Factors affecting post-mortem production of equine ICSI blastocysts

Abstract

In vitro fertilization of equine oocytes by intracytoplasmic sperm injection (ICSI) has enabled post-mortem production of equine embryos. This procedure makes it possible to secure one last offspring from mares that must be suddenly euthanized. Currently, little is known about how clinical factors influence the outcome. The aim of this study was to evaluate the effect of the following clinical factors on the chance of a successful session (≥1 embryo) and blastocyst rates after post-mortem ICSI: Mare age, number of collected oocytes per mare, location of euthanasia (at our clinic/in the field), duration of ovary transport, method of ovary removal (during anaesthesia, after pentobarbital, after bolt gun), reason for euthanasia (acute life-threatening conditions requiring immediate euthanasia vs. non-acute conditions, where euthanasia could be postponed). The study was performed retrospectively and included ovaries from 65 euthanized mares in the period between 2019-2022. The mares, mainly Warmbloods, were aged between 1 to 26 years old (average 14.4 years old). If euthanasia was performed in the field, the ovaries were transported to our clinic (0.5 to 14 hours) in sterile saline at 22-32⁰C. The oocytes were collected by scraping each follicle. After overnight holding, the oocytes were matured for 30 hours. The mature metaphase II oocytes were inseminated by ICSI with the desired semen and after7-10 days of culture, blastocysts were vitrified. In total, 842 oocytes were collected from 65 mares (range: 1 to 47 oocytes per mare, average = 13), 744 oocytes were placed in maturation medium and the remaining degenerated oocytes were discarded. A total of 65% (481/744) of the oocytes reached the MII stage and were injected, and 11.4% (55/481) of these reached the blastocyst stage within 10 days after ICSI. The proportion of successful sessions was 37%. The factors that were shown to affect the chance of a successful session, were the number of collected oocytes per mare and the reason for euthanasia (acute vs. non-acute; multiple logistic regression, p < 0.05). The other examined factors did not affect the chance of a successful session (multiple logistic regression, p > 0.05). When comparing mares euthanized for non-acute reasons to mares euthanized for acute reasons, there was a higher chance of a successful session (50%, 16/38 vs. 20.7%, 6/29, respectively, p = 0.02, Fisher's exact test) and a higher blastocyst rate (16.2%, 44/272 vs. 5.3%, 11/209, p = 0.0001, Fisher's exact test) in mares euthanized for non-acute reasons. This study reports that post-mortem, in vitro embryo production can be performed on mares euthanized both in the field and at the ICSI clinic, but with a lower chance of a successful session for mares euthanized due to acute life-threatening conditions.