Submission Number: KAT-4-25-16

Abstract Number: 363

TOWARD DEFINED PHYSIOLOGICAL EMBRYO CULTURE MEDIA: REPLACEMENT OF BSA WITH RECOMBINANT ALBUMIN.

Kathy Hooper, Michelle Lane* and David K Gardner*

Colorado Center for Reproductive Medicine, Englewood, CO, USA ¹

Abstract:
The development of defined embryo culture media is attractive as it will lead to the standardization of culture conditions and facilitate studies on embryo development. To this end synthetic macromolecules such as polyvinylalcohol have been used to replace serum albumin. Although such an approach eliminates the variation inherent when using a blood product, it removes an important physiological component of the medium, albumin. With the introduction of recombinant serum albumin, it is now possible to prepare defined physiological media.

The effect of recombinant human serum albumin (rec-HSA) on the development and differentiation of bovine embryos was determined. In vitro matured and fertilized bovine oocytes were cultured in medium G 1.2 for 72h, followed by culture in medium G 2.2 for a further 72h. Embryos (n = 392) were cultured at 38.5^oC, in 5% O₂, 6% CO₂ and 89% N₂. Media contained either 8 mg/ml BSA or 2.5 mg/ml rec-HSA. On cessation of culture, blastocyst development was assessed and blastocyst cell numbers determined through differential nuclear staining. Differences between treatments were determined by generalized linear modeling using GLIM.

Blastocyst development (% of cleaved embryos) on day 7 post-insemination was equivalent in the presence of either BSA (40%) or rec-HSA (37%). The addition of 0.5 mM citrate to media had no effect on blastocyst cell number when BSA was the protein source (control 143 6; plus citrate 138 5). However, the presence of citrate was associated with a significant increase in cell number in the presence of rec-HSA (control 123 7; plus citrate 146 5, P < 0.01). The increase in cell number in the presence of citrate was observed in both inner cell mass and trophectoderm.

The higher cell numbers obtained when using rec-HSA with citrate supports the findings of previous reports that the mitogenic effects of different batches of BSA can be attributed to citrate contamination. The ability to use recombinant serum albumin with citrate in embryo culture systems should lead to the standardization of physiological embryo culture conditions. .

Keywords: bovine, citrate, culture

This abstract is being presented at: 8:00 AM in session:
Preimplantation Embryo/Fetus