EFFECT OF PRETREATMENT WITH ROSCOVITINE ON IN VITRO MATURATION OF EQUINE OOCYTES FROM SMALL OVARIAN FOLLICLES AND SUBSEQUENT NUCLEAR TRANSFER EFFICIENCY

• M.J. SANSINENA, D. HYLAN, A. KLUMPP, B. REGGIO, D. PACCAMONTI, R.S. DENNISTON AND R.A. GODKE

Buenos Aires, Argentina

Congreso; World Equine Veterinary Congress (WEVA); 2003

Asociacion Argentina Veterinaria Equina

 Oocyte maturation, both nuclear and cytoplasmic, is an important aspect in the development of somatic cell nuclear transfer procedures for the horse. Cytoplasmic oocyte maturation involves the synthesis of proteins from nuclear and mitochondrial transcripts used during early embryonic development. Roscovitine, a specific kinase inhibitor, has been shown to inhibit germinal vesicle breakdown without compromising further developmental competence in the cow (Mermillod et al., 2000). The objective of this study was to evaluate the effect of roscovitine prematuration in oocytes from small equine ovarian follicles and to use these in vitro matured oocytes for equine nuclear transfer procedures. Twenty-nine mixed breed, cycling mares were aspirated for 6 consecutive times during the breeding season. Mares were treated with Regumate™ for 14 d prior to transvaginal ultrasound-guided aspiration (TUGA). Seven days prior to oocyte collection, follicle ablation was performed on all mares to initiate a new wave of follicles. Follicles were classified at the time of aspiration as pre-ovulatory (not used in this experiment), subordinate (>20 mm diameter) or small (<20 mm diameter). A total of 217 oocytes were recovered from 346 follicles for an overall recovery rate of 63%. When classified according to follicular size, 71 subordinate follicles yielded 24 oocytes (34% recovery), whereas 193 oocytes were recovered from 275 small follicles resulting in a recovery rate of 70%. Oocytes recovered from subordinate follicles were subjected to in vitro maturation (IVM) in TCM-199, 15% estrous mare serum, 5 mg/ml of FSH, 10 mg/ml of LH and 1 mg/ml of E2 for 36 h.  Oocytes aspirated from small follicles were randomly allocated to IVM only or pre-treated for 48 h in roscovitine (50 mM) prior to IVM. Mature, metaphase-II oocytes were selected for nuclear transfer procedures based on morphology, membrane integrity and cytoplasmic condition.  Although a higher percentage of oocytes collected from subordinate follicles were suitable for enucleation (75%), this was not significantly different from the percentage of oocytes enucleated from the small follicles in either the IVM or the roscovitine + IVM treatment groups (45% and 57%, respectively).  There was no difference in the number of enucleated oocytes that were reconstructed among the three treatments, with an overall reconstruction rate of 71%.  However, significantly more couplets fused with ova from subordinate and small (IVM only) treatments compared with the small (roscovitine + IVM) treatment (71%, 55% and 0%, respectively). This could be due to the fact that oocytes from the subordinate and small (IVM only) groups were held in vitro for 36 h whereas oocytes from the small (roscovitine + IVM) group were held in vitro for 84 h (48 h roscovitine prematuration plus 36 h IVM). This prolonged culture may affect the membrane stability, and therefore explain the lack of fusion in this treatment. In conclusion, roscovitine prematuration did not affect the maturation rate of oocytes from small follicles and could be used as a tool to transport or hold equine oocytes prior to IVM. Further research is needed to determine the effects of prolonged culture periods of oocyte membrane integrity.