260 PRELIMINARY RESULTS OF IN VITRO FERTILIZATION FOR THE ENDANGERED MURCIANO-LEVANTINA BOVINE BREED

The Murciano-levantina cattle from the Spanish Southeast belong to a bovine endangered breed under the special protection of FAO (WWL-DAD:3)*. Their characteristics of rusticity, longevity, docility and disease resistance make the recovery of the breed interesting. The objective of the present work was to determine the efficiency of in vitro bovine embryo production using oocytes collected from postmortem ovaries fertilized with frozen-thawed sperm obtained from either commercial or Murciano-levantina bulls. Cumulus-oocyte complexes were matured for 24 h in TCM199 with 10% FBS, 2 mM L-glutamine, 0.2 mM sodium pyruvate, 0.3 μg mL−1 LH and 5 μg mL−1 FSH. Oocytes presumed matured were fertilized with Percoll-prepared sperm (750 000/mL) in IVF-TALP, as described by Parrish et al., (1988 Biol. Reprod. 38, 1171–1180). Zygotes (at 18 h) or two-cell-stage embryos (at 48 h) were stained with Hoechst 33342 (10 μg mL−1; 20 min) and assessed by means of epifluorescence microscopy. The experiment was replicated on 4 occasions. The results of penetration (PEN-%), average number of spermatozoa inside the oocyte (S/O), monospermy (MON-%), putative embryos (zygotes) with two pronuclei (2 PN-%) and percentages of cleavage at 48 hours were recorded (Table 1). The results show that early embryos can be obtained with no differences in monospermy and cleavage rates regardless whether the sperm source was commercial or Murciano-levantina (P ≥ 0.05). *Lista Mundial de Vigilancia para la Diversidad de los Animales Domesticos (1997). http://www.fao.org/docrep/V8300S/V8300S00.htm. Table 1 In vitro fertilization results with one commercial and one Murciano-levantina bull