Use of Confocal Microscopy to Evaluate Equine Zygote Development After Sperm Injection of Oocytes MaturedIn VivoorIn Vitro

Abstract Confocal microscopy was used to image stages of equine zygote development, at timed intervals, after intracytoplasmic sperm injection (ICSI) of oocytes that were maturedin vivoorin vitro. After fixation for 4, 6, 8, 12, or 16 h after ICSI, zygotes were incubated withα/βtubulin antibodies and human anticentromere antibody (CREST/ACA), washed, incubated in secondary antibodies, conjugated to either Alexa 488 or Alexa 647, and incubated with 561-Phalloidin and Hoechst 33258. An Olympus IX81 spinning disk confocal microscope was used for imaging. Data were analyzed usingχ²and Fisher’s exact tests. Minor differences in developmental phases were observed for oocytes maturedin vivoorin vitro. Oocytes formed pronuclei earlier when maturedin vivo(67% at 6 h and 80% at 8 h) thanin vitro(13% at 6 and 8 h); 80% of oocytes maturedin vitroformed pronuclei by 12 h. More (p=0.04) zygotes had atypical phenotypes, indicative of a failure of normal zygote development, when oocyte maturation occurredin vitroversusin vivo(30 and 11%, respectively). Some potential zygotes from oocytes maturedin vivohad normal phenotypes, although development appeared to be delayed or arrested. Confocal microscopy provided a feasible method to assess equine zygote development using limited samples.