In vivo culture of bovine embryos and quality assessment of in vivo vs. in vitro produced embryos

Routine access to the bovine oviduct for in vivo culture accomplishes various demands on embryo production for scientific as well as commercial purposes. The experiments conducted in the present study focused on the efficiency of recovery methods after temporary in vivo culture of bovine embryos in oviducts of the homologous species using transvaginal endoscopy (Experiment I) and on the quality assessment of recovered blastocysts (Experiment II). In Experiment I in vitro matured oocytes were fertilized, cultured for 1 to 3 days and transferred unilaterally into the ipsilateral oviducts of 54 heifers by the means of transvaginal endoscopy. After 4 to 6 days of in vivo culture embryos were re-collected either by non-surgical flushing of uterine horns (U-group) or by combined flushing of the oviducts and uterine horns (OU-group). In total the recovery rate was 38.4% (780/2029). After flushing at day seven, 106 blastocysts (blastocyst rate: 13.6% ) were found. The additional 24 h of in vitro culture (day eight) resulted in 153 blastocysts (blastocyst rate: 19.6% ). The recovery rate in the OU-group was twice as efficient as in the U-group (390/1358 vs. 390/671, P < 0.01). The recovery rates among the different stages of transferred embryos did not differ significantly; likewise cross-effects among the stages and the recovery methods were nonsignificant. The recovery methods (P < 0.001) and the interaction between the recovery methods and the stages of transferred embryos (P < 0.01) had an influence on blastocyst yields on day seven (U-group 37/1358 vs. OU-group 69/671) and day eight (U-group 48/1358 vs. OU-group 105/671). In Experiment II embryo quality was assessed by the survival rate of blastocysts after freezing in ethylene glycol. Day seven embryos were produced in vitro (in vitro group D7) or by IVM/IVF followed by a combined culture procedure (2 to 3 days in vitro prior to 4 to 5 days in vivo) (in vivo group D7) or after superovulation and collection at day seven (superovulation group). Embryos from in vitro group D7 re-expanded only for 6 h after thawing, embryos from in vivo group D7 and superovulation group were alive for 24 h and 72 h of culture, respectively. Only embryos derived by superovulation showed hatching activity. Blastocysts from the in vitro group D7 and the in vivo group D7 that were held in culture medium for additional 24 h (day eight) showed an analogous post-thawing culture behaviour. In conclusion, the results of the present study demonstrated that some embryos transferred for in vivo culture remain in the oviduct even at day seven. Hence, combined flushing of oviducts and uterine horns after in vivo culture in the bovine oviduct is necessary for effective embryo re-collection. The quality of recovered embryos after temporary in vivo culture assessed by cryotolerance was in-between those produced in vitro or recovered after superovulation.