204 Effect of euthanasia solution on oocyte developmental competence and fertilisation: a bovine model to determine toxicity in equine gametes
S. Martin-Pelaez A , A. de la Fuente B , M. Verstraete C , Z. Rabow D , S. Meyers B and P. Dini AA Department of Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis, CA, USA
B Department of Anatomy, Physiology, & Cell Biology, School of Veterinary Medicine, University of California, Davis, CA, USA
C Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium
D National Institute of Health West Coast Metabolomics Center, University of California, Davis, CA, USA
Reproduction, Fertility and Development 35(2) 231-231 https://doi.org/10.1071/RDv35n2Ab204
Published: 5 December 2022
© 2023 The Author(s) (or their employer(s)). Published by CSIRO Publishing on behalf of the IETS
Postmortem and pre-euthanasia gamete retrieval provides a last opportunity to preserve the genetic value of horses by removal of the ovaries and oocyte collection. Pentobarbital is the most common euthanasia agent; however, its effect on developmental competence of retrieved oocytes has not been determined. Here, we aimed to evaluate the presence and concentration of pentobarbital in follicular fluid (FF) post-euthanasia and investigate its effect on developmental competence of oocytes using a bovine IVF model. The concentration of pentobarbital was measured by gas-chromatography/mass-spectrometry in FF from mare ovaries 24 h post-euthanasia (n = 10), immediately after euthanasia (n = 10), and from ovariectomies (negative control; n = 10). Pentobarbital was detected in all FF samples with an average concentration of 56.5 mg/L, while it was not detected in the negative control group. Next, bovine cumulus-oocyte complexes were collected from slaughterhouse ovaries and oocytes were held in holding media with pentobarbital for 6 h (maximum duration that oocytes can be viable in a postmortem ovary). Oocytes were divided into three groups for incubation: pentobarbital at a concentration of 60 mg/L (H60, n = 196); at 164 mg/L (pentobarbital concentration in serum; H164, n = 215); or no pentobarbital (control; n = 212). After the holding, the oocytes were matured and fertilised in vitro, followed by in vitro culture to blastocyst stage. The cumulus expansion grade, cleavage rate, and blastocyst formation rate were compared among the groups using a Wilcoxon test, followed by paired wise comparison. Grade one cumulus expansion (multiple expanded layers of non-degenerated cumulus cells) was higher in the controls (53.5%) than H60 (23.5%) and H164 (13.0%; P < 0.001); while H164 showed the highest number of the Grade three (unexpanded cumulus cells) oocytes (39%; P < 0.001), followed by H60 (24%), and the controls (13%). Cleavage rate was higher in the controls than H164 (64% vs 44%; P < 0.01) and showed a trend between the controls and H60 (64% vs 52.5%; P = 0.06). Blastocyst rates (blastocyst/cleaved oocytes) was not different among the groups; with a numerically higher rate in the controls (29%, 11–53%; median, min-max), followed by H60 (25%, 0–44%) and H164 (24%, 0–58%). Here, we showed that pentobarbital reaches the FF immediately after euthanasia, exposing oocytes to this drug. This exposure had an effect on the maturation and cleavage rates. Interestingly, the IVP success rate in oocytes exposed to pentobarbital was highly variable, with some replicates yielding no embryos. These findings mark an important milestone in equine postmortem gamete retrieval, as ovary collection post-euthanasia can yield a similar number of IVP embryos than pre-euthanasia collected ovaries, although the success can vary from zero to normal embryo production rates.
