Application of Assisted Reproductive Technologies (ART to Clinical Practice)

1. Introduction

In the past decade, assisted reproductive technologies (ART) such as oocyte transfer, intracytoplasmic sperm injection (ICSI), and nuclear transfer (cloning) have become effective enough for clinical use. ART such as oocyte transfer and ICSI allow production of foals from mares that cannot provide an embryo by standard embryo-transfer procedures. For the equine practitioner, application of ART can range from simply sending ovaries to a laboratory after a mare’s untimely death to setting up complete oocyte and embryo culture systems in a practice. This paper outlines some of the increasingly common ways in which ART may be applied to equine clinical practice, including ongoing developments in embryo biopsy for pre-implantation genetic diagnosis and embryo vitrification.

2. Shipment of Ovaries Postmortem

It is possible to recover oocytes from ovaries up to 24 h postmortem and produce foals from those oocytes by either ICSI or oocyte transfer. Although little critical information exists in this area, data suggest that oocytes recovered less than 6–8 h after the mare’s death are more likely to produce embryos, and the embryos that are produced yield pregnancies that are more likely to be carried to term.^1,2 

When ovaries arrive at the laboratory, oocytes are recovered from all visible follicles. Because these oocytes are from follicles that were not ready to ovulate, the oocytes are immature; they are placed in culture for 24–36 h to mature in vitro. After culture, about 50% of oocytes are expected to mature, and these can be used for (1) oocyte transfer (surgical transfer to the oviduct of an inseminated recipient mare), (2) ICSI and then surgical oviductal transfer of cleaved embryos 24 h after ICSI, or (3) ICSI and in vitro culture to the blastocyst stage followed by transcervical transfer to a recipient mare (either immediately or after vitrification or freezing³). If multiple oocytes are recovered, the third procedure (ICSI and culture of oocytes to the blastocyst stage) would seem to be the treatment of choice, because this will allow transfer of each developing embryo to a separate recipient mare. In contrast, when surgical transfers are performed, the cost and labor involved necessitate transfer of multiple oocytes into single recipients, thus incurring the risk of multiple pregnancies in one recipient and no pregnancies in another recipient.

Removal of Ovaries and Transport to the Laboratory

To ship excised ovaries for oocyte transfer or ICSI, the ovaries should be removed from the mare as soon as possible postmortem; however, delays in recovery from the mare of up to 5 h postmortem have been associated with production of foals from shipped ovaries.⁴ We have obtained embryos and pregnancies from mares euthanized with barbiturate overdose and from mares from which the ovaries were removed under general anaesthesia (inhalant or xylazine/ketamine) before euthanasia. We suspect that euthanasia by potassium chloride overdose may reduce oocyte viability. After removal, ovaries should not be refrigerated. If transport time is less than 2 h, ovaries should be kept at body temperature (98°F or 37°C). For longer transport times, ovaries should be cooled to room temperature (about 65–70°F or 20°C). The ovaries should be packaged to minimize temperature fluctuation during shipment. For shipment by car, ovaries may be placed in a styrofoam container with ballast (bottles or bags of water at the appropriate temperature) around them.

For shipment by air, the currently recommended method of packaging uses an Equitainer.a The coolant cans should be warmed (for short transport) or at room temperature (for longer transport) for 24 h before shipment. If coolant cans at the appropriate temperature are not available, then the coolant cans should be removed from the Equitainer and a ballast (e.g., a rectal sleeve filled with water at the appropriate temperature) placed in the receptacle in the Equitainer. The ovaries should be bagged separately, either in normal saline or if none is available, in a closed plastic bag and placed on the ballast. Another ballast at the appropriate temperature should be placed on top of the ovaries. Frozen coolant cans, as are typically used with transported semen, should not be used.

For best results, ovaries should be received within 6 h of the mare’s death or removal of the ovaries from a live mare. Semen from the desired stallion should be shipped to the laboratory to arrive no later than the day after the ovaries are received. If oocyte transfer is being performed, the semen should be of high fertility, because the recipient mare will be inseminated; semen of low quality, including frozen semen, should be avoided. If ICSI is being performed, fresh, cooled-shipped, or frozen semen may be used.

Reported rates of production of late pregnancies/foals with oocytes recovered from clinical cases post-mortem per donor mare are 8/25 (32%) by oocyte transfer¹ and 10/16 (63%) by ICSI and in vitro embryo culture to the blastocyst stage followed by transcervical transfer.⁵

3. Shipment of Oocytes Postmortem

In the case of postmortem production of foals, to avoid the reduction in viability associated with pro- longed shipment of ovaries to the laboratory, as noted above, it is possible for the practitioner to recover oocytes from the ovaries and ship these to the laboratory. We have identified a medium, EH medium (Table 1), that allows holding of oocytes overnight at room temperature without a reduction in the rate of oocyte maturation or embryo development.⁶ EH medium is a mixture of 40% M199/ Hank’s/Hepes and 40% M199 with Earle’s salts with 25 μg/ml gentamycin and 20% fetal calf serum.
 

To recover oocytes from the follicles of excised ovaries, aspiration with a needle and syringe, as used in cattle, should not be used; this results in a low recovery rate and stripping of the majority of the cumulus from the oocytes.⁷ This is because of the tighter and stronger attachment of the oocyte to the follicle wall in the horse than in other species.⁸ Although there are many ways to recover oocytes from excised ovaries, the following is the procedure that is used in our laboratory, which has been associated with good meiotic and developmental competence of the recovered oocytes.^6,9,10  [...]