133 EFFECTS OF FROZEN STORED CULTURE MEDIA ON PRE-IMPLANTATION DEVELOPMENT OF PARTHENOTES AND CLONED EMBRYOS IN PIGS

Abstract

The present study was designed to examine if frozen storage of porcine zygote medium (PZM3) plus 3 mg mL^–1 BSA (Yoshioka et al. 2002 Bio. Reprod. 66, 112–119) is feasible to culture pig embryos produced by parthenogenetic activation and somatic nuclear transfer. Slaughterhouse-derived sow cumulus–oocyte complexes (COCs) were matured in TCM199 supplemented with 10% porcine follicle fluid, 5% cattle serum, 10 IU mL^–1 eCG, 5 IU mL^–1 hCG, 0.8 mm L-glutamine and 0.05 mg mL^–1 gentamicin at 38.5°C, 100% humidity and 5% CO₂ in air. For activation, cumulus cells were removed after 42 to 44 h of maturation, and the denuded oocytes with 1st polar body were activated with a double 160 V mm^–1, 100 μs direct pulse followed by culture in PZM3. Each experiment was replicated at least three times. Data were expressed as mean ± SEM and analyzed by using chi-square module in SPSS 11.0, with P < 0.05 denoting significant difference. In Experiment 1, after preparation, liquid PZM3 was aliquoted to 50 mL falcon centrifuge tubes. Randomly, half of the tubes with PZM3 were put into –80°C freezers, and the rest were placed into 4°C refrigerator. Within one week after storage, a tube of frozen PZM3, while that stored at 4°C served as control, was warmed at 38.5°C in CO₂ incubator, and more than three 4-well culture dishes were then made with 400 μL PZM3 in each well and balanced for at least 4 h in the incubator before experiment. The results showed that both cleavage (78/93, 83.9 ± 1.2% v. 87/103, 84.5 ± 1.8%, P > 0.05) and blastocyst (60/93, 65.2 ± 2.1% v. 65/103, 63.1 ± 3.8%, P > 0.05) rates were similar between frozen-warmed PZM3 and control, as was total cell numbers per blastocyst (50 ± 7 v. 47 ± 5, P > 0.05) between groups. In Experiment 2, we used somatic cloned embryos to investigate the effect of frozen-warmed PZM3 on pre-implantation development of such embryos. Our results indicated that no significant difference in rates of cleavage (68/95, 71.5 ± 5.1% v. 78/100, 78.1 ± 1.9%, P > 0.05), blastocyst formation (33/95, 34.6 ± 7.6% v. 78/100, 38.2 ± 3.5%, P > 0.05) and total cell numbers per blastocyst (40 ± 11 v. 48 ± 9, P > 0.05) was found between the test and control groups, designed the same as in Experiment 1. In Experiment 3, we tested whether PZM3 in frozen storage for 5 months was able to support in vitro development of parthenotes comparable to freshly-made ones. PZM3 after frozen storage for 5 months was warmed using the same method as Experiment 1, and the newly made PZM3 within 1 week of storage at 4°C acted as control. The results showed that although the cleavage (135/138, 97.8 ± 2.7% v. 117/129, 90.7 ± 3.1%, P > 0.05) and blastocyst (104/138, 75.4 ± 1.6% v. 84/129, 65.1 ± 2.3, P > 0.05) rates in control group were both slightly higher than that in the test group, no statistical differences was observed. We also found no significant difference in total cell numbers per blastocyst (48 ± 7 v. 46 ± 6, P > 0.05) between groups. Taken together, our results imply that frozen storage of PZM3 is feasible, and of practical value for culture pig embryos.