Procedures for In Vitro Production of Bovine Embryos
The procedures for in vitro production [IVP; i.e. in vitro maturation (IVM), in vitro fertilization (IVF), and in vitro culture (IVC)] of embryos described here are based on procedures developed in other laboratories at the University of Wisconsin (Parrish et al., 1986), University of Guelph (Xu et al., 1992), and University of Missouri (Hernandez-Ledezma et al., 1993). These procedures as used by our group have been published previously (Edwards et al., 1997; Paula-Lopes et al., 1998). Keep in mind that the protocols described here are not fixed but rather constantly evolve as new developments take place. Therefore, practitioners of IVP will be well advised to experiment with the procedures used, especially after reading of improvements made by other laboratories. This protocol is organized by day of the protocol with d 0 being the day of fertilization.
DAY -2
PREPARATION FOR IN VITRO PRODUCTION OF EMBRYOS
Materials and Equipment Needed
2 x 1 L sterile saline (transport saline)
1 aliquot of stock 16 (Pen-Strep 10 ml) per bottle of saline
3 to 4 bottles of oocyte collection medium (OCM) (400 ml)
1 aliquot of stock 11 (glutamine 4 ml) per bottle of OCM
1 aliquot of stock 15 (Pen/Strep 4 ml) per bottle of OCM
1 aliquot of stock 4 (BSS Hep 8 ml) per bottle of OCM
1 aliquot of oocyte maturation medium (OMM) (87 ml)
1 aliquot of stock 8 (gentamicin 1 ml) per aliquot of OMM
1 aliquot of stock 3 (BSS 10 ml) per aliquot of OMM
1 aliquot of stock 6 (Folltropin 125 ml) per aliquot of OMM
1 aliquot of stock 10 (glutamine 1 ml) per aliquot of OMM
200 ml of Stock 5 (estradiol) per aliquot of OMM
1 ml of Stock 2 (sodium pyruvate) per aliquot of OMM
Laminar flow hood
Procedure
1. The day before the collection of ovaries, prepare and place at 4°C until use.
a) Saline Pen/Strep -add 1 aliquot (10 ml) of Stock 16: pen/strep to each liter of transport saline
b) OCM Supplements - add supplements to OCM as described
c) OMM Supplements - add supplements to OMM as described
2. Place saline (0.5 L/container) into two clean containers (Fig. 1). Place both containers in a thermos and leave at room temperature for use the next day.
Figure 1. This container is an example of one that is suitable for transportation of ovaries. Almost any leak-proof plastic container will work.
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