220 EFFECT OF ZINC SUPPLEMENTATION DURING IN VITRO MATURATION ON EQUINE BLASTOCYST RATES AFTER INTRACYTOPLASMIC SPERM INJECTION

Abstract

Use of intracytoplasmic sperm injection (ICSI) in horses has growing clinical and research importance; however, little is known of factors affecting efficacy of this system. Supplementation of zinc during in vitro maturation (IVM) has been shown to increase oocyte glutathione levels, decrease reactive oxygen species, and increase blastocyst rates in vitro in cattle and pigs, but has not been evaluated in the horse. In this study, we examined the effect of zinc supplementation during IVM on rates of maturation and blastocyst formation after ICSI. Oocytes were collected from follicles ≥5 mm in diameter in live mares. Blood serum and follicular fluid from 15- to 30-mm follicles were collected from 3 mares for zinc analysis by flame atomic absorption spectrometry. Oocytes were held overnight at room temperature (Choi et al. 2006 Theriogenology 66, 955–963), and then randomly assigned to IVM in the presence of 1 of 4 concentrations of added zinc (0.0, 0.5, 1.0, and 1.5 μg mL^–1), added as ZnSO₄·7H₂O. The oocytes were cultured for IVM in M199 with Earle’s salts, 5 mU mL^–1 FSH, and 10% fetal bovine serum (FBS) for 30 h. The oocytes were then denuded of cumulus and those with a polar body subjected to ICSI with frozen-thawed sperm. Presumptive zygotes were cultured in a commercial human embryo culture medium (LifeGlobal; http://www.lifeglobalgroup.com/), supplemented with 10% FBS, under 6% CO₂, 5% O₂, and 89% N₂ at 38.2°C. On Day 5 (Day 0 = injection day), embryos were evaluated for presumptive cleavage and transferred to medium with 20 mM added glucose. Blastocyst formation was evaluated on Days 7 to 11. Data were analysed by Fisher’s exact test. The mean zinc concentrations in mare serum and follicular fluid were 0.50 and 0.44 μg mL^–1, respectively. The mean zinc concentration of the FBS was 2.70 μg mL^–1, and that of IVM medium containing 10% FBS were 0.28, 0.80, 1.23, and 1.68 μg mL^–1 for the 0.0, 0.5, 1.0, and 1.5 μg mL^–1 added-zinc treatments, respectively. The rates of oocyte maturation were not significantly different among treatments (43/61, 70%; 46/70, 66%; 45/71, 63%; and 49/70, 70%, respectively). Neither cleavage rates (81–92%) nor blastocyst rates (12/42, 29%; 9/43, 21%, 8/44, 18%, and 15/48, 31%, respectively) differed significantly among treatments. However, the proportion of blastocysts that developed on Day 7 out of total blastocysts was higher for the combined added-zinc treatments (0.5, 1, and 1.5 μg mL^–1) than for the treatment with no added zinc (15/32, 47% v. 1/12, 8%, respectively; P < 0.05). These results indicate that supplementation of zinc to the IVM medium used did not influence equine oocyte maturation or blastocyst development rates but may have improved embryo quality, as reflected in earlier blastocyst development. The more subtle response seen, compared with that reported in other species, may be because the 10% FBS resulted in zinc concentrations in the basal medium (0.28 μg mL^–1) only slightly lower than that in equine follicular fluid (0.44 μg mL^–1).

This work was supported by the Link Equine Research Endowment Fund, Texas A&M University, and by the Clinical Equine ICSI Program, Texas A&M University.