143 Embryo transfer of embryos generated by intracytoplasmic sperm injection

Intracytoplasmic sperm injection (ICSI) has become a valuable tool to address not only male factor infertility, but also gene editing, a more conservative use of sperm, and the fertilization of vitrified oocytes. However, ICSI in cattle has previously had limited success and typically features additional sperm treatments and post-injection artificial activation. These additions further complicate and limit application of ICSI in research or commercially. We evaluated if embryos produced by modified injection methods could be used to generate viable embryos from fresh and vitrified bovine oocytes without sperm treatment or post-injection activation. For these experiments, abattoir derived in vitro-matured oocytes were denuded and subjected to ICSI or vitrified for later injection. For vitrification, oocytes were selected and incubated in a series of media including of M199 with Earle’s salts, 10 mM HEPES, 20% FBS, and stepwise increase of DMSO and ethylene glycol before vitrification on a cryolock and storage in liquid nitrogen. For injection, vitrified oocytes were warmed in a series of DPBS sucrose solutions and incubated in a maturation medium for 1 h. Frozen semen was thawed, separated via density gradient, and transferred into a 7% polyvinylpyrrolidone solution for injection. Fresh and vitrified oocytes underwent either a standard ICSI procedure or an injection with multiple modifications including, but not limited to, breaking of the sperm tail at various loci, increased force while breaking the oolemma, greater amount and force of cytoplasm aspiration, and more vigorous injection of the sperm into the cytoplasm. Presumptive zygotes were placed into IVC directly after injection, assessed for cleavage at 24 h, and blastocyst formation after 7 days. Results for cleavage and blastocyst formation were gathered and analysed via one-way analysis of variance with Tukey’s post hoc testing. Modified ICSI of fresh oocytes resulted in 82.6% (223/270) cleavage and 31.8% (86/270) blastocyst development. Similarly, modified ICSI of vitrified oocytes resulted in 78.9% (291/369) cleavage stage embryos and 33.9% (125/369) blastocyst development. No differences were seen between fresh and vitrified ICSI groups. Conventional ICSI of fresh and vitrified oocytes resulted in 20.9% (33/158) and 22.2% (30/135) cleavage and 3.2% (5/158) and 3% (4/135) blastocyst development, respectively. To demonstrate the viability of these embryos, embryo transfer (ET) was performed using unhatched Day 8 modified-ICSI generated embryos from fresh (7) and vitrified (3) oocytes. A total of 10 cows underwent a 7-day co-sync with CIDR synchronization protocol followed by ET 8 days later. Pregnancy checks were conducted at least 60 days post ET. A total of three cows maintained viable pregnancies, two derived from vitrified oocytes and one from fresh oocytes. Our results demonstrate viable embryos can be generated with ICSI from fresh and vitrified bovine oocytes without pre-injection sperm treatments or artificial activation protocols.

Support was provided by ACRES/LSU Collaborative Research Program, LSU School of Veterinary Medicine.