Effect of pentobarbital as a euthanasia agent on equine in vitro embryo production.
Abstract: Postmortem and pre-euthanasia oocyte retrieval provides the last opportunity to preserve the genetic material in mares. Pentobarbital (PB) is the most common euthanasia agent; however, its effect on the developmental competence of oocytes has not been determined. Here, we evaluated the concentration of PB in equine follicular fluid (FF) and investigated its effect on the developmental competence of oocytes using a bovine IVF model to overcome the low availability of equine oocytes. The concentration of PB was measured by gas-chromatography/mass-spectrometry in FF collected from mare ovaries immediately after euthanasia (n = 10), 24 h post-euthanasia (n = 10), and from the ovaries collected by ovariectomy (negative control; n = 10). The serum concentration of PB was also evaluated as a positive control. PB was detected in all FF samples with an average concentration of 56.5 μg/ml. Next, bovine cumulus-oocyte complexes (COC) were held in holding media with PB for 6 h at 60 μg/ml (H60, n = 196), 164 μg/ml (H164, n = 215) or without PB (control; n = 212). After holding, the oocytes were matured and fertilized in vitro, followed by in vitro culture to the blastocyst stage. The cumulus expansion grade, cleavage rate, blastocyst rate, embryo kinetic rate and the blastocyst cell numbers were compared among the experimental groups of bovine COC. Higher rates of Grade 1 cumulus expansion were found in controls (54%, 32-76%; median, min-max) in comparison to H60 and H164 (24%,11-33% and 13%, 8-44%; P < 0.001). The cleavage rate was higher in the controls than in H164 (64% vs. 44%; P 0.05) in comparison to the laboratory-established rate during the same timepoints. Overall, we showed that PB reaches the FF immediately after euthanasia, exposing oocytes to this drug. This exposure affected cumulus expansion and cleavage rates in a bovine model, suggesting initial damage caused by PB that may not completely impede the formation of embryos, although lower overall embryo numbers might be obtained.
Copyright © 2023 Elsevier Inc. All rights reserved.
Publication Date: 2023-04-05 PubMed ID: 37084499DOI: 10.1016/j.theriogenology.2023.04.002Google Scholar: Lookup
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Summary
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This research investigates the effects of pentobarbital, a common euthanasia agent, on the developmental abilities of horse oocytes (the cells that develop into eggs for reproduction) both immediately after death and in the 24 hours following. Using a bovine in vitro model due to a lack of available equine oocytes, the researchers found that while the drug impacted the initial stages of embryo development, it did not completely stop the formation of embryos.
Introduction and Background
- The research focused on the effect of pentobarbital (PB), a common euthanasia agent used on horses, on their oocytes – the cells responsible for embryonic development.
- Death of horses and use of euthanasia provides the last chance to preserve their genetic material, particularly through oocytes.
- An existing gap in research was that the impact of PB on the developmental abilities of the oocytes was not known.
Methods and Outcomes
- The research measured PB’s concentration in the equine follicular fluid (the fluid that surrounds the oocyte in the ovary), immediately after death, and 24 hours later.
- The study also controlled for negative and positive scenarios by taking samples from horse ovaries removed through ovariectomy before euthanasia and also evaluating the concentration of PB in serum.
- It was found that PB was present in all follicular fluid (FF) samples, hinting at exposure of oocytes.
In-Vitro Experiment and Results
- To study the impact of this exposure to PB, the researchers used a bovine in vitro model.
- The bovine cumulus-oocyte complexes (COC) were cultured with PB in varying concentrations and compared to COC without PB.
- The oocytes were then matured and fertilized in vitro, followed by in vitro culture to the blastocyst stage.
- The parameters such as cumulus expansion grade, cleavage rate, blastocyst rate, embryo kinetic rate, and blastocyst cell numbers were compared between the experimental groups.
- The results showed a higher rate of Grade 1 cumulus expansion and a higher cleavage rate for controls (without PB) compared to those exposed to PB.
- However, there was no significant difference in blastocyst rates and total cell number among the PB-exposed and non-exposed.
- In a preliminary study, when equine oocytes were exposed to PB, there was a lower maturation rate in comparison to unexposed oocytes.
Conclusions
- The study concludes that while PB impacts the initial stages of embryo development, it does not outright prevent the formation of embryos.
- The presence of PB in FF suggests that oocytes are exposed to this drug immediately after euthanasia and a 24-hour setting.
- It suggests that using oocytes for genetic preservation even after death using PB as a euthanasia agent may result in a less successful outcome due to the detrimental effect of PB on oocyte development.
Cite This Article
APA
Martin-Pelaez S, Rabow Z, de la Fuente A, Draheim P, Loynachan A, Fiehn O, Meyers S, Lyman C, Dini P.
(2023).
Effect of pentobarbital as a euthanasia agent on equine in vitro embryo production.
Theriogenology, 205, 1-8.
https://doi.org/10.1016/j.theriogenology.2023.04.002 Publication
Researcher Affiliations
- Department of Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis, CA, USA.
- West Coast Metabolomics Center, University of California, Davis, CA, USA.
- Department of Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis, CA, USA; Department of Anatomy, Physiology, & Cell Biology, School of Veterinary Medicine, University of California, Davis, CA, USA.
- College of Veterinary Medicine, Auburn University, Auburn, AL, USA.
- Veterinary Diagnostic Laboratory, Department of Veterinary Science, University of Kentucky, Lexington, KY, USA.
- West Coast Metabolomics Center, University of California, Davis, CA, USA.
- Department of Anatomy, Physiology, & Cell Biology, School of Veterinary Medicine, University of California, Davis, CA, USA.
- College of Veterinary Medicine, Auburn University, Auburn, AL, USA.
- Department of Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis, CA, USA. Electronic address: pdini@ucdavis.edu.
MeSH Terms
- Animals
- Horses
- Female
- Male
- Cattle
- Pentobarbital / pharmacology
- Semen
- Euthanasia, Animal
- Oocytes
- Embryo, Mammalian
- Blastocyst
- In Vitro Oocyte Maturation Techniques / veterinary
- In Vitro Oocyte Maturation Techniques / methods
- Fertilization in Vitro / veterinary
Conflict of Interest Statement
Declaration of competing interest The authors do not have any conflict of interest to declare.
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