Effect of Early Medium pH on Equine Embryo Development in vitro After Intracytoplasmic Sperm Injection

Intracytoplasmic sperm injection (ICSI) and in vitro embryo culture are used to produce equine embryos for both clinical and research use, but little information is available on factors affecting embryo development in this system. In vivo, stallion sperm are exposed to a high pH (7.7 to 8.0) in the uterine fluid,1 and in most species oviductal pH equals or exceeds that of the uterus. Accordingly, marked sperm protein tyrosine phosphorylation occurs only when the environmental pH approaches or exceeds 8, suggesting that this may be the physiological pH of the equine oviduct.1 If so, the new zygote is likely also exposed to a high pH in the period immediately following fertilization. We hypothesized that exposure to high pH (~ 8) during sperm preparation and the first 2 h incubation of zygotes after ICSI may be beneficial to fertilization and cleavage rates. For this study, to maximize the ability to demonstrate an effect of treatment on early embryo development, we utilized sperm from a stallion shown to have suboptimal cleavage rates after ICSI under standard ICSI conditions (63%, vs. 95% for control stallion).2 Oocytes were obtained by ultrasound-guided transvaginal follicle aspiration from live mares. In vitromatured oocytes were injected with sperm via Piezo drill, then presumptive zygotes were cultured in human embryo culture medium (LifeGlobal; GB) containing 10% FBS; 20 mM glucose was added at Day 5. Embryos were examined for cleavage on Day 5 and for blastocyst development on Days 7 to 11. A high-pH and a standard-pH treatment were used. To achieve this, sperm preparation (swim-up) and the first 2 h of embryo culture were performed in bicarbonate-containing media (modified CZB and GB, respectively) incubated either in air (high-pH treatment) or in 5% CO2 in air (standard-pH treatment). Media were equilibrated in their respective incubator for a minimum of 2 h before use. The pH values of incubated media were 8.0-8.4 (air) vs. 7.4-7.5 (5% CO2 in air). Following the 2-h culture period, all embryos were placed in standard conditions (6% CO2, 5% O2 and 89% N2) for the remainder of culture. There was no significant difference in cleavage rates between the high-pH and standard-pH treatments (22/30, 73% and 19/25, 76%, respectively). The blastocyst rate per injected oocyte in the high-pH treatment was significantly lower than that in the standard-pH treatment (0 vs. 24%, respectively; P < 0.01). High pH (8 to 8.4) during sperm swim-up and the first 2 h of embryo culture did not improve cleavage rates, and was detrimental to further embryo development.