In vitro penetration of swine oocytes by homologous spermatozoa: Distinct systems for gameteʹs co-incubation and oocyteʹs cryopreservation

In vitro penetration (IVP) of swine oocytes by homologous spermatozoa was evaluated in two experiments using four boars as semen donors. In experiment 1, the IVP rate and the number of penetrating spermatozoa (PSP) were compared using three co-incubation systems for vitrified oocytes and fresh sperm: (1) 35 mL petri dishes in a CO2 incubator, (2) 35 mL petri dishes in bags (submarine system) and (3) glass flasks partially submerged in water bath with the same gas mixture used for the bag system. Mean PSP was 8.2 ± 10.1 and the IVP rate was 90.5%. The PSP differed across all systems (P = 0.0006): 15.5 ± 0.5 for flasks, 6.3 ± 0.4 for CO2, and 3.9 ± 0.4 for bags. The IVP rate for flasks (95.0%) was greater (P = 0.01) than for CO2 and bags (90.8% and 85.0%, respectively), but it did not differ between flasks and CO2 for three boars (P > 0.05). In experiment 2, co-incubation was done as described for glass flasks in experiment 1. The IVP rate and PSP were compared for cryopreserved oocytes: either vitrified in open pulled straws (OPS), or frozen in cryotubes. Mean PSP was 5.4 ± 6.5 and IVP rate was 89.6%. Both PSP and IVP rate were greater (P < 0.0001) for oocytes frozen in cryotubes (7.0 ± 0.3% and 95.8%, respectively) than those frozen in OPS (3.7 ± 0.3% and 83.4%, respectively), with no differences found for three boars (P > 0.05). In summary, successful IVP of swine oocytes by homologous spermatozoa can be achieved using gametes incubated in glass flasks and oocytes frozen in cryotubes.