80 USE OF THE SHORT DISPOSABLE NEEDLE SYSTEM FOR FOLLICLE ASPIRATION IN MARES

To achieve success in equine somatic cell nuclear transfer (SCNT), it is important to obtain recipient oocytes of good quality. Transvaginal ultrasoundguided follicle aspiration (TVUFA) is one of the methods to obtain recipient oocytes in equine SCNT, but the commercial long double-lumen needle for TVUFA in large animals is not currently purchasable. The aims of the present study were (1) to compare the recovery rate of short disposable needle system (14G) with that of the long double-lumen needle (12G) and (2) to investigate the developmental competency of recovered oocytes after SCNT and embryo transfer (ET). A real-time ultrasound scanner (Mylab30 vet, Esaote, Italy) equipped with a 7.5-MHz convex array transducer (model EC123) housed in a plastic vaginal device with stainless steel needle guidance was used for TVUFA from pre-ovulatory follicles 25 to 40 mm in diameter on synchronized thoroughbred mares between 6 and 9 years of age. Two types of needles were used: (1) a 12G double-lumen long needle (V-EOAD-1260L; Cook, Brisbane, Australia); (2) a 14G single-lumen disposable needle (2.1 × 80mm; Bovi-vet, Kruuse, Denmark) inserted with 18G inner needle (0.8 × 600 mm) using stainless steel connector and tube. Recovered oocytes were matured in vitro in TCM-199 with 5 mU mL-1 FSH (Folltropin-V, Bioniche, Belleville, ON, Canada) and 10% fetal bovine serum (Sigma, St. Louis, MO, USA) for 12 to 16 h. Matured oocytes were enucleated and electrically fused with equine skin fibroblasts (2.25 kV cm-1, 20µs, 2 pulses). Fused couplets were activated with 5 µM ionomycin for 4 min followed by 5 h culture in 1 mM 6-DMAP. Immediately following SCNT procedures, cloned embryos were surgically transferred to the oviducts of recipient mares (n = 2 to 5 embryos per recipient) that had ovulated within 24 hours before the transfer. An initial pregnancy examination was performed using transrectal ultrasonography between Days 14 and 16 (Day 0 = surgery). The recovery rate of the short disposable needle (n = 89, 44.1%) was slightly increased compared with that of the long needle (n = 34, 29.8%), but the difference was not significant. Nineteen SCNT embryos were transferred to 8 mares, and 1 mare is maintaining pregnancy for 60 days. The results of this study demonstrated that our short disposable needle system could be used instead of the commercial long needle and in vivo development of oocyte recovered with the 14G needle could be maintained after nuclear transfer and embryo transfer.