyst rate.
• Four live foals were produced from 17 transferred blastocysts (24%) using metaphase II oocytes and extended activation delays.

Conclusions:

• Utilizing equine oocytes immediately upon reaching metaphase II (MII) is crucial for successful NT.
• Implementing a longer delay between oocyte reconstruction and activation enhances developmental competence in equine NT.
• These findings provide a foundation for improving efficiency in equine somatic cell nuclear transfer (SCNT).