Use of oocytes selected by brilliant cresyl blue staining enhances rabbit cloned embryo development in vitro

Abstract

SummaryRabbits play an important role in people’s lives due to their high nutritional value and high-quality hair that can be used as raw material for textiles. Furthermore, rabbits are an important animal model for human disease, as genome-edited animals are particularly valuable for studying gene functions and pathogenesis. Somatic cell nuclear transfer (SCNT) is an important technique for producing genome-edited animals and it has great value in saving endangered species and in clone stem cell therapy. However, the low efficiency of SCNT limits its application, with the selection of suitable rabbit oocytes being crucial to its success. In the present study, we collected oocytes from ovarian follicles and stained them with 26 μM brilliant cresyl blue (BCB). We then matured the oocytes in vitro and used them for SCNT. Comparison of the BCB-positive oocytes with BCB-negative oocytes and the control group showed that the BCB-positive group had a significantly higher maturation rate (81.4% vs. 48.9% and 65.3% for the negative and control groups, respectively), cleavage rate (86.6% vs. 67.9% and 77.9%), blastocyst rate (30.5% vs. 12.8% and 19.6%), total number of blastocysts (90±7.5 vs. 65.3±6.3 and 67.5±5.7), and inner cell mass (ICM)/ trophectoderm (TE) index (42.3±4.2 vs. 30.2±2.1 and 33.9±5.1) (P<0.05). The BCB-positive group had a significantly lower apoptosis index (2.1±0.6 vs. 8.2±0.9 and 6.7±1.1 for the negative and control groups, respectively) (P<0.05). These findings demonstrate that BCB-positive oocytes have a higher maturation ability and developmental competence in vitro, indicating that BCB staining is a reliable method for selecting oocytes to enhance the efficiency of SCNT.

Extracellular vesicles and melatonin benefit embryonic develop by regulating reactive oxygen species and 5‐methylcytosine

“…Apoptosis assays were performed using the DeadEnd Fluorometric TUNEL System (Promega) as previously described. 28 Briefly, embryos (n = [15][16][17][18][19][20] were fixed in 4% paraformaldehyde for 2 hours at RT, permeabilized with 0.5% Triton X-100 for 5 minutes at RT, and incubated in FITCconjugated dUTP and terminal deoxynucleotidyl transferase at 37°C in the dark for 1 hour. The end-labeling reaction was terminated using 2X SSC in the dark at RT for 15 minutes.…”

Melatonin Protects Rabbit Somatic Cell Nuclear Transfer (SCNT) Embryos from Electrofusion Damage

“…Up to now, electrofusion was the most commonly used method for most SCNT studies 8,9,18,19 . Regarding the piezo microinjection method, researchers were concerned about the risk of mercury contamination during the injection using micropipettes 20 .…”

“…SCNT was conducted as previously described 8 . Oocytes containing the first polar body were selected and stained with 7.5 μg/mL Hoechst 33342 in Earle's balanced salt solution-complete medium (Thermo Fisher Scientific; Waltham, MA, USA) for 20 min.…”

“…Another is virus fusion using inactivated hemagglutinating virus of Japan (HVJ), however, the potential risk of HVJ to the embryo development remains unclear; the biological safety of this virus is questionable and the fusion rate is not especially high. Electrofusion is a method that has been widely used with a good success rate 8,9 . One benefit of using electrofusion is that the physical parameters of the technique can be fixed accurately and are easily reproducible.…”

Tauroursodeoxycholic acid (TUDCA) enhanced intracytoplasmic sperm injection (ICSI) embryo developmental competence by ameliorating endoplasmic reticulum (ER) stress and inhibiting apoptosis

“…Apoptosis detection was performed using the DeadEnd Fluorometric TUNEL System (Promega, Madison, WI) as previously described [24]. Briefly, blastocysts were fixed in 4% paraformaldehyde (at RT for 2 h), permeabilized in 0.5% Triton X-100 (at RT for 5 min), and incubated with FITCconjugated dUTP and terminal deoxynucleotidyl transferase in the dark (37 °C for 1 h).…”