Optimization of parthenogenetic activation of rabbit oocytes and development of rabbit embryo by somatic cell nuclear transfer

Abstract

The present study explored a suitable parthenogenetic activation (PA) procedure for rabbit oocytes and investigated the developmental potential of somatic cell nuclear transfer (SCNT) embryos using rabbit foetal fibroblasts (RFFs). The electrical activation had the optimal rate of blastocyst (14.06%) when oocytes were activated by three direct current (DC) pulses (40 V/mm, 20 μs each) followed by 6-dimethylaminopurine (6-DMAP) and cycloheximide (CHX) treatment; the blastocyst rate of ionomycin (ION) + 6-DMAP + CHX (12.07%) activation was higher than that of ION + 6-DMAP (8.6%) activation or ION + CHX (1.24%) activation; there was no significant difference in blastocyst rate between ION + 6-DMAP + CHX and DC + 6-DMAP + CHX groups. The blastocyst rate of ION + 6-DMAP + CHX-activated oocytes in the basic rabbit culture medium (M-199) + 10% foetal bovine serum (FBS; 14.28%) was higher than that in buffalo conditioned medium (5.75%) or G1/G2 medium (0), and the blastocyst rate was increased when M-199 + 10% FBS was supplemented with amino acids. Refreshing culture medium every day or every other day significantly increased the blastocyst rate. Treatment of donor cells with 0.5% FBS for 3-5 days increased blastocyst rate of SCNT embryos (33.33%) than no serum starvation (22.47%) or 0.5% FBS treatment for 6-9 days (23.61%); the blastocyst rate of SCNT embryos derived from nontransgenic RFFs was higher than that derived from transgenic RFFs by electroporation. The blastocyst development ability of SCNT embryos derived from RFFs by electroporation (32.22%) was higher than that of liposome (19.11%) or calcium phosphate (20.00%) transfection, and only the embryos from electroporation group have the EGFP expression (24.44%). In conclusion, this study for the first time systematically optimized the conditions for yield of rabbit embryo by SCNT.

Combined Exogenous Activation of Bovine Oocytes: Effects on Maturation-Promoting Factor, Mitogen-Activated Protein Kinases, and Embryonic Competence

“…However, our analysis revealed no significant differences in the proportions of cleaved embryos and total blastocysts, indicating that there were no variations in embryonic development between the groups being compared. Previous studies in other species have reported that combined inhibition of protein synthesis and phosphorylation improved in vitro developmental potential [50][51][52][53][54][55]. They were even able to produce offspring in horses using somatic cell nuclear transfer [47,48].…”

“…Moreover, the parthenotes activated with ANY or CHX do not activate the JNK and P38 signaling pathways related to cellular damage [19]. With a view to improving the efficiency of the chemical activation of mammalian oocytes, several studies have reported the dual combination of oocyte-activating compounds (cycloheximide and DMAP), particularly in horses [46][47][48], pigs [49,50], buffalos [51], goats [52], and rabbits [53,54]. Thus, the combined use of chemical inhibitors raises a valid strategy for improving oocyte activation efficiency and subsequent embryo development.…”

Sperm‐borne proteins improve rabbit cloning efficiency via regulating embryonic cleavage and epigenetics

“…However, PLCζ and other sperm proteins are completely missing in the cloned embryo. Ca 2+ oscillations can be simulated by chemical or electrical action in cloned embryos to activate them, but this often results in inferior activation, abnormal division, and poor development [32, 33]. Injection of soluble sperm extracts into parthenogenetic embryos can improve activation and development [34–37].…”

The Creation of a Multiallele Knockout Genotype in Rabbit Using CRISPR/Cas9 and Its Application in Translational Medicine

“…CRISPR/Cas9 technology in animal transgenesis has undergone great improvements in recent years. Prior to the employment of this technique, it was a big challenge to knockout a gene in rabbits because of the lack of suitable embryonic stem cells [19][20][21] and the low efficiency and difficulties associated with rabbit somatic cell nuclear transfer [22]. Now, with the almost completely sequenced rabbit genome and expertise available, CRISPR/Cas9 is a relatively robust technology that can be used to knockout almost any gene in rabbits.…”