The Importance of Testing Semen Prior to Use During In Vitro Fertilisation (IVF)

The most accurate way to measure the fertilisation ability of semen in vitro is to test it using abattoir-derived cumulus-oocyte-complexes (COCs). While often sperm has been assessed for motility and morphology when collected and cryopreserved, sometimes internal issues (eg. DNA fragmentation) that cannot be visualised by microscopy can severely impact IVF results.

These results can then be used by producers to finalise in vitro mating’s of commercially collected COC/oocytes from their valuable donor cows and heifers. It will also assist in the preparation of enough semen straws per bull at the time of IVF.

Taking the time to test semen using abattoir-derived COCs prior to commercial use in vitro could ultimately result in economic benefits for the service provider. Embryo production rates, and more importantly, pregnancy and calving outcomes from these oocytes will also be maximised.

Ovum Pick Up (OPU) and In Vitro Maturation (IVM)

When handling oocytes, temperature and pH fluctuations should be minimised. Therefore it is recommended to use incubators,  heated stages and plates to warm all medium and consumables. We recommend these items should be calibrated to 38.5°C.

Using an appropriately buffered medium will limit pH fluctuations which is why we have designed our entire media suite to have exceptional pH stability due to the combination of buffers that we use. We also have transport maturation media options that can be used without the requirement of gas during in vitro maturation (IVM). Transporting gas cylinders across the country can be logistically challenging so ask us about our alternatives to suit your requirements!

It is also recommended to use consumables that are embryo safe and non-toxic. Bovine embryos are more sensitive than mouse embryos therefore performing Bovine Embryo Assays are recommended instead of Mouse Embryo Assays for testing toxicity of plasticware and mediums used for in vitro production of bovine embryos.

When searching and selecting for cumulus oocyte complexes (COCs) they should be selected based on evenly coloured cytoplasms and a complete layer of cumulus cells as a minimum (although sometimes there is no choice based on the quality of COCs/oocytes collected). An important tip is to wash COCs thoroughly in order to remove blood and excess granulosa cells following OPU and before transporting them back to the laboratory in IVM media.  Each step of COC/oocyte collection and IVM is critical to setting up successful fertilisation and embryo production, contact us at admin@artlabsolutions.com to discuss how we can maximise your cattle breeding opportunities.

The Importance of Embryo Assay Testing Your Laboratory Consumables

Ensure your consumables are “embryo-safe”by using Mouse Embryo Assay (MEA) and/or Bovine Embryo Assay (BEA) approved consumables.

Contaminants can be introduced into your laboratory through new batches of oils, media and general plasticware which is why MEAs and BEAs can provide quality assurance. You will notice that many IVF laboratory products available for purchase are already validated by a MEA. However, for unvalidated products you may like to arrange a MEA and/or BEA to confirm batch quality.

Contamination of oils can cause considerable issues for your embryo results. Many IVF laboratories use micro drops under oil for embryo production, to protect the media from osmotic and pH changes when transitioning to-and-from the incubator environment. Oil is susceptible to contamination by volatile organic compounds, hydroperoxides and other oil-breakdown contaminates if not stored optimally over time.  If you aliquot your freshly purchased batch of oil, we recommend using glass vessels. Individual contaminants can be difficult to identify, but these can cause poor or variable results when using unscreened oils. We recommend IVF laboratories source their oil either from human IVF media companies or purchase specific MEA tested oil.   

New batches of plasticware are another potential source of contaminants that may cause variation to your IVF results. The majority of plasticware used in the IVF laboratory will be polystyrene or polycarbonate which are more porous to water vapour than mineral oil. It is therefore favourable to use an overlay of oil with these types of plastics to prevent water evaporation from your media during embryo culture, and maintain osmolarity and pH of the media. MEA and/or BEA tested plasticware can give you confidence that the consumables you are using are “embryo-safe” which is a must before beginning expensive and time-consuming embryology work. 

At ART Lab Solutions our bovine media is both MEA and BEA tested to ensure it is toxicity free and performs consistently between batches. ART Lab Solutions can facilitate your MEA or BEA testing requirements for oils and other laboratory consumables and products if required. For more information contact us at admin@artlabsolutions.com.

Looking for IVF laboratory management tips? Keep reading…

Regular maintenance of your laboratory and it’s equipment is important. Maintenance includes calibrating equipment regularly (daily if possible otherwise a couple times a week), keeping track of batches of media, culture oils and laboratory consumables being used and being aware of potential sources of volatile organic compounds (VOCs). While some of this tracking can seem mundane and unnecessary when things are going well, you will be glad you did when or if you need to troubleshoot when things aren’t going so great.

INCUBATORS

All CO2 incubators should be regularly calibrated for both CO2 and temperature. Benchtop incubators should also be regularly serviced to ensure gas lines and valves are working optimally, and heating is calibrated to the optimal temperature. Temperature monitoring and calibration should also regularly be performed on all microscope heated stages, heating blocks and portable incubators.

REFRIGERATORS

The temperatures of refrigerators should also be observed regularly. Sometimes the temperature at the back of fridges can be as low as 0°C and cause freezing. Freezing media to store is very detrimental to the stability of particular components that make up the media and should therefore be avoided.

OIL & MEDIA

The batch of oil and the glassware/plasticware in which oil and media are stored or aliquoted into should be recorded. For our media, we do not recommend aliquoting into smaller amounts for storage, as every new container can have a residual contaminant. Instead just remove the volume you need within 24 hours of use for equilibration.

