87 BACK TO THE DRAWING BOARD: IN VITRO CULTURE OF BOVINE EMBRYOS FOR FREEZING

Abstract

Despite hundreds of scientific papers published, no system has resulted in in vitro embryos comparable to those produced in vivo. We hypothesised that assembling the most pertinent elements of IVF studies into one system would result in a highly efficient in vitro culture system. Here we report the in vitro production of bovine embryos using a culture system with strict environmental conditions that produces very-good-quality embryos at high rates. This system consists of a sequential culture system with media composition based on recent reports that characterise the bovine female reproductive tract (Hugentobler et al. 2007 Mol. Reprod. Dev. 74, 445–454; Hugentobler et al. 2007 Theriogenology 68, 538–548; Hugentobler et al. 2008 Mol. Reprod. Dev. 75, 496–503). This system uses a 3-step culture media to prevent toxicity resulting from ammonium accumulation and nutrient depletion and also to adjust the component concentrations to support embryo needs at different developmental stages. Fatty acid-free BSA is used as the protein source and the culture is in droplets under high-quality paraffin oil at 38.5°C under 6.8% CO₂, 5% O₂ and 88.2% N₂. Numerous other aspects were investigated to limit embryo stresses (Lane et al. 2008 Reprod. Fertil. Dev. 20, 23–32) during manipulations, including the use of mini-incubators and very-high-purity gas combined with stringent laboratory practices. In the first year using this new embryo production system, 2839 oocytes were fertilized, resulting in a transferable blastocyst rate of 51%. Of the 1448 embryos produced, 779 were transferred fresh at our facility with pregnancy rates of 55 and 49% at 28 and 60 days, respectively. Pregnancy rates were directly related to the quality of the embryos transferred as 61% of grade 1 embryos transferred induced a pregnancy at Day 28, compared with 41% of grade 2 embryos. Pregnancy induction is not the only indication of good embryo quality. As is well-documented, in vitro-produced bovine embryos do not tolerate slow freezing, so vitrification was applied to surmount this intolerance. However, this is difficult to apply to industry because direct transfer of vitrified embryos is challenging. We hypothesised that the improvement of embryo culture would result in embryos that could tolerate slow freezing. Grade 1 blastocysts (n = 229) were frozen in 1.6M ethylene glycol and 0.1 M sucrose using standard slow freezing procedures. A very high proportion (91%) of frozen–thawed in vitro-produced embryos re-expanded after 24 h of culture with a good quality inner cell mass. Subsequently, 45 grade 1 blastocysts were frozen and transferred, giving pregnancy rates of 58% at Day 60. In conclusion, combining good-quality culture media and conditions resulted in the production of in vitro embryos that were very efficient at inducing pregnancies and tolerating slow freezing, which makes it now possible to consider direct transfer of frozen in vitro-produced bovine embryos.