4-cell embryo (Photo credit Felix et al., U-PA (New Bolton))

Frozen Semen for Standard IVF?

Frozen Semen for Standard IVF - 4-cell embryo (Photo credit Felix et al., U-PA (New Bolton))

4-cell embryo
Photo credit: Felix et al., U-PA (New Bolton)

In September of 2022, Felix et al. (University of Pennsylvania, New Bolton) published a paper which detailed the use of fresh semen for successful standard in-vitro fertilization in the equine[1] with poor results when using frozen semen, so a question to arise out of that research was, with refinement, is it possible to use frozen semen for standard IVF in the equine?

Until the work previously published by the University of Pennsylvania (New Bolton) team, “true” IVF – where sperm and the oocyte are introduced together in a petri dish and essentially left to their own devices to achieve fertilization – has had extremely limited success. By “limited success”, we mean 2 foals total, which were produced in the early 1990s by Palmer et al. in France! By changing protocols and media, the New Bolton team achieved a 90% fertilization rate, with 74% of the fertilized eggs giving rise to blastocysts, which in turn produced 3 live foals. This was achieved using fresh semen.

Because of the limitations previously seen with true equine IVF, a dramatic increase in recent years has been seen with the use of ICSI – intracytoplasmic sperm injection. In this procedure a single sperm is injected directly into the oocyte to artificially achieve fertilization. One advantage of this method is that a minimal number of sperm are required, and so frozen semen can be used even if the supply is limited. With the development of the new true IVF technique, the question arises “can frozen semen be used for standard IVF in the equine?” At ISER XIII two of the researchers from the original team – Drs. Felix and Hinrichs – presented a paper detailing their work providing an answer to that question.

In order to achieve satisfactory results with the new equine frozen semen true IVF technique, it was recognized that the sperm needed to be of “good” quality, so a selection process was required. The research looked at three different physical selection methods: swim-up, colloid centrifugation and microfluidics. Thawed semen from a single stallion was used in each method. In the swim-up group, 200 µL of thawed semen was used for processing; in the colloid centrifugation group, 400 µL; and in the microfluidic group 500 µL.

After undergoing the various selection procedures, the resulting sperm were standardized by being extended to a concentration of 1 million sperm/mL in 50 µL, and incubated in vitro-matured equine cumulus-oocyte complexes were then added to the droplets containing the sperm. All were then further incubated before being transferred to embryo culture medium for 32h. At the end of that period, the oocytes were evaluated to determine if cleavage with two or more normal nuclei were present, and if so were deemed “fertilized”.

The microfluidic selection process yielded the best fertilization (cleavage) rate at 71.9% (23/32), swim-up producing only 22.9% (8/35) and colloidal centrifugation lagging further behind at 16.1% (5/31).

Although further review is required to determine ongoing development to live foal production, this technique shows that the use of frozen semen for standard IVF in the equine is feasible. With refinement, it is possible that the volumes used in this research can be reduced – possibly rivalling those currently used with ICSI – making this a possible alternative to that more expensive technique.

(Felix M, Hinrichs K. 2023. Selection of frozen-thawed semen for standard in vitro fertilization. JEVS 125:104648)

Felix MR, Turner RM, Dobbie T, Hinrichs K. 2022. Successful in vitro fertilization in the horse: production of blastocysts and birth of foals after prolonged sperm incubation for capacitation. Biology of Reproduction, 107(6):1551-1564