Macromolecular crowded culture medium formed by addition of polyvinylpyrrolidone (PVP; molecular weight = 360 000), positively influences the viability, growth, and development of bovine oocytes. Owing to its apparently various effects, uncovering the specific mechanisms of crowding responsible for these outcomes is important. The present study was conducted to determine the effects of crowding on oocytes with a particular focus on the intimacy of contacts between oocyte and cumulus/granulosa cells. Growing mouse oocyte-granulosa cell complexes were cultured for 10 days in a modified α-minimum essential medium, supplemented with PVP at a concentration of 0%, 1%, 2%, or 3% (w/v). Although the complexes developed in all groups, 2% and 3% PVP medium induced a substantial morphological modification, and a larger proportion of oocytes associated with cumulus cells survived in 3% PVP medium than in the 0% or 1% PVP medium. No significant difference was found in the frequencies of polar body extrusion (78–88%) and blastocyst formation (approximately 40%) after in vitro fertilization among the experimental groups. Confocal laser scanning microscopy indicated a higher number of transzonal processes reaching the oocyte from cumulus cells in 2% PVP medium than in 0% PVP medium. Transmission electron microscopy depicted close adhesion of the oocyte with cumulus cells in 2% PVP medium —bearing a resemblance to their in vivo counterparts— and loose adhesion in 0% PVP medium. In conclusion, we found that a mechanism for the action of crowded conditions involves the strengthening of contacts and communication between oocytes and companion cumulus/granulosa cells.

This study was aimed at evaluating the effects of multi-layered cumulus cells (MCCs) during vitrification and in vitro fertilization (IVF) of mature bovine oocytes and embryogenesis after IVF. The rates of cleavage and blastocyst formation were higher in vitrified and fertilized oocytes with MCCs than in denuded oocytes (P < 0.05), but were comparable to the rates in fresh oocytes with MCCs or without (denuded). When the MCC-enclosed oocytes were denuded before IVF, blastocyst formation rate reduced compared with that in vitrified oocytes with MCCs (P < 0.05). This suggested that the MCCs surrounding the mature bovine oocytes play important roles during cryopreservation: protecting them against freezing and promoting their survival and development post IVF, thereby increasing the success rates of IVF and embryonic development. Herein, we showed for the first time that calves could be produced using only 14–19 vitrified mature oocytes with MCCs from the ovaries of individual cows post slaughter.

For examining pig ovaries, which have complex structures, laparoscopy is a useful technique, but requires general anesthesia; therefore, it cannot be performed repeatedly within a short period of time. We report a transvaginal endoscopy-based technique for conducting ovarian examinations without general anesthesia. Sows were sedated in pig stalls. Using a colonoscope, the vaginal wall was punctured with a trocar. To avoid the trocar being caught in the broad ligament of the uterus or the connective tissue around the vagina, the trocar was inserted close to the external uterine os and between the 2:00 and 3:00 or the 9:00 and 10:00 positions (in a clockwise direction). Then, a urethroscope was inserted into the abdomen, and an examination was carried out after the ovaries had been moved towards the urethroscope camera via rectal palpation. This less invasive procedure may allow repeated examinations and will increase our understanding of ovarian dynamics in pigs.