It has been almost 20 years since I started an assisted reproduction program at Colorado state University. The purpose of that program was to develop techniques to preserve equine genetics. It is beyond the scope of this article to discuss all the goals of that program but I would like to focus on production of foals from old mares and stallions via in vitro technology (aka test tube horse).
Ovum Pickup and Intracytoplasmic Sperm Injection
The first “test tube” horse named, Fire Cracker, was produced at CSU in 1996. Since that time thousands of in vitro embryos have been produced, transferred and resulted in foals. Intracytoplasmic Sperm Injection (ICSI) has become a viable technique for producing foals from mares with reproductive problems. It is also useful for stallions that have low sperm numbers or have died and that only limited frozen semen is available. From my knowledge, there are 4 commercial facilities and two universities that currently offer ICSI. These include Equigea (operated out of Peterson Smith Equine Reproduction Center in Ocala Florida, an SBS Affiliate Laboratory), Equine Medical Service in Missouri, and In Foal Inc. in California, EquiEmbryo in Texas (Young Ho Choi) as well as Colorado State University and Texas A& M University. Although there seems to be interest across the US for clinics and Universities to add more ICSI facilities, it is unlikely that there will be very many new facilities established. The startup cost for facilities and equipment can be several hundreds of thousands of dollars and even more daunting is finding a trained technician to perform the ICSI. It takes a lot of trial and error to become good at ICSI even though some of the technicians have come out of the human field. There seems to be more art than science involved. Also, the number of candidates is limited since the majority of the ICSI procedure and in vitro embryo production is driven by mare infertility or stallion infertility.
Initially, most oocytes were collected from mature pre-ovulatory follicles from hormonally treated mares. Typically, once collected by transvaginal follicle aspiration, the mature oocyte needs to be injected within 12 hours. Most of these mature oocytes are collected and injected at the same facility but if the oocyte is shipped it must be maintained in special culture medium and shipped in a portable incubator designed to maintain the proper gas mixture. A major breakthrough at Texas A&M was the development of procedures for collection and shipment of immature oocytes. These immature oocytes are less sensitive to temperature and culture conditions and are generally shipped in a simple embryo holding medium in a device that holds the temperature close to room temperature. Once these oocytes arrive at the ICSI facility they are placed in culture medium in a CO2 incubator and cultured for 24-30 hr to allow maturation.
OPU and ICSI in the Industry Today
The procedure of aspirating small follicles has stimulated interest by the veterinarian to learn the technique of transvaginal oocyte collection (Ovum pickup, OPU) and shipment of immature oocytes to an ICSI facility. The OPU technique requires minimal equipment, and with proper instruction as well as lots of practice sessions, can be learned by most veterinarians experienced in reproduction. Based on publications from several ICSI facilities, the number of embryos produced is 0.7-1.0 embryo per OPU session of small follicles. This rate similar or greater than what one would obtain with flushing mares for embryos. These in vitro embryos can either be shipped back to the referring veterinarian for transfer, transferred into recipients at the ICSI facility or frozen. The advantage of freezing is that the embryos can be thawed and transferred when recipients are available. For additional information about freezing embryos please see our article, Cryopreservation of Equine Embryos.
Some interesting trends in in vitro embryo production have come out of the only large commercial ICSI facility in Europe, Avantea in Cremona Italy. They produced over 800 ICSI embryos in 2016. Nearly all were produced from OPU of immature follicles. Mares had OPU performed in Utrecht University and other locations and oocytes shipped to Avantea in Italy. It seems as though the European sport horse trainer/breeder prefers OPU/ ICSI over standard embryo transfer since the mare does not have to be examined with ultrasound as often and does not have to be bred or flushed. The mare only comes to the clinic every 2-3 weeks to have her eggs collected from follicles on the ovaries. One of the reasons that such large numbers of in vitro embryos are produced by Avantea is the willingness of the breeders to present the mares almost year around. Oocyte collection in the non-breeding season appears to be just as successful as during the breeding season. Obviously if embryos are produced in the off season then they must be frozen until recipient mares begin cycling the following season or later. Pregnancy rates of frozen/ thawed, in vitro produced embryos, have been reported by this group to be 55-60%.
There are things to consider when deciding between traditional ET and OPU/ICSI. They include cost, early embryonic death (EED) and the number of oocytes you may obtain with OPU.
A traditional ET pregnancy costs approximately $5000.00 which includes the recipient mare. This is lower in comparison to OPU/ICSI. With the recipient mare included the process of OPU/ICSI can cost $7500.00-$8000.00 for relatively fertile animals or close to $10,000.00 for mares which require multiple cycles. These are estimates only as the fees will vary between facilities. However, each group typically charges separately for OPU, maturation, ICSI, blastocyst formation, etc.
Early Embryonic Death
When performing embryo transfer, one can expect less than 10% EED. This is lower in comparison to OPU/ICSI where you can expect up to 20% EED.
Number of Oocytes per cycle
The number of oocytes retrieved from OPU depends on the number of follicles present on the mare’s ovaries at the time of the procedure. Usually a 50% oocyte recovery per follicle is expected with OPU or approximately 6-7 oocytes and 0.7-1.0 blastocysts per OPU. For example, if six oocytes are recovered from OPU, 4 will mature, and 25% of those will become blastocysts. Resulting in one blastocyst per OPU.
Time will tell if we can convince veterinarians and breeders in the United States to produce ICSI embryos year around by using freezing technology. Also, can the mindset of the breeder be changed where they prefer OPU/ ICSI over embryo transfer for normal reproductively health mares and utilize the entire year to produce embryos?If you like this article you may also be interested in the following articles: