In vitro production of ICSI derived horse blastocysts

Abstract

In vitro production of horse embryos via intracytoplasmic sperm injection (ICSI) is a useful clinical and research technique. However, current rates of blastocyst production are still sub-optimal. Here, we compared results from our commercial ICSI program to evaluate whether slight modifications in protocols can result in improved blastocyst rates. Firstly, we evaluated the time cumulus oocyte complexes (COCs) can be maintained in holding medium before starting in vitro maturation (IVM) (Group A: 4-10 hours and Group B: overnight). Secondly, we evaluated the effects of using a microfluidic device (Zymöt) for the preparation of sperm samples for ICSI (Group C). This device comprises a microporous membrane that allows to select spermatozoa in base of their motility, avoiding samples centrifugation. This could eventually reduce DNA fragmentation and oxidative stress, which have been associated with conventional sperm preparation protocols. Frozen-thawed cut straws with semen from different stallions were used and prepared either with a gradient and swim-up protocol or with the Zymöt microfluidic device. COCs were recovered from immature follicles using transvaginal ultrasound-guided follicle aspiration (TVA) procedures. After collection, COCs were transferred to holding medium for 4 to 10 hours or maintained in holding medium overnight at room temperature (22–24°C) before starting IVM. In vitro maturation was performed in T-199 Earle's medium supplemented with 5 mU/ml FSH, 50 ng/ml EGF and 10% FBS. After IVM, oocytes were denuded from cumulus and those showing a polar body were subjected to piezo-ICSI (PiezoXpert, Eppendorf). Spermatozoa were first immobilized with strong piezo pulses and then injected individually in the matured oocytes after penetrating the zona pellucida and oolemma with low intensity pulses.The injected oocytes were then cultured in a mix of KSOM-DMEM/F12 medium supplemented with 6% FBS, which was refreshed on Day 5 of development. Embryo culture was performed in humidified benchtop incubators at 6.5% CO2 and 5%O2 at 38.2°C. A Chi-square test was used to compare results.