163 IMPROVEMENT OF IN VITRO PRODUCTION OF PIG EMBRYOS BY SYNCHRONISATION OF OOCYTE MEIOTIC MATURATION WITH CYCLOHEXIMIDE

Abstract

It is suggested that the relatively high rates of polyspermic fertilization and poor development of pig embryos produced in vitro are caused by asynchronous oocyte maturation. We have recently shown that pre-treatment of pig oocytes with cycloheximide (CHX) is an efficient way of synchronizing their meiotic maturation in vitro. However, it is not known whether this procedure affects fertilization or further development. The present study examined the effects of CHX-synchronised meiotic maturation on subsequent embryo development and the response to FSH. Pig ovaries were collected from a local abattoir. Cumulus-oocyte complexes (COCs) were aspirated from 3–5 mm diameter follicles with a translucent appearance and extensive vascularization. COCs were first pre-incubated in defined maturation medium (DM; M199 with Earle’s salts, 25 mM HEPES and sodium bicarbonate, 3 mM L-glutamine, 0.1% (w/v) BSA, 0.57 mM cysteine, 10 ng mL⁻¹ EGF, 0.2 μg mL⁻¹ pLH, 100 μ mL⁻¹ penicillin and 0.1 mg mL⁻¹ streptomycin) or in DM supplemented with 50 ng mL⁻¹ pFSH (DMF) and 5 μg mL⁻¹ CHX for 12 h. COCs were then further cultured in the same DM without CHX for 24–30 h or in DMF for 36 h. For controls, COCs were cultured conventionally in DM for 42 h or DMF for 48 h. After removal of cumulus cells, all cultured oocytes were inseminated with ejaculated sperm at a final concentration of 300 000 mL⁻¹ for 6 h. The IVF medium was modified Tris-buffered medium containing 0.1% BSA, 20 μM adenosine and 0.2 mM reduced glutathione. Putative embryos were cultured in NCSU23 without glucose but supplemented with 4.5 mM Na lactate and 0.33 mM Na pyruvate for 2 days. Cleaved embryos were further cultured in normal NCSU23 for 4 days. IVM and IVF were performed in 5% CO₂ in air and IVC in 5% CO₂, 5% O₂, 90% N₂, all at 39°C and 95% RH. Three replicates with DM, with or without CHX, and one with DMF, with or without CHX, were performed with 30–50 oocytes in each replicate. Statistical comparisons were by t-test. The result with DM showed that the rate for normal cleavage at 2 days after insemination of CHX-treated oocytes (40.6 ± 3.8%) was similar to that of controls (40.4 ± 3.5%). However, the proportion developing to healthy blastocysts at Day 6 was significantly higher in the CHX-treated group (16.9 ± 1.2%) than in controls (9.6 ± 1.3%; P < 0.05). A significantly higher number of Day 2-cleaved embryos from CHX-treated oocytes developed to the day 6 blastocyst stage compared with controls (44.7 ± 5.0% and 22.3 ± 2.4%, respectively; P < 0.05). Supplementation of the basic maturation medium with pFSH increased the rate of cleavage in both CHX-treated oocytes (73.2%) and controls (76.9%) and increased the proportions developing to healthy blastocysts at Day 6 (CHX-treated: 39.0%; control: 11.5%). We conclude that oocytes pre-treated with CHX retain their developmental competence and that meiotic synchronization with CHX improves the efficiency of in vitro production of pig embryos. (Supported by BBSRC 42/S18810.)