Difference in Sensitivity to Culture Condition Between In Vitro Fertilized and Somatic Cell Nuclear Transfer Embryos in Pigs

Abstract

Abstract. We evaluated the developmental competence of somatic cell nuclear transfer (SCNT) embryos using in vitro embryo culture systems. Embryos were cultured in NCSU-23, NCSU-23 supplemented with essential and non-essential amino acids (NCSU-23aa) or modified PZM-5 supplemented with BSA instead of PVA (mPZM-5). The rates of blastocyst formation were significantly higher in the mPZM-5 group than in the other groups, regardless of the method of embryo production (38.0 vs. 25.3 or 29.1% for IVF, 18.2 vs. 8.7 or 9.4% for SCNT, respectively). The mean cell numbers of IVF and SCNT blastocysts were also significantly higher in mPZM-5 than in the other groups (62.0 vs. 42.3 or 43.0 for IVF, 46.5 vs. 29.4 or 31.3 for SCNT, respectively). Next, the embryos were cultured in mPZM-5 from days 0 to 4 and then in mPZM-5 (P/P), NCSU-23 (P/N) or NCSU-23aa (P/Naa) until day 6. The rates of blastocyst formation were similar among the 3 two-step culture systems in both embryo groups (36.2, 34.2, and 33.6% for IVF, 20.8,14.1, and 17.2% for SCNT, respectively). The mean cell number in the IVF and SCNT blastocysts was significantly lower in P/N than in P/ P and P/Naa (46.5 vs. 63.5 and 68.7 for IVF, 29.3 vs. 45.5 and 39.7 for SCNT, respectively). Next, we examined the effect of media on apoptosis in IVF and SCNT blastocysts. The apoptosis indices in the blastocysts derived from either NCSU-23 or mPZM-5 were analyzed by TUNEL assay. The apoptosis index of the SCNT blastocysts was significantly lower in mPZM-5 than in NCSU-23 (8.8 vs. 13.6%), whereas no such difference was observed between groups in the IVF embryos (5.1 vs. 4.4%). These data suggested that SCNT embryos were more easily affected by culture environment compared with IVF embryos, offering the possibility to further enhance the developmental competence of SCNT embryos by developing more appropriate culture conditions in pigs. Key words: Apoptosis, In vitro embryo culture, Miniature pig, Nuclear transfer (J. Reprod. Dev. 55: [299][300][301][302][303][304] 2009) loned pigs by somatic cell nuclear transfer (SCNT) might be used as improved domestic livestock. However, their use is not limited to this. Owing to the many similarities in between the anatomy and physiology of pigs and humans, these cloned pigs can be used for xenotransplantation and as improved models for studying human physiology and disease [1,2]. Moreover, in particular, miniature pigs offer several advantages: their small body size enables space-saving and facilitates better control of specific pathogens. They therefore represent attractive options for use in medical applications and related technologies [3]. Thus, miniature pig cloning by SCNT might also prove useful in many biomedical applications [4]. Although it has been successful recently, its efficiency is still low, as is the case in other animals [5]. One of the reasons for the very low success rate of miniature pig cloning is the limitations of the in vitro production (IVP) system for pigs, including in vitro maturation (IVM) and their subsequen...

Improving the Quality of Miniature Pig Somatic Cell Nuclear Transfer Blastocysts: Aggregation of SCNT Embryos at the Four‐cell Stage

“…2002)] and cultured in PZM‐3 at 38.5°C in 5% CO 2 in air. The rate of monospermic penetration of IVF embryos was 61.5% (56/91) of penetrated oocytes (Yamanaka et al. 2009).…”

“…2007), diploid complement retention using cytochalasins (Sugimura et al. 2008) and in vitro culture medium (Yamanaka et al. 2009).…”

Promising System for Selecting Healthy In Vitro–Fertilized Embryos in Cattle

“…Detection of apoptosis-positive cells with the TUNEL assay was performed as described by Yamanaka et al [26]. Blastocysts were washed three times in PBS supplemented with 0.1% polyvinylalcohol (PVA; Sigma) and fixed in 2% (w/v) paraformaldehyde (Sigma) and 0.2% (v/v) Triton X-100 in PBS for 40 h at room temperature.…”

Acetylation Level of Histone H3 in Early Embryonic Stages Affects Subsequent Development of Miniature Pig Somatic Cell Nuclear Transfer Embryos

“…Coincubation of oocytes with sperm was carried out for 6 h post-insemination. The oocytes were then washed 3 times, and 20-30 oocytes were cultured in 100-μl drops of modified porcine zygote medium (PZM)-5 [24,25] for 6 days at 38.5 C in 5% CO2 in air. The rates of cleavage and blastocyst development were assessed on days 2 and 6 of in vitro culture, respectively.…”