the cortical reaction (Cran & Cheng, 1986; Kim et al., 1996). In the pig, the female genital tract limits the number of spermatozoa reaching the site of fertilisa- tion in order to maintain a ratio of 1:1 (Hunter, 1998). Once penetrated, the oocyte protects itself via the cor- tical reaction, which prevents penetration by multiple spermatozoa (Kim et al., 1996). The cortical reaction occurs when the cortical granules fuse with the overly- ing oolemma and release their contents into the peri- vitelline space. This reaction provokes changes to the zona pellucida and prevents polyspermy. There could be differences between the cortical reaction of the oocytes matured in vivo and those matured in vitro (Cran & Cheng, 1986), the latter showing an incorrect distribution of cortical granules that would explain the incomplete release upon sperm entry (Kim et al., 1996). Also, ageing oocytes are more sensitive to polyspermy (Hunter, 1988, 1991; Grupen et al., 1997) because their Introduction Successful methods for in vitro maturation (IVM) and in vitro fertilisation (IVF) of pig oocytes have been developed quite recently in comparison with other species (Cheng et al., 1986; Nagai & Moor, 1990). The major problem in pig embryo production is the high incidence of polyspermy (Nagai et al., 1984; Hunter, 1990). The term polyspermy, meaning the entry of more than one spermatozoon into the cytoplasm of an oocyte, usually causes very early death of the zygote (Hunter, 1991), and may result from a malfunction of 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 Zygote 8 (May), pp 139–144. © 2000 Cambridge University Press Printed in the United Kingdom All correspondence to: Marc-André Sirard, Centre de Recherche en biologie de la Reproduction, Département des Sciences Animales, Pavillon Comtois, Université Laval, Ste- Foy, Québec, Canada, G1K 7P4. Tel: +1 (418) 656 7359. Fax: +1 (418) 656 3766. e-mail: Marc-Andre.Sirard@CRBR.ulaval.ca Influence of oviductal cells and conditioned medium on porcine gametes Mariève Bureau, Janice L. Bailey and Marc-André Sirard Centre de Recherche en Biologie de la Reproduction, Département des Sciences Animales, Université Laval, Ste-Foy, Québec, Canada Date submitted: 6.11.99. Date accepted: 20.12.99 Summary The aim of this study was to optimise porcine in vitro fertilisation (IVF) with cryopreserved semen with the exploitation of the oviduct secretion. The oocytes were cultured in NCSU37 supplemented with db- cAMP (1 mM), porcine follicular fluid (pFF; 10%), cysteine (0.1 mg/ml) and β-mercaptoethanol (25 μM) for 44 h (the first 20 h with 10 IU/ml hCG and PMSG). The oviductal epithelial cells (OEC) were cultured in TCM-199 medium (with 10% FCS, 0.2 mM pyruvate and 50 μg/ml gentamicin) for 48 h. To determine the effects of OEC and conditioned medium, oocytes were separated into five groups for the last 3 h of maturation and placed in: fresh maturation medium (controls), OEC-cNCSU with OEC in the maturation medium for 24 h; OEC-fNUSU with fresh OEC in maturation medium; cTCM with TCM-199 conditioned with OEC for 48 h; or fTCM with fresh TCM-199. Results indicate that OEC-cNCSU and OEC-fNCSU increase the number of oocytes reaching the two pronucleus (2PN) stage (p < 0.01) and decrease the polyspermy rate (p < 0.01) compared with controls. The rates are significantly lower than controls when cTCM and fTCM were used (p < 0.01). As regards blastocyst rates, an increase was observed in the OEC- cNCSU and cTCM groups (p < 0.05). For the second experiment, spermatozoa were incubated with OEC in IVF medium (mTBM medium supplemented with 0.1% BSA) without caffeine for 4 h prior to IVF. Results indicate that sperm treatment with OEC increases the 2PN rate (p < 0.01) compared with controls and reduces the polyspermy rate (p < 0.01). In conclusion, our study shows that co-incubation of OEC with both oocytes and sperm before IVF reduces polyspermy rates and improves embryo development. Keywords: Conditioned medium, Fertilisation, Maturation, Oviductal cells, Porcine