Developmental Competence of equine MI oocytes following in vitro maturation culture

Abstract

With an increasing demand for the in vitro production (IVP) of equine embryos over the past 10 years, the value of each oocyte recovered has also increased. Intracytoplasmic sperm injection (ICSI) is performed to fertilize the oocytes reaching MII stage of nuclear maturation, which is determined by microscopic evidence of an extruded polar body (PB). Cytoplasmic maturation, during which replication of organelles, proteins and RNA's necessary for postfertilization development occurs, is more difficult to evaluate. However, equine oocytes contain a large amount of cytoplasmic lipids and the migration and polarization of lipid granules can be microscopically visualized in mature oocytes. Furthermore, the distribution of lipids may reflect a level of cytoplasmic capacitation and developmental competence, although the cleavage rate of equine oocytes fertilized following maturation culture does not appear to be significantly different based on lipid polarization (Ambruosi et al. Therio 2009; 71:1093-1104). The present study evaluated the migration and polarization of cytoplasmic lipids (granularity) following in vitro maturation to determine if blastocyst development can occur in the absence of PB extrusion. Cumulus-oocyte-complexes (COC; n=411) obtained from ovarian follicles by transvaginal aspiration were stored in a holding medium overnight at 20°C before undergoing incubation at 38.5°C in 50ul drops of equilibrated maturation media as described by Foss. et al (EVJ Supp 2013;45:39-43). After 28.5 hours, oocytes were denuded followingexposure to 80IU hyaluronidase and evaluated for the presence of an extruded PB as well as lipid reorganization. The cytoplasm of MI oocytes that did not exhibit PB extrusion was graded according to the percentage with a dark granular appearance. All oocytes were fertilized by conventional ICSI with thawed spermatozoa from ICSI-fertile stallions. Fertilized oocytes were cultured as described by Foss et al (EVJ Suppl 2013;45:39-43). When examined under an inverted 1000X light microscope, 66% (N=273/411) of the denuded oocytes exhibited an intact membrane with an extruded polar body and were considered mature (MII), while 26% (N=106/411) of the oocytes without a visible polar body were classified as immature (MI). Of the MI oocytes injected, 23% (N=24/106) were deemed to have attained some level of cytoplasmic capacitation (Cc=40-70% granularity). A cleavage rate of 25.5% was recorded for all injected MI oocytes regardless of Cc status. Of the 24 MI oocytes with some Cc, nine (37%) cleaved and continued to develop, and seven of those developed to the blastocyst stage. Of the 82 MI oocytes that did not demonstrate Cc, 18 (22%) cleaved but no blastocysts (0%) were obtained. This study found a blastocyst developmental potential of 6.6% per total MI's injected and 29% per MI oocytes with at least a 40-70% Cc. Therefore, injecting oocytes that do not show nuclear maturation, yet have reached a level of cytoplasmic capacitation, may still result in blastocyst development.