Cloned Foal Born from Postmortem-Obtained Ear Sample Refrigerated for 5 Days Before Fibroblast Isolation and Decontamination of the Infected Monolayer Culture.

This research article details the process of cloning a polo horse from an ear skin sample, taken postmortem and then refrigerated for five days. The successful attempt resulted in a healthy foal genetically identical to the donor mare.

Sample Collection and Preservation

• The study began with the collection of ear skin samples from a six-year-old mare that had died due to complications from postcolic surgery. The goal was to preserve the horse’s genetics for cloning.
• The ear skin samples were then stored in an equine embryo transfer medium at 4 degrees Celsius for five days, a step crucial for maintaining the viability of the cells.

Treatment of Samples and Cell Culturing

• The samples underwent trypsin digestion – a process that breaks down proteins – to allow for the establishment of fibroblast cultures. However, all cultures showed significant signs of bacterial infection.
• To salvage the project, the researchers rigorously and repeatedly cleaned the cells before individually cultivating them in four 96-well dishes. This led to the successful growth of new monolayer fibroblast cultures in a few wells that had remained uncontaminated.

Somatic Cell Nuclear Transfer and Embryo Implantation

• These uncontaminated cells were then used to perform somatic cell nuclear transfer, a crucial method for cloning. This resulted in the derivation of six Day 7 blastocysts from 14 reconstructed zygotes.
• Four of these blastocysts were transferred into four naturally cycling mares on the fifth day following ovulation. This process resulted in two successful pregnancies: one resulted from a fresh blastocyst transfer, the other from a vitrified (or frozen) blastocyst transfer.

Result and Evaluation

• The vitrified embryo transfer led to the birth of a healthy foal. The foal was at the time of the report 21 months old and was found to be genetically and phenotypically identical to the donor mare, thus confirming the success of the postmortem cloning process.