STERILE SOLUTIONS FOR EMBRYO TRANSFER

Embryo transfer in cattle, sheep, goats and also in horses is an expanding business in many parts of the world. The technology of assisted reproduction has large potential for the management of controlled breeding programs particularly in the cattle industry. Furthermore, the technology allows for the transfer of disease-free embryos in the frozen state from one country to another in order to introduce new genetic material and build up a gene pool at a rate which would not be possible by any other means.

The traditional approach of hormone-controlled superovulation and non-surgical recovery of embryos ("flushing") is most widely used and does not require sophisticated laboratories and highly specialized staff. The production of embryos by in vitro fertilization and in vitro maturation on the other hand is more efficient because many more embryos can be produced, but it is also a far more demanding technique and is at present only used routinely in a few laboratories. The in vitro methods will be of particular value in the preservation of wildlife.

Highveld Biological has been involved in the manufacture of sterile solutions for flushing, manipulating, freezing, thawing and short term maintenance of embryos from bovine, sheep, goats and horses for almost 10 years; the general application of this technology in South Africa is due to the technical input and service to the veterinary practitioners which this company is able to provide.

Below is a short protocol for the use of buffer solutions, media and sera used in embryo transfer:

Embryo Transfer Medium (ETM) containing bovine serum albumin fraction V (special formulations are available for sheep and horse).

Dulbecco's Phosphate Buffered Saline (D-PBS) containing fetal calf serum (flushing medium).

Before flushing, warm the respective medium to room temperature. If D-PBS is used in combination with 1% fetal calf serum (b), melt units of 1ml or 5ml serum and add to 100 ml or 500 ml of flushing medium.

After harvesting, the embryos should be manipulated into petri-dishes containing holding medium with 10% serum. Under a stereo microscope the embryos are graded according to international standards and separated into 5 groups: stage 1 to 4 are embryos suitable for transfer, stage 5 are unfertilized ova.

Embryos are rinsed free of debris in 4 changes of washing buffer; they are then either transferred into a suitably prepared cow using holding medium, or processed for frozen storage with or without a trypsinization stage.

A 0.25% solution of trypsin in Ca-and Mg-free D-PBS is sometimes used to sanitize the zona pellucida of the embryo which may harbor possible infectious agents like bacteria, mycoplasma or viruses; this procedure together with careful washing in D-PBS or holding medium is considered reasonably efficient to render specific-pathogen-free embryos which are then suitable for export. Expensive isolation and identification methods for infectious agents including the polymerase chain reaction (PCR), may have to be employed if requested by regulatory agencies. The ultimate aim is to avoid any possible risk of disease transmission through embryo transfer.

a) Freezing Media A and B contain increasing amounts of glycerol.
b) Direct Transfer Medium (DT Medium) contain ethylene glycol.

The sanitized embryos may be manipulated into a petri-dish containing freezing medium A (5% glycerol), equilibrated for 5 minutes, then changed to freezing medium B (10% glycerol) and transferred in this buffer to straws for freeze storage in liquid nitrogen. An alternative and simpler method is to transfer the embryos directly from the holding medium into a medium containing 0.75 or 1.5M ethylene glycol (DT Medium).

a) Thawing Media I to IV contain decreasing amounts of glycerol.
b) Direct Transfer Medium (DT Medium) contain ethylene glycol.

If thawing media I to IV are used, 35 mm petri-dishes are filled with 20ml of each medium and the contents of not more than 3 melted straws are dispensed into thawing medium I. The embryos are moved carefully through the thawing media I to IV, leaving them to equilibrate in each medium for 5 to 6 minutes. To avoid cross dilution of the carefully adjusted thawing media, use new dishes with fresh media for the contents of every two to three straws.

For the DT method, the straws are removed from liquid nitrogen, thawed and the content immediately transferred into the recipient animal. This method is much easier and as successful as the 4 stage method.

If the transfer of the embryos has to be delayed for any reason, they should be transferred into a more complex nutrient culture medium Ham's F12 with fetal calf serum where they can stay up to 4 hours at room temperature and up to 12 hours in a 37°C incubator.

Buffers and media contain antibiotics and must be stored at 4°C; sera and antibiotic solutions are stored frozen.

All solutions should be handled under sterile conditions in a laminar air flow cabinet. If this is not possible, bottles once opened, must be considered non-sterile and the content should be discarded after 6 to 8 hours.

Solutions which are stored consistently at 4°C have a shelf life of at least 12 months, frozen items can still be used after 24 months, provided they have not been defrosted.

PRODUCTS FOR EMBRYO TRANSFER AVAILABLE FROM HIGHVELD BIOLOGICAL

403	Ethylene glycol	210-H	Holding medium
L10-F1	Ethylene glycol in DPBS	L10	Ovum Transfer medium
201-F	Flushing medium	L10-cm	OTM (equine)
210-F	Freezing medium	P10	Ovum medium (powder)
L08-E	Ham's F10 (embryo culture)	L10-T	Thawing medium