136 Effect of laser-induced sperm membrane damage on preimplantation development of horse ICSI embryos

In the field of equine reproductive biotechnology, optimizing success rates of intracytoplasmic sperm injection (ICSI) remains crucial for improving breeding outcomes, genetic advancements, and animal welfare. One of the challenges associated with ICSI is the need to disrupt the membrane of a viable spermatozoon before injection. Plasma membrane damage has been shown to be an essential process in sperm immobilization before ICSI and plays a vital role in sperm-triggered oocyte activation. Therefore, sperm immobilization before ICSI is thought to be routine protocol and necessary for efficient fertilization. Laser systems have been an integral part of routine human IVF laboratory work for many years and laser-induced membrane sperm damage is a technique that has been tested in some species. However, its effectiveness in equine ICSI remains to be investigated. This study assessed how utilizing laser to disrupt the sperm membrane of motile sperm affected the preimplantation development of horse ICSI embryos. Cumulus–oocyte complexes were collected by local veterinarians through transvaginal ultrasound-guided follicle aspiration, placed in holding medium for 24 h, matured in vitro for 28 h, and cultured for 10 days. Frozen semen from two proven ICSI stallions (non-pooled) was thawed, and sperm cells were selected based on normal morphology and motility. Details of oocyte maturation and embryo culture media are available in Gambini et al. (2020 PLoS One 15, e0238948). A piezo drill was not used in our experiments. Control ICSI was performed by pressing the tail of the spermatozoon to the bottom of the dish with the injection pipette and then quickly withdrawing the pipette until a bend in the middle of the tail was observed. For the laser experimental group, the spermatozoon was immobilized by one laser pulse (RI Saturn 5 laser system) of ~200 μJ applied to the end of the tail of a motile sperm, as far away from the sperm head as possible. Curling of the sperm tail was observed after the pulse. Fisher’s exact test was used to assess statistical differences. A total of 90 matured oocytes were injected, with 47 in the control group and 43 in the laser group. Embryos were assessed for cleavage and blastocyst formation. In the control group, 65.96% of the injected oocytes cleaved, while 79.07% of the laser group cleaved. Blastocyst formation rates on Day 8 were 27.66% for the control group and 20.93% for the laser group. By Day 10, 38.30% of the control group embryos had reached the blastocyst stage, compared with 30.23% in the laser group. No statistical differences were observed between the groups. No stallion effect was observed. These results suggest that laser-induced sperm membrane damage does not significantly affect the overall efficiency of ICSI blastocyst formation in horse. Additionally, we proved that this technology is safe for immobilizing sperm and showed that it could also be used to select a live sperm from a sample of immotile spermatozoa currently being tested by our research group.

This research was supported by Maria Vasas Foundation UQFGIBBI-2020001519.