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. 

 

SETTING-UP AND QUALITY CONTROLLING A LAB USING ABATTOIR-DERIVED OOCYTES, THE BOVINE EMBRYO ASSAY.

You decide to set-up your own cattle IVF lab, and you have undergone a bovine IVF course (ART Lab Solutions can deliver this to you as well!). In any IVF lab, quality control is critical to success, but how can you measure if your lab is functioning well? Enter the Bovine Embryo Assay (BEA)!

In some ways, it is used similarly to the Mouse Embryo Assay (MEA) conducted by clinical IVF labs and medical device manufacturers for ‘toxicity’ assessment when introducing a new product, chemical, or disposable into the lab. However, routinely performed BEA’s also gives a picture of how the lab and the embryologists are performing. The rationale for regular BEA’s is that, unlike the MEA tests, there is an enormous variability in embryo production from different bull-cow combinations.  This is typical in bovine IVF.

Recording the results of your BEA continuously provides you with critical evidence to assist disappointed clients, enables you to look at seasonal factors, and notifies when the lab does have a string of unusual results, both bad and good!

Try to minimise the variability within the BEA as much as possible.  A major source of variation lies with different bulls.  Try to amass a large amount of semen straws from the one bull, even better from the one bull collected over a short period.  If you need to change bull semen source, validate the frozen semen of several other bulls at once and see which ones give you a similar development rate, but make sure this validation occurs at least three times.

As for sourcing cow oocytes, this requires access to an abattoir which could be difficult depending on distances, access regulations and costs.  An alternative is to obtain maturing COCs under Transport IVM conditions; contact us if you need assistance with this.  With abattoir-sourced ovaries, consistency is key wherever possible, especially around breed and pregnancy status.

Transporting ovaries back to the IVF lab is best within 6 h of slaughter, commonly held at 30-35°C, with an optimal time from slaughter to oocyte aspiration of 4 h.  We recommend using Phosphate Buffered Saline and vacuum flasks for this purpose. The number of oocytes entering into a BEA test to make it meaningful would normally be at least 150 COCs.  However, this may not be feasible, and therefore any number above 50 oocytes is useful, but again, numbers must be recorded.

ART Lab Solutions will provide the standard operating procedure on request, along with the complete suite of bovine in vitro embryo production media.

 Contact admin@artlabsolutions.com for more information.