1202 Elena Ruggeri et al.


Tremoleda et al., 2003). Our first observationsweredone4h after ICSI, and in most presumptive zygotes, the maternal metaphase II spindle with aligned chromosomes, first polar


body, and sperm headwere imaged. In the study by Tremoleda et al. (2003), oocytes remained at metaphase II directly after sperm injection, with signs of oocyte activation noted in most oocytes at 6h and leading to the second meiotic division with extrusion of the second polar body. We observed the anaphase to telophase transition, prior to extrusion of a second polar body, by 4h in approximately one quarter of injected oocytes. In our study, IVO seemed to develop to pronuclei


sooner after ICSI than IVM, with the highest percentages of pronuclei noted after ICSI at 6 and 8 h for IVO, in com- parison with 12 and 16 h for IVM. Within pronuclei were distinct areas with no CREST anticentromere staining, sig- nifying NPB (Fig. 2a). The timing of pronuclei formation agreed with previous reports using IVM oocytes, with half of sperm-injected oocytes reaching the pronuclear stage by 12 h, while the remaining presumptive zygotes were still in earlier stages of development (Tremoleda et al., 2003). In another study, half of the sperm-injected IVM oocytes had two pronuclei at 20h after ICSI, but none had two pronuclei at 10h after ICSI (Tremoleda et al., 2003). A significant percen- tage (36%) of these sperm-injected, IVMoocytes did not cleave into two cells until 48h after ICSI (Tremoleda et al., 2003). In our laboratory, we would expect cleavage or indicators or impending cleavage for IVO and IVM oocytes by 24h after ICSI (Carnevale, personal communication, 2017). The timing of pronuclei formation for IVO and IVM


could be impacted by many factors other than maturation method, including differences in sperm, procedures, inherent oocyte quality, and total time of maturation. However, oocyte quality and cytoplasmic maturation could also differ between IVO and IVM oocytes (Sutton et al., 2003; Gilchrist & Thompson, 2007). Oocytes collected from all follicles from excised ovaries represent a heterogenous group of oocytes in different stages of competence, matura- tion, and atresia (Hinrichs, 1991). In contrast, oocytes col- lected from dominant, maturing follicles are in a relatively consistent stage of maturation, with ovulation of a meta- phase II oocyte anticipated ~38 h after administration of induction drugs (Figueiredo et al., 2011). In this study, IVO oocytes were collected during the process of maturation, probably at metaphase I, and cultured in medium for the completion of maturation, in a system that has been proven to result in developmentally competent oocytes (Carnevale et al., 2004). In contrast IVM oocytes were collected from follicles of various sizes and from ovaries of mares of unknown ages or stages of their reproductive cycle or preg- nancy. Oocytes collected from small- or medium-sized,


Figure 3. Abnormal morphologies of potential equine zygotes after intracytoplasmic sperm injection include (a) premature chromosome condensation, with female spindle (top left) and male chromosomes flanked by a spindle structure (lower right); (b) multiple pronuclei; (c) sperm chromatin induced ectopic polar body, represented by the two sets of chromosomes undergoing anaphase; (d) multipolar spindle; (e) scattered maternal chromo- somes (blue, DNA; red, centromeres and kinetochores; green, tubulin).


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