Further to this, all new batches of oil, media and laboratory consumables should be tested via a mouse embryo assay as a means to rule out any toxicity which may be present in the new batch. A toxic batch of consumables, media or oil can have detrimental effects on your embryos therefore ruling this out at the beginning before any research/commercial work is performed is a must.

EXPIRY DATES

Be aware of expiry dates. Once a bottle has been opened, our media expires after 2 weeks. The opening and/or expiry dates should be clearly and accurately labelled as each bottle is opened.

VOCS

Introduction of VOCs can occur through many ways. Changing cleaning reagents or gas cylinders are common sources of VOCs. We recommend using trusted cleaning reagents such as 7X detergent; always following dilution instructions. Gas cylinders should be medical grade and also carbon filtered.

By being aware and taking the measures mentioned above you will be able to carefully analyse any critical-to-less critical changes that have been made to the laboratory during a period. Of course, for any further trouble-shooting queries contact admin@artlabsolutions.com.

Ovum Pick Up (OPU) vs Multiple Ovulation Embryo Transfer (MOET)

There are many advantages and disadvantages to choosing the right method of embryo production for your cattle as both technologies have their place.

A few highlights of performing OPU on your donors can save both time and money. OPU does not utilise any hormone injections, thus minimising cost, and can be replicated as regularly as every 4 days. Whereas MOET requires bringing donors into the yards twice per day, for 3 or 4 days in a row which can be quite laborious, especially in a centre managing several programs over consecutive days.

Furthermore, OPU-IVF allows for excellent semen economy. The same straw of semen can often be used over hundreds of oocytes from multiple donors. Another advantage is that if there are large numbers of oocytes collected, they can also be split and fertilised by more than one bull in vitro.

If you would like to have a chat about OPU-IVF procedures, please contact our team directly at admin@artlabsolutions.com for more information.

Preparing Donors & Recipients for Ovum Pick Up (OPU) & Embryo Transfer

Good cattle embryos will only get you so far in your quest for pregnancy success which is why preparing the donors and recipients are important in maximising your breeding opportunities.

Preparation of your cattle should begin 6-8 weeks before collecting any oocytes or embryos, and for the transfer of embryos. Preparations include any relevant vaccinations, internal (worms and fluke etc.) and external (ticks, flies, lice etc.) parasite treatments, and injectable trace minerals. The necessary vaccinations will vary depending on your location.

In Australia cows and heifers should be vaccinated against clostridial diseases as well as Leptospirosis, Vibriosis, and Pestivirus to prevent early and late-term pregnancy losses. Parasite treatment while very necessary, can be damaging to oocytes and embryos. Treating cows and heifers 6-8 weeks prior to OPU or embryo flushing will enable time for several follicular waves to have occurred, reduce chemicals which may be present within the system, and thus lead to more viable oocytes and embryos.

A 6-8 week preparation time is ideal for providing an injectable dose of minerals: especially Zinc, Manganese, Copper and Selenium. These minerals are all important for cellular functions throughout the body and early embryo development. In addition Zinc, Manganese, Copper and Selenium are used in the production of antioxidants that protect cells from oxidative damage. Injectable mineral treatments enable faster, more efficient absorption than oral options and are ideal during periods of high energy demands such as pre-joining and calving. Lick blocks are still a good year-round option for rumen health.

Ideally cattle should be gaining in body condition rather than losing body condition. Heifers should meet critical mating weights (~55-65% of mature weight, usually 12-14 months of age) prior to undergoing estrous synchronisation. Once the correct preparation of donors and recipients are in place, it is now ideal to begin oocyte/embryo collection procedures.

Contact admin@artlabsolutions.com for more information.

 

Communication is key to fixing problems

At ART Lab Solutions we work with you to troubleshoot any issues which may arise during the implementation of a new culture media system. Different culture media requires different methods and tweaking to get it right. We work with our clients every step of the way to achieve optimal handling techniques and set laboratory conditions to the correct standard. Your success is our success.

Get in touch with our team today at admin@artlabsolutions.com to start your journey with our complete bovine media IVP suite

Media are designed to work with a specific protocol.

Have you tried a new commercial media system that appears to work in other labs, but you were dissatisfied with the results?  It is not that the media was ‘bad’, but that the differences in protocol between what is practised versus what is recommended by the media provider is not the same.  This is particularly so for IVM, the most sensitive part of the IVP process.  This sensitivity is due to the higher demands for substrates in the media by cumulus-oocyte complexes relative to denuded zygotes and embryos, simply because of the large number of cumulus cells co-incubated that significantly impact media formulation over the maturation period.  Evaluation of different IVM media systems using abattoir-derived COCs is further likely to distort results, as these COCs will have far more cumulus compared with OPU-derived COCs.

Over many years, ART Lab Solutions has been developing their IVP system with commercial embryo producers.  Yes, we validate our media with an independent source of abattoir-derived oocytes (and a 1-cell mouse embryo assay as well), but the proof for commercial operators is OPU-derived oocytes.  We have serviced numerous commercial operators, both Australian and International for many years with whom have been achieving pregnancy results of around 45% from thousands of embryos, from a large variety of cattle breeds and during all seasons. If you have any concerns about your commercial or research operations please contact us 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