Molecular biological methods for monitoring oocyte maturation and in vitro fertilization in bitches.

: Oocytes were collected from ovaries of bitches, at various stages of the oestrous cycle, after routine sterilization. Cumulus-enclosed oocytes were cultured for 0-72 h in a modified M-199 medium containing 10% oestrous bitch serum and 20 micrograms oestradiol ml-1. For oocytes surrounded by two or more layers of cumulus cells, an increase in the expression of mRNA transcripts for zona pellucida glycoprotein 3 (ZP3) was seen and reached peak levels after 48 h culture in vitro. Histological assessment showed that 39% of these oocytes had extruded their first polar body after 24 h culture in vitro. When these in vitro matured oocytes were transferred to oviduct cell monolayers and inseminated with fresh dog spermatozoa in Medium-199 supplemented with 10% fetal calf serum and 50 mg gentamicin sulfate ml-1, penetration of the zona pellucida started 1 h after insemination for oocytes that had been cultured for 48 and 72 h. At 12 h after insemination both male and female pronuclei were seen in 37.5% and 20% of the oocytes incubated for 48 and 72 h, respectively. No further development was seen.