A Simple Microaspiration Technique for Isolating Somatic Cells from Cryopreserved Equine Semen as Nuclear Donors for Cloning

We aim to show that somatic cells from cryopreserved equine semen can be useful to recover individuals of high genetic value. Our current challenge is to be able to select somatic cells with intact diploid DNA to be used as nuclear donor. To begin, remove the equine semen straws from the liquid nitrogen tank.

Expose them to air for 10 seconds, and thaw them in a water bath at 37 degrees Celsius for 30 seconds. Take a 250-microliters sample of the thawed semen. Dilute it with one milliliter of supplemented DMEM-F12 medium.

Incubate the semen mixture at 37 degrees Celsius for 30 minutes. Pipette out 25 microliters from the incubated dilution into a tube, and dilute it in 975 microliters of DPBS supplemented with 0.1 milligram per milliliter polyvinyl alcohol or polyvinyl pyrrolidone. Gently mix the suspension.

Maintain the diluted semen mixture at 37 degrees Celsius throughout the entire cell collection process. Place a Petri dish lid containing diluted equine semen on the microscope stage to prepare for somatic cell collection. Select a micropipette of suitable diameter for somatic cell collection and connect it to the mechanical aspiration system.

Carefully insert the micropipette into the micro drop and align it with the dish lid base. Once the pipette aspirates culture medium, begin individual capture of somatic cells. Focus on the bottom of the micro drop, where various cell morphologies are visible.

Position the micropipette tip next to each somatic cell, and aspirate under negative pressure. Collect all round and elongated cells, regardless of size. After collecting 10 cells, deposit them into a 10-microliter micro drop located on the upper surface of the dish lid.

Retain all collected somatic cells within this micro drop during the entire collection process. Once the desired number of somatic cells is collected, perform a simple washing procedure using micro drops to remove sperm, debris, and microbial contaminants. Using a second dish lid, perform a sequential three-step washing of somatic cells.

Use a micropipette to transfer the somatic cells into the first drop, and allow them to settle for three to five minutes. Transfer the cells from the first drop to the second drop using the same procedure. Repeat the transfer once more and place the somatic cells into the third drop.

Finally, deposit the isolated somatic cells into a prepared micro drop designated for somatic cell nuclear transfer. The micro aspiration technique successfully recovered somatic cells from cryopreserved equine semen, with capture rates ranging from 317.7 to 424.7 cells per hour across three stallions. Stallion number 3 showing the highest yield.

Five distinct somatic cell types were identified based on size and morphology. We identified a population of somatic cells that differs in morphology and viability in cryopreserved equine semen, which may be useful as nuclear donor. The cells can be selected based on their morphology, and the procedure does not require the establishment of primary culture.

We will focus on improving the viability of these cells after bulking equine semen.