123 LONG-TERM PRODUCTION OF IN VITRO-MATURED PORCINE OOCYTES AND EMBRYOS

Abstract

Although in vivo matured, ovulated, or both, oocytes provide the finest genetic material for use in assisted reproductive technologies (ART), their en masse production requires livestock production facilities, staff and associated overhead, is expensive and labor intensive, their harvest involves surgical or laparoscopic expertise, and large yields needed for en masse daily embryo production are cumbersome and very costly. In vitro-matured (IVM) oocytes have long been a practical gamete source for ART, including in vitro fertilization, ICSI and cloning. Rather than using conventional IVF to produce embryos, we employ in vitro oocyte activation for the production of diploidized parthenogenetic embryos, removing problems associated with variable embryo production due to polyspermic inseminations. In this way, we can produce a repeatable and consistent supply of mature oocytes, advanced embryos, or both, used in product testing, quality control, transgenic or cloned (or both) embryo production, in vitro development controls, as well as in-house culture control embryos for customer scientific data sharing. In this study, we observe mature oocyte and parthenogenetic embryo production over a complete year as control information for our laboratory. Additionally, colleagues may use these data for comparison in their own scientific mission. At least 3 times a month for 12 consecutive months, ovaries were collected from mature females at an abattoir and transported to our laboratory. Cumulus–oocyte complexes were aspirated from 4–6 mm follicles with an 18-gauge needle fixed to a vacuum pump system. Only COC surrounded by two or more layers of compact cumulus investment and containing oocytes of equal size were placed into a commercial TCM-199-based IVM system (Minitube of America Inc., Verona, WI, USA). After 42 h IVM, mature oocytes were isolated from their expanded cumulus and subjected to chemical (ionomycin/DMAP) parthenogenetic activation based on US Patent 5,496,720. Embryos were cultured 120 h in NCSU-23, then cultured for an additional 48 h in NCSU-23 (no BSA) supplemented with 10% FBS. A minimum of 1504 premium and 4604 standard oocytes (Minitube of America Inc.) were placed into IVM. Both premium (1364, 90.7%) and standard (4061, 88.2%; P > 0.05) oocytes are used to produce mature oocytes (MO). Of 781 premium MOs made into diploidized parthenogenetic embryos, 459 (58.8%) developed into blastocysts (61.3 cells/embryo). Of 2068 standard MO made into diploidized parthenogenetic embryos, 914 (44.2%; P < 0.05) developed into blastocysts (64.7 cells/embryo). En masse in vitro maturation of oocytes can supply a repeatable and consistent supply of mature oocytes for use in assisted reproductive technologies. These MO have the developmental potential to form blastocysts in vitro and enable scientists to infer developmental competence of in vitro-produced embryos for research and commercial use.