Embryo culture drop size – is there an optimum?

We recently had an excellent question from one of our clients, who wanted to know if our recommendation of 5 micro-litres (uL) of medium per embryo cultured was critical or not, or could it be changed to suit their IVP embryo production system.  Firstly, our protocol recommendations are based on our long-history of research and commercial application with the media formulations we have, and this is why we provide our preferred protocol.

The optimal medium volume per embryo cultured really depends on several factors: breed of cattle; culture vessel; oxygen concentration (we ALWAYS recommend 5-7% oxygen for all stages of embryo culture); numbers of embryos being cultured in one container or drop; medium buffering system; use of oil and rate of evaporation in the absence of oil; use of additives such as growth factors or serum.

Given all of that, we err on the cautionary side and recommend a relatively large volume per embryo cultured, given that we recommend our sequential medium system.   Experimentally, we culture 4-5 embryos per 20uL microdrop under paraffin oil (MEA/BEA tested).  If you want to explore using larger embryo groups, you may find reducing the volume size from 5uL/embryo to even as low as 2 uL/embryo could be beneficial.  We would not recommend even lower media: embryo ratios.  One reason we use 4-5 embryos/20uL is that at transition to VitroBlast medium, we group the best embryos with each other – this provides more            opportunity for the best embryos to benefit from the autocrine/paracrine growth factors    produced by the embryos themselves.  These will also be the most metabolically active, and therefore will be more ‘draining’ of the medium’s substrates.  This will also be a factor if you have residual cumulus cells that remain on the embryo and even plate down. These additional cells will definitely ‘drain’ the medium of substrates.  It wouldn’t be an issue at 5uL per embryo, but may at low volume/embryo grouping.

As always, we cannot guarantee results and take no liability for your embryo production and transfer outcomes.

If you have a question for the ART Lab Solutions team contact us directly at admin@artlabsolutions.com. 

 

July’s Tip of the Month – Oil on Oil

Each month we will give a tip on how to improve your results, sourced either from the literature or our own research.

The Oil on Oil

Many IVF labs use micro drops under oil for embryo production, to protect the media from osmotic change and reduce the degree of pH change when transitioning to-and-from the incubator environment.  However, many embryologists would like to eliminate oil from their IVF systems; its messy and is a source of impurities that may negatively impact embryo development and quality.  So why is oil the preferred option to protect the media from osmotic and pH changes, especially when using with microliter micro drops of media?
Mineral oil is unique in its properties, because its hydrophobic (‘excludes water’) nature but allows gas to exchange between atmosphere and media
Oil is virtually impermeable to water vapour and even under 38-39 degrees temperature, there is undetectable osmotic change over the culture peiod.  Most plasticware used for IVF is either polystyrene or polycarbonate, and these are more porous to water vapour than mineral oil.  This goes unnoticed, as the amount lost is related to the surface area, of which the largest is the media-oil interface.  Surround media with nothing but plastic at a high surface-area to volume ratio will cause osmotic change over several days.
Silicon oil is also less hydrophobic and enables water vapour to be lost.
This hydrophobic property of mineral oil is combined with an impressive property for gas exchange. Although not as efficient as direct aqueous equilibration, gas exchange through mineral oil occurs readily, enabling sufficient gas exchange to maintain pH equilibria when using bicarbonate/CO2 buffering.
Contamination of oil is an issue. In particular, oil is susceptible to contamination by volatile organic compounds, hydroperoxides and other oil-breakdown contaminates, especially if not stored well over time.  Oil is best stored in glass, especially if aliquotting from a freshly purchased batch. Testing to identify individual contaminants is difficult, but these can be the cause of either poor or variable results if using unscreened oils. We recommend IVF laboratories source oil either from human IVF media companies or purchase specific mouse embryo assay tested oil.
ART Lab Solutions can facilitate your MEA testing requirements for oils and other laboratory consumables and products if required.
For more information on this month’s tip, contact admin@artlabsolutions.com