184 Effect of Concentration and Exposure Duration of Fetal Bovine Serum on Parthenogenetic Development of Porcine Follicular Oocytes

Abstract

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

Deoxynivalenol and Deepoxy-Deoxynivalenol- Induced Alterations in Theca Cell Function as a Major Cause of Infertility in Dairy Cows

“…Bovine theca cells were cultured in serumfree conditions that maintain testosterone, progesterone secretion and responsiveness to LH [10]. Bovine ovaries were obtained at the slaughterhouse from adult cows, independently of the stage of the estrous cycle, and transported to the laboratory at 30 ºC in phosphatebuffered saline (PBS) containing penicillin (100 IU) and streptomycin (100 µg/mL) [11]. Follicles (4-6 mm diameter) were bisected within the ovarian stroma, gently scraped to remove granulosa cells, and the theca 'shells' were peeled from the ovarian stroma with forceps.…”