Intracytoplasmic sperm injection of frozen‐thawed bovine oocytes and subsequent embryo development

Abstract

Oocyte cryopreservation and intracytoplasmic sperm injection (ICSI) are advantageous to expand their usefulness in genetic engineering. Oocytes matured for 22 hr were vitrified in droplets of cryoprotectants (3.2 M ethylene glycol (EG), 2.36 M dimethyl sulfoxide (DMSO), 0.6 M sucrose) on copper electron microscope (EM) grids. After being warmed, the oocytes were cultured in IVM medium for an additional 2 hr. Sperm treated with dithiothreitol were utilized for ICSI. Oocytes injected with sperm were activated by combination of ionomycin with cycloheximide (CHX). The ICSI oocytes were compared for the rates of pronuclear formation, development, cell number, and the ratio of ICM to those of fresh ICSI and IVF control. The proportion of 2PN formation was significantly higher in IVF control (Group 1) than those in other treated groups. Among the treated groups a significant lower 2PN formation was observed in IVF-frozen-thawed than in ICSI-fresh and frozen-thawed groups. Cleavage rates in IVF-frozen-thawed and ICSI-frozen-thawed groups were significantly lower than those of IVF control and ICSI-fresh groups. In ICSI groups, the rates of cleavage and blastocyst in fresh oocytes were significantly higher than in frozen-thawed. Development rates into blastocysts in the ICSI-fresh and frozen-thawed groups were significantly lower than that of IVF control. Total cell number was significantly lower in both frozen-thawed IVF and ICSI groups than those in IVF-control and ICSI-fresh groups. However, the rates of the remaining cells that were found in the ICM were significantly higher in both frozen-thawed IVF and ICSI than in the IVF-control and ICSI-fresh groups. The results indicated that frozen-thawed bovine oocytes were suitable for ICSI procedure.

Improved preimplantation development of bovine ICSI embryos generated with spermatozoa pretreated with membrane-destabilizing agents lysolecithin and Triton X-100

“…In fact, a higher blastocyst formation rate was achieved with LL treatment compared with the control without treatment being one of the highest blastocyst rates published so far in the bovine species [5,14,[44][45][46]. These results are also consistent with a previous study in rats showing a higher blastocyst formation rate in sperminjected oocytes generated with spermatozoa treated with LL and TX [27] but disagree with a previous report in mice in which no differences in embryonic development was observed with these treatments [22].…”

Sperm capacitation pretreatment positively impacts bovine intracytoplasmic sperm injection

“…For this reason, we analyzed embryo quality to address whether or not our sperm pretreatments impact later development. Evaluation of embryo quality—in terms of cell count, cell allocation to the inner cell mass versus trophectoderm, and the apoptotic cell index—showed no differences between the treatments and the control group, and our data were comparable to those described previously for bovine ICSI (Arias et al, ; Jo et al, ; Rho et al, ; Zambrano et al, ). Thus, pretreatment with MβCD supports sperm decondensation after injection, and neither MβCD or IBMX pretreatments affect the quality of bovine ICSI‐derived embryos.…”

Intracytoplasmic Sperm Injection after Vitrification of Immature Oocytes in Follicular Fluid Increases Bovine Embryo Production

“…Our data also show increased results of Vitri-ICSI. Conversely, previous findings in the bovine [17] did not find any increment in embryo development after MII oocyte vitrification and ICSI, though under their conditions the cleavage rates increased. This study shows that: the composition of the base solution for oocyte vitrification can improve the embryo production outcome, and ICSI improves the embryo production for previously vitrified oocytes.…”