Validation of a heterologous fertilization assay and comparison of fertilization rates of equine oocytes using in vitro fertilization, perivitelline, and intracytoplasmic sperm injections.
Abstract: IVF in horses is rarely successful. One reason for this could be the failure of sperm to fully capacitate or exhibit hyperactive motility. We hypothesized that the zona pellucida (ZP) of equine oocytes prevents fertilization in vitro, and bypassing the ZP would increase fertilization rates. Limited availability of equine oocytes for research has necessitated the use of heterologous oocyte binding assays using bovine oocytes. We sought to validate an assay using bovine oocytes and equine sperm and then to demonstrate that bypassing the ZP using perivitelline sperm injections (PVIs) with equine sperm capacitated with dilauroyl phosphatidylcholine would result in higher fertilization rates than standard IVF in bovine and equine oocytes. In experiment 1, bovine oocytes were used for (1) IVF with bovine sperm, (2) IVF with equine sperm, and (3) intracytoplasmic sperm injections (ICSIs) with equine sperm. Presumptive zygotes were either stained with 4',6-diamidino-2-phenylindole from 18 to 26 hours at 2-hour intervals or evaluated for cleavage at 56 hours after addition of sperm. Equine sperm fertilized bovine oocytes; however, pronuclei formation was delayed compared with bovine sperm after IVF. The delayed pronuclear formation was not seen after ICSI. In experiment 2, bovine oocytes were assigned to the following five groups: (1) cumulus oocyte complexes (COCs) coincubated with bovine sperm; (2) COC exposed to sucrose then coincubated with bovine sperm; (3) COC coincubated with equine sperm; (4) COC exposed to sucrose, and coincubated with equine sperm; and (5) oocytes exposed to sucrose, and 10 to 15 equine sperm injected into the perivitelline (PV) space. Equine sperm tended (P = 0.08) to fertilize more bovine oocytes when injected into the PV space than after IVF. In experiment 3, oocytes were assigned to the following four groups: (1) IVF, equine, and bovine COC coincubated with equine sperm; (2) PVI of equine and bovine oocytes; (3) PVI with equine oocytes pretreated with sucrose; and (4) ICSI of equine oocytes. Oocytes were examined at 24 hours for cleavage. No equine oocytes cleaved after IVF or PVI. However, ICSI conducted with equine sperm treated with dilauroyl phosphatidylcholine resulted in 85% of the oocytes cleaving. Sperm injected into the PV space of equine oocytes did not appear to enter the ooplasm. This study validated the use of bovine oocytes for equine sperm studies and indicates that failure of equine IVF is more than an inability of equine sperm to penetrate the ZP.
Copyright © 2014 Elsevier Inc. All rights reserved.
Publication Date: 2014-04-16 PubMed ID: 24815920DOI: 10.1016/j.theriogenology.2014.04.002Google Scholar: Lookup
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- Comparative Study
- Journal Article
- Research Support
- Non-U.S. Gov't
- Validation Study
Summary
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This article presents a study investigating the challenges in successful in vitro fertilization (IVF) in horses, exploring whether the Zona Pellucida (ZP) of equine oocytes impedes fertilization and if bypassing it can improve the rates of fertilization.
Overview of the Research
- The researchers used bovine (cattle) oocytes, which are more readily available, to carry out a heterologous oocyte binding assay, which gauges how well equine sperm binds to and perforates a non-equine oocyte.
- The study involved three main experiments in which bovine oocytes were used in various ways involving both bovine and equine sperm, as well as equine and bovine oocytes with intracytoplasmic sperm injections (ICSIs).
- The research sought to understand whether bypassing the Zona Pellucida (ZP) — the outer layer of the oocyte, or egg — with perivitelline sperm injections (PVIs) could lead to a higher rate of successful fertilisation than standard IVF. The PV space is the gap between the cell membrane of the oocyte and the ZP.
Key Experiments and Findings
- In the first experiment, the researchers used bovine oocytes for IVF with bovine sperm, IVF with equine sperm, and for IVF with intracytoplasmic sperm injections (ICSIs) with equine sperm. It was observed that equine sperm could fertilize bovine oocytes, although the formation of the pronuclei, the first sign of successful fertilisation, was delayed compared to bovine sperm. This delay wasn’t observed with ICSI.
- In the second experiment, researchers investigated five conditions using bovine oocytes and sperm. They found that equine sperm was marginally more likely to fertilize bovine oocytes when injected into the PV space than after standard IVF.
- The third experiment involved equine and bovine oocytes subjected to different treatments, including IVF and PVI and the application of ICSI of equine oocytes, with equine sperm treated with dilauroyl phosphatidylcholine. The outcome suggested that only ICSI resulted in cleavage (the first stage of cell division after fertilisation) of the oocytes, with the percentage of oocytes cleaving being as high as 85%.
- The results also showed that injecting equine sperm into the PV space did not seem to ensure the entry of sperm into the ooplasm (the cytoplasm of oocytes). This finding indicated the failure of equine IVF is not simply due to the incapacity of equine sperm to penetrate the ZP.
Conclusion
- The study validates the use of bovine oocytes as a representation for equine sperm studies as well as highlighting that the unsuccessful rates of equine IVF might extend beyond the inability of equine sperm to go through the ZP.
Cite This Article
APA
Sessions-Bresnahan DR, Graham JK, Carnevale EM.
(2014).
Validation of a heterologous fertilization assay and comparison of fertilization rates of equine oocytes using in vitro fertilization, perivitelline, and intracytoplasmic sperm injections.
Theriogenology, 82(2), 274-282.
https://doi.org/10.1016/j.theriogenology.2014.04.002 Publication
Researcher Affiliations
- Equine Reproduction Laboratory, Department of Biomedical Sciences, Colorado State University, Fort Collins, Colorado, USA.
- Equine Reproduction Laboratory, Department of Biomedical Sciences, Colorado State University, Fort Collins, Colorado, USA.
- Equine Reproduction Laboratory, Department of Biomedical Sciences, Colorado State University, Fort Collins, Colorado, USA. Electronic address: elaine.carnevale@colostate.edu.
MeSH Terms
- Animals
- Cattle
- Coculture Techniques
- Fertilization / physiology
- Fertilization in Vitro / veterinary
- Horses / physiology
- Male
- Oocytes / physiology
- Phosphatidylcholines / pharmacology
- Sperm Injections, Intracytoplasmic / veterinary
- Spermatozoa / physiology
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