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Abstract. This study was conducted to evaluate the effects of exposing porcine ovaries to 30-33 C during transportation for 4 h and subsequently room temperature (25 C) for 6 h of storage on in vitro maturation (IVM) and subsequent parthenogenetic development of oocytes collected from the ovaries. After IVM, oocytes having a tight oopalsm membrane and no signs of degeneration were exposed to Dulbecco's phosphate-buffered saline (DPBS) with 7% ethanol (v/v) for 7 min to induce parthenogenetic activation. Moreover, we also determined whether exposure of the collected oocytes to room temperature for 1, 2 and 4 h in DPBS or porcine follicular fluid (pFF) affected parthenogenetic development. When porcine ovaries were stored after transportation, oocytes collected from the stored ovaries showed a significantly higher rate of degeneration after 65 h of IVM (58.4%) and a significantly lower rate of cleavage after parthenogenetic activation (40.1%) than oocytes collected from ovaries immediately after transportation (38.9% and 47.4%, respectively). However, there was no significant difference in developmental rates to the morula and blastocyst stages between these two groups (14.4% and 14.3%, respectively). The duration of preservation, 1, 2, and 4 h, of oocytes in DPBS did not affect parthenogenetic development. In contrast, when preserved for 4 h in pFF, the developmental rates of the oocytes were significantly decreased. This suggested that some factor(s) in follicular fluid affects the developmental rate of oocytes with the passage of time in ambient conditions. These results suggest that even after 6 h storage of ovaries, oocytes having normal morphology after IVM have the same rate of parthenogenetic development as oocytes collected from ovaries just after 4 h of transportation, except for a lower cleavage rate, and that exposure of oocytes to room temperature for 4 h in DPBS does not affect their parthenogenetic developmental competence.

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The aim of the present experiment was to examine hatching rate as a testing tool of porcine embryo viability before early-stage embryo transfer, such as zygotes or 2-cell stage embryos. We evaluated the optimal concentrations and exposure durations of fetal bovine serum (FBS) on porcine parthenotes. Ovaries were obtained from prepubertal gilts at a local abattoir and brought to the laboratory in physiological saline with antibiotics at 30–33°C. The ovaries were washed and wiped, and then cumulus–oocytes complexes (COCs) in the follicular fluid were aspirated from surface-visible follicles (2–6 mm in diameter) with a 10-mL syringe fitted with an 18-gauge needle. After being washed 3 times with modified phosphate-buffered saline (DPBS; GIBCO, Grand Island, NY, USA) containing 0.3% BSA, the COCs were suspended in maturation medium, NCSU-23 containing 10% (v/v) porcine follicular fluid, 10 ng mL−1 epidermal growth factor (EFG; Sigma-Aldrich Corp., St Louis, MO, USA), 10 µg mL−1 follicular stimulating hormone (FSH; Sigma), 35 µg mL−1 luteinizing hormone (LH; Sigma), 1 mg mL−1 cysteine (Sigma, USA), 100 IU mL−1 penicillin G, and 100 µg mL−1 streptomycin sulfate (GIBCO). After 24 h, the COCs were transferred to the same medium without hormones. The oocytes matured for 48 h were denuded. The oocytes with a visible polar body were selected and returned to the maturation medium without hormones. After 65 h of maturation, oocytes were exposed to PBS with 7% ethanol (v/v) for 7 min, and then the oocytes were washed and treated in TCM-199 containing 5 µg mL−1 cytochalasin B (Sigma) for 5 h at 38.5°C in an atmosphere of 5% CO2 and 95% air with high humidity. After cytochalasin B treatment, the presumptive parthenotes were cultured in PZM-5 medium (IFP, Japan) and cleavage of the parthenotes was assessed at 72 h of activation. Normally cleaved parthenotes were cultured for 8 days to evaluate their ability to develop to the blastocyst and hatching stages. The FBS was added at Day 4 or 5 with concentrations of 2.5, 5, or 10%. The blastocyst rates ranged from 39.1 to 70% in each treatment. However, the hatching rate was dramatically decreased in the non-addition group. In this experiment, the developmental potential may be estimated before embryo transfer by an in vitro culture system up to the hatching stage. Table 1. Effect of concentration and exposure duration of FBS on parthenogenetic development of porcine follicular oocytes