In Vitro Fertilization of In Vitro-Matured Equine Oocytes: Effect of Maturation Medium, Duration of Maturation, and Sperm Calcium Ionophore Treatment, and Comparison with Rates of Fertilization In Vivo after Oviductal Transfer
Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine, Texas A&M University, College Station, TX 77843-4466, USA.Biology of Reproduction (Impact Factor: 3.32). 07/2002; 67(1):256-62. DOI: 10.1095/biolreprod67.1.256
Three experiments were conducted to evaluate the effect of oocyte and sperm treatments on rates of in vitro fertilization (IVF) in the horse and to determine the capacity of in vitro-matured horse oocytes to be fertilized in vivo. There was no effect of duration of oocyte maturation (24 vs. 42 h) or calcium ionophore concentration during sperm capacitation (3 microM vs. 7.14 microM) on in vitro fertilization rates. Oocytes matured in 100% follicular fluid had significantly higher fertilization (13% to 24%) than did oocytes matured in maturation medium or in 20% follicular fluid (0% to 12%; P < 0.05). There was no significant difference in fertilization rate among 3 sperm treatments utilizing 7.14 microM calcium ionophore (12% to 21%). Of in vitro-matured oocytes recovered 40-44 h after transfer to the oviducts of inseminated mares, 77% showed normal fertilization (2 pronuclei to normal cleavage). Cleavage to 2 or more cells was seen in 22% of oocytes matured in follicular fluid and 63% of oocytes matured in maturation medium; this difference was significant (P < 0.05). We conclude that in vitro-matured horse oocytes are capable of being fertilized at high rates in the appropriate environment and that in vitro maturation of oocytes in follicular fluid increases fertilization rate in vitro but reduces embryo development after fertilization in vivo. Further work is needed to determine the optimum environment for sperm capacitation and IVF in the horse.
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- "collected and transferred into the oviducts of inseminated recipient mares, they are consistently fertilized and result in normal embryo development . In addition, intracytoplasmic sperm injection (ICSI) is used in equine clinical settings in a few laboratories and results in fertilization and embryo development   . "
ABSTRACT: In vitro fertilization (IVF) in horses is rarely successful. One reason for this could be failure of sperm to fully capacitate or exhibit hyper-active 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 (PVI) with equine sperm capacitated with dilauroyl phosphatidylcholine (PC12) 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 (ICSI) with equine sperm. Presumptive zygotes were either stained with 4',6-diamidino-2-phenylindole (DAPI) from 18 to 26 h at 2 h intervals or evaluated for cleavage 56 h following addition of sperm. Equine sperm fertilized bovine oocytes; however, pro-nuclei formation was delayed compared to bovine sperm after IVF. The delayed pro-nuclear formation was not seen following ICSI. In Experiment 2, bovine oocytes were assigned to five groups: 1) cumulus oocyte complexes (COC) co-incubated with bovine sperm, 2) COC exposed to sucrose then co-incubated with bovine sperm, 3) COC co-incubated with equine sperm, 4) COC exposed to sucrose and co-incubated 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 four groups: 1) IVF, equine and bovine COC co-incubated 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 24h for cleavage. No equine oocytes cleaved after IVF or PVI. However, ICSI conducted with equine sperm treated with PC12 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.
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- "Finally , under our experimental conditions , we observed that during equine IVF , we obtained IVF rates of 62% , after pre - incubation of oocytes with oviductal fluid . This result is interesting , considering that conven - tional IVF has only limited success in equine species ( Palmer et al . 1991 , Dell ' aquila et al . 1996 , Hinrichs et al . 2002 , McPartlin et al . 2009 , Mugnier et al . 2009 ) . We chose the technique developed by McPartlin et al . ( 2009 ) in which spermatozoa were hyperactivated using procaine . In this study , they obtained IVF rates of 61% but their results do not seem to be repeatable , and we obtained 37% under the same conditions ( our control group ) ."
ABSTRACT: Oviductal environment affects preparation of gametes for fertilization, fertilization itself, and the subsequent embryo development. The aim of this study is to evaluate the effect of oviductal fluid and the possible involvement of Deleted in Malignant Brain Tumours 1 (DMBT1) on in vitro fertilization (IVF) in porcine and equine species that represent divergent IVF models. We first performed IVF after pre-incubation of oocytes with or without oviductal fluid supplemented or not with antibodies directed against DMBT1. We showed that oviductal fluid induces an increase of the monospermic fertilization rate, and that this effect is cancelled by the addition of antibodies, in both porcine and equine species. Moreover, pre-incubation of oocytes with recombinant DMBT1 induces an increase of the monospermic fertilization rate in the pig, confirming an involvement of DMBT1 in the fertilization process. The presence of DMBT1 in the oviduct at different stages of the oestrus cycle was shown by Western blot and confirmed by immunohistochemical analysis of ampulla and isthmus regions. The presence of DMBT1 in cumulus-oocyte complexes was shown by Western blot analysis, and the localisation of DMBT1 in the zona pellucida and cytoplasm of equine and porcine oocytes was observed using immunofluorescence analysis and confocal microscopy. Moreover, we showed an interaction between DMBT1 and porcine spermatozoa using surface plasmon resonance studies. Finally, a bioinformatics and phylogenetic analysis allowed us to identify the DMBT1 protein as well as a DMBT1-like protein in several mammals. Our results strongly suggest an important role of DMBT1 in the process of fertilization.
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- "Oocytes beginning maturation in vivo are competent for fertilization and development into early pregnancies after transfer into the oviducts of inseminated mares, regardless whether they are collected directly before the anticipated time of ovulation or cultured in vitro for approximately 12 hours before transfer  . Oocytes matured in vitro have also resulted in high fertilization rates , pregnancies, and offspring after transfer into inseminated recipients  . "
ABSTRACT: The development of methods to produce embryos in vitro in the horse has been delayed compared with other domestic species. Oocytes can be collected from excised ovaries or from the small or preovulatory follicles of live mares. Intracytoplasmic sperm injection is the only reliable method to fertilize equine oocytes in vitro. Intracytoplasmic sperm injection-produced embryos can be transferred into the oviducts of recipient mares or cultured to the morula or blastocyst stage of development for nonsurgical embryo transfers into recipients' uteri. Embryos cultured in vitro have some morphological differences compared with embryos collected from the mares' uteri. Most notably, the embryonic capsule does not form in culture, and the zona pellucida fails to expand completely. However, embryo produced in vitro can result in viable pregnancies and healthy offspring.
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