Bovine in vitro embryo production – What happens after OPU?

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Published on: September 26, 2017

Daniela Demetrio, DVM, MS
Ruann and Maddox Dairy – Riverdale, CA – USA – demetriodvm@yahoo.com

After ultrasound guided ovum pick up (OPU), the oocytes must go through in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC) to produce a viable embryo.

In vitro maturation

The oocyte aspiration dish is washed after OPU and the oocytes found are placed in a 6-well dish with 1000µl of search media. They are washed at least 3 times and classified in grade 1, 2, 3 or degenerated.  Oocytes with multiple layers of cumulus cells and a regular ooplasm (grades 1 and 2) have a better chance to produce a viable embryo.

The oocytes must go through in vitro maturation for 18 to 24 hours to become competent for fertilization.

Maturation is done in tubes inside a portable incubator (38.5oC) or in dishes inside an incubator (38.7oC, 5% CO2 in a humidified atmosphere). Usually the cumulus cells surrounding the oocyte expand after maturation.

In vitro fertilization

Matured oocytes are removed from the IVM tube or dish, washed once in the IVF media and moved to the fertilization dish (50µl media drop covered with 800µl oil – 38.7oC, 5% CO2 in a humidified atmosphere).

A straw of semen is thawed and its contents are placed on top of the percoll medium (silica-based colloidal medium), and centrifuged to separate the live spermatozoa from the cryoprotectant, seminal plasma and dead spermatozoa. The supernatant is removed and the remaining spermatozoa are washed with IVF media and centrifuged once again. The concentration on the final pellet has to be evaluated to determine the final insemination volume. The oocytes are inseminated with 10 to 20µl of the spermatozoa solution. Fertilization takes 8 to 12 hours.

 

In vitro culture

After fertilization, the cumulus cells surrounding the zygotes are removed by pipetting (also known as stripping). Zygotes are washed and placed into droplets of culture media (70µl media drop covered with 800µl oil – 38.7oC, 5% CO2 and 5% O2 in a humidified atmosphere). Sometimes the first cleavage is noted.

 

 

On day 3 of culture, morulas with 8-16 cells should be present and the cleavage rate is calculated (total cleaved divided by the total oocytes in IVC).Zygotes with less than 4 cells and unfertile are removed from the drop. Approximately 90% of the media is removed and replaced by new IVC media containing different nutrients necessary for this phase.

 

 

On day 5 of culture, compact morulas and early blastocysts should be present and an estimate of the final embryo production can be made. Approximately 90% of the media is removed and replaced by new IVC media containing different nutrients necessary for this phase.

 

 

 

On day 6 of culture, compact morulas and blastocysts should be present. The growth dynamics of the embryos seem to be highly correlated with the sire used for in vitro fertilization. At Ruann and Maddox we transfer day 6 blastocysts depending on recipient availability.

 

 

 

On day 7 of culture, the embryos should be transferred fresh into the synchronized recipients or cryopreserved. Expanded, hatching or hatched blastocysts have a higher chance of making a pregnancy than embryos that are still on the morula stage.

 

 

 

 

 

 

 

 

 

 

There are several ways to perform each step of the process and the description above is an example of how it is done at Ruann and Maddox Dairy (Riverdale, CA).  The oocytes and embryos shown in the pictures are from Ruann Holstein Donor cows.

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