32 CRYOPRESERVATION OF DONOR CELLS FOR NUCLEAR TRANSFER: EFFECT OF CELL FREEZING METHOD ON THE EFFICIENCY OF SOMATIC CELL NUCLEAR TRANSFER IN PIGS

Abstract

Donor cell quality is one of the most important factors affecting somatic cell nuclear transfer (SCNT) in mammals. Many studies have been carried out to improve the donor cell characteristics in nuclear transfer, including studies on cell type, cell cycle stage, cell passage, and handling of donor cells before the SCNT. Even though most SCNT work is done with donor cells that have been previously frozen and thawed, no studies have been conducted to evaluate the effect of the cell freezing rate on the SCNT efficiency. The objective of this experiment was to evaluate the effect of the cell freezing method on development of pig SCNT embryos in vitro. Fibroblasts were collected from a 29-day-old female fetus, suspended in DMEM-F12 + 40% fetal bovine serum (FBS) + 10% dimethyl sulfoxide (DMSO), and placed in 1.6-mL cryovials for freezing. Vials were randomly assigned to two treatments: In treatment 1, cells were frozen at a controlled rate of 1�C/min in a programmable machine (P) until -40�C, and then plunged into liquid nitrogen (LN2; -196�C). In treatment 2, the traditional system (T), vials were placed in a styrofoam box and left overnight in a freezer at -80�C. The next day samples were plunged into LN2 (196�C). For each treatment, cells were thawed and cultured until confluence before being used for SCNT. Cells were used at passages 2 and 6. Cumulus-oocyte complexes (COCs) were aspirated from slaughterhouse ovaries and cultured for 39 h in TCM 199 supplemented with 10% porcine follicular fluid (pFF), 5 �g/mL insulin, 10 ng/mL epidermal growth factor (EGF), 0.6 mM cysteine, 0.2 mM pyruvate, 25 �g/mL gentamycin and 5 �g/mL each of equine and human chorionic gonadotropin (eCG and hCG). Oocytes were stained with bisbenzimide and enucleated in manipulation media with 7.5 �g/mL cytochalasin B by removing the first polar body and metaphase plate by means of a 16-�m beveled glass pipette. Cells from each treatment were injected into the perivitelline space of recipient enucleated oocytes and fused by two DC pulses of 140 V for 50 �s in fusion media. The fusion rate was evaluated 1 h later, and reconstructed oocytes were activated by two DC pulses of 120 V for 60 �s. After activation, oocytes were placed in bicarbonate-buffered NCSU-13 with 0.4% BSA and cultured at 38.5�C, 5% CO2 in a humidified atmosphere. Embryos were observed for cell cleavage at Day 2, and blastocyst development rate and cell number counting were done at Day 7 of culture. Every experiment was repeated three times. The temperature descending rate for P was slower and more linear (1�C/min vs. 2�C/min) than for the T method. Fusion rate was not significantly affected (P < 0.05) by the freezing method when they were evaluated either individually at each passage or accumulated regardless the passage (78.9 � 3.6% vs. 79.4 � 6.3%) for P and T, respectively. The same trends were observed for cleavage (61.2 � 5.2% vs. 64.3 � 5.2%), blastocyst development (4.2 � 1.8% vs. 5.0 � 2.8%), and number of cells at the blastocyst stage (19.4 � 3.1 vs. 19.8 � 6.2) for P and T, respectively. The present findings indicate that blastocyst development after SCNT does not differ when fetal fibroblasts donor cells are frozen by the two methods tested.