Bisection of equine embryos

Animal Reproduction Laboratory, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, Colorado 80523, USA
Equine Veterinary Journal (Impact Factor: 2.29). 06/2010; 21(S8):129 - 133. DOI: 10.1111/j.2042-3306.1989.tb04697.x

ABSTRACT Three experiments were designed to evaluate methods of bisecting equine embryos. Viability of demi-embryos was compared to whole embryos in vitro and in vivo. In Experiment 1, Day 6 embryos were bisected either using a standard micromanipulation technique or a manual split of embryos that were adhered to a petri dish. Viability was assessed by culture in vitro. Bisected embryos in both groups increased in size during culture and developed normally. In general, manual bisection was accomplished with less difficulty and time than the micromanipulation method. Eleven additional embryos were bisected manually and both halves transferred surgically into the same recipient (Experiment 2). More pregnancies were obtained from transfer of twin whole embryos (16/22) compared to bisected, demi-embryos (5/22). In Experiment 3, 30 embryos were bisected manually; 15 were encapsulated in agar chip. Both halves were transferred non-surgically to each recipient. Fifteen whole embryos were transferred non-surgically to recipients as controls. The number of vesicles per original embryo was greater for whole embryos than for demi-embryos in agar chips.

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    ABSTRACT: Blastocysts (5 unexpanded, 5 expanding, 2 hatched) were recovered non‐surgically between 144 and 192 h after detection of ovulation. Unexpanded and expanding embryos were cultured (TCM199 + 10% foetal bovine serum) for 10 days. Mean diameter was 168.0 μm for unexpanded and 251.0 μm for expanding blastocysts, respectively. Unexpanded embryos started to hatch from the zona pellucida (ZP) when their diameter reached 405.4 μm (embryonic age 240.0 h). Four embryos squeezed through a small opening in the ZP whereas 1 embryo gradually tore open the ZP. Three embryos completed the hatching process within 96–120 h. In contrast, expanding embryos started to hatch at a mean diameter of 499.8 μm (embryonic age 192.0 h). Four embryos escaped from the ZP within 12 h. One embryo squeezed through the ZP but did not shed the ZP entirely during culture. In‐vitro hatched embryos continued to expand spherically until reaching a maximum mean diameter of 1141.7 μm (unexpanded embryos) and 2765.0 μm (expanding embryos), except for 1 embryo which attached to the bottom of the culture dish. In completely hatched embryos, no capsule could be detected at x200 magnification after staining. Three embryos which did not entirely hatch from the ZP until the end of culture had a capsule remaining inside the ZP. In 1 case, a thin acellular layer was detected in the shed ZP after 0.3% trypsin treatment. Both freshly recovered hatched blastocysts (diameter 640 μm, 697 μm) had a clearly visible capsule around the trophectoderm cells. Equine blastocysts not only lost their zona pellucida but also their capsule during the hatching process in vitro.
    Equine Veterinary Journal 06/2010; 25(S15):91 - 94. · 2.29 Impact Factor
  • Equine Veterinary Journal 06/2010; 21(S8):92 - 100. · 2.29 Impact Factor
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    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.
    Journal of Equine Veterinary Science 07/2012; 32(7):367-371. · 0.62 Impact Factor