Proceedings of the 5th International Symposium on EQUINE ...

Havemeyer Foundation Monograph Series No. 3NUCLEAR TRANSFER EMBRYOS IN THE HORSEB. C. Reggio, M. Sansinena, R. A. Cochran, A. Guitreau, J. A. Carter,R. S. Denniston and R. A. GodkeDepartment <strong>of</strong> Animal Science, Louisiana State University, Baton Rouge, Louisiana 70803, USAINTRODUCTIONAfter <strong>the</strong> recent births <strong>of</strong> healthy foals producedfrom intracytoplasmic sperm injection by thislaboratory (Cochran et al. 1998), an attempt wasmade to produce cloned horses embryos usingsimilarly harvested oocytes from live mares(Meintjes et al. 1995) (Part I). Oocyte availabilityis one <strong>of</strong> <strong>the</strong> major limitations in conductingnuclear transfer studies in many species. In <strong>the</strong>horse, oocyte collection is cumbersome and <strong>of</strong>tenseasonal, thus potenially limiting to <strong>the</strong> success <strong>of</strong>a nuclear transfer program. In a study conductedby Dominko et al. (1999) bovine oocytes wereused as recipients <strong>of</strong> nuclear transfer <strong>of</strong> variousmammalian species (rat, cow, sheep, pig, monkeyand rat). Regardless <strong>of</strong> <strong>the</strong> species <strong>of</strong> donorfibroblasts used in this study, all coupletsprogressed through <strong>the</strong> first cell cycle, and somereconstructed embryos developed to <strong>the</strong>blastocysts stage. Since <strong>the</strong> timing <strong>of</strong> <strong>the</strong> first twocleavage divisions in bovine in vitro-producedembryos corresponded more closely to <strong>the</strong> timing<strong>of</strong> cleavage in o<strong>the</strong>r <strong>the</strong> donor nucleus species,bovine oocytes were used in <strong>the</strong> second part <strong>of</strong> thisstudy (Part II).Part I. A group <strong>of</strong> 6 mixed breed mares weretreated daily with 0.044 mg/kg altrenogest(Regumate) <strong>the</strong>n subjected to a follicle reductionvia transvaginal ultrasound-guided aspiration onDay 21. Oocytes were <strong>the</strong>n collected every 10 days beginning on Day 31 and matured in vitro inTCM-199 supplemented with oestrous mare serumfor 36 h. Mature oocytes were enucleated byremoving <strong>the</strong> first polar body and secondmetaphase plate using Hoechst 33342 stain (5µg/ml) and epifluorescent microscopy to confirm<strong>the</strong> complete removal <strong>of</strong> maternal DNA. A singledonor cumulus cell recovered from <strong>the</strong> pool <strong>of</strong>mature oocytes was <strong>the</strong>n inserted into <strong>the</strong>perivitelline space <strong>of</strong> each cytoplast. Enucleationand reconstruction were conducted in 2 µg/ml <strong>of</strong>cytochalasin B (CB) using a Nikon Diaphotmicroscope equipped with H<strong>of</strong>fman objectives andNarishige micromanipulators. Donor cells andrecipient cytoplasts were fused with 2 electricalpulses (3.5 kV/cm, 30 µs) in a 0.3 M mannitolfusion buffer containing 0.1 mM MgSO4, 0.5 mMHEPES and 1 mg/ml <strong>of</strong> BSA (pH 7.0; 25°C).Immediately following <strong>the</strong> electr<strong>of</strong>usionprocedure, couplets were incubated for 1 h in 7.5mg/ml <strong>of</strong> CB, washed and held in P-1 culturemedium (Irvine Scientific) for 9 h at 38°C.Couplets (n = 36) were <strong>the</strong>n randomly assigned toone <strong>of</strong> two treatments: activation (A) or noactivation (B). Activation was performed at 45 hpost-maturation with 5 µM ionomycin followed bya 4 h treatment in 2 mM 6-dimethylaminopurine.Nonactivated oocytes were maintained in P-1medium supplemented with 10% FBS.From 142 follicles aspirated (n = 5 aspirations)(Table 1), 74 cumulus enclosed oocytes wereTABLE 1: Aspiration, recovery and maturation <strong>of</strong> equine oocytes <strong>of</strong> live maresAspirated Recovered Matured* Reconstructed*142 74 (52%) 48 (65%) 36 (75%)*Percentage <strong>of</strong> oocytes matured and reconstructed based on <strong>the</strong> number <strong>of</strong> oocytes recovered.45