We are growing!

ART Lab Solutions is proud to announce that Madeline Batsiokis has joined our team. Madeline has extensive experience working with cattle oocytes and embryos in commercial and research laboratories. She has worked in and set-up laboratories across Kenya, Spain, Brazil, China and within Australia, and trained many staff members to a full proficiency as cattle embryologists. 

Madeline completed her Masters of Philosophy with the thesis titled “Bovine in vitro embryo production (IVP):assessment of two new approaches to improve the efficiency of embryo production”. 

We welcome Madeline Batsiokis to the team and trust she will be an asset for our existing and new clients.

Real results. Real success.

Real results. Real success.
22,000 oocytes, 50% conversion of zygotes to blastocysts, 33% blastocyst rate from oocytes. Sexed semen. ART Lab Solutions media.
Thank you to our valued clients for providing your photos and results. We love providing you with a quality media product that enables you to achieve your goals!

This could be you. Contact us at admin@artlabsolutions.com to get started with a complete suite of cattle IVF media.

Is there an optimal interval between OPU’s?

I was talking to a client about OPU and what frequency should oocyte aspiration occur.  It’s not the first time I have had a conversation around this topic.  It seems there is no real consensus about frequency – most I speak with think weekly or fortnightly are acceptable.  But is there an optimal time interval?  Arguably, one of the most remarkable studies ever undertaken on this subject was conducted by a New Zealand team, published way back in 1999 (Hagemann et al., Molecular Reproduction and Development 53: 451-8).   They determined the day of ovulation by ultrasound (Day 1 of the oestrous cycle, which equalled the first day when the largest (dominant) follicle was no longer observed), then slaughtered 50 cows divided over 4 different days – Days 2, 7, 10 and 15 of the cycle.  This coincided with 2 follicular waves (Day 2 and 7 were the 1st wave, Day 10 and 15 were the 2nd wave), but also when follicular growth (Day 2 and 10) or when a dominant follicle (Day7 and 15) was present.  For each pair of ovaries collected, they hand-dissected every follicle equal to 3mm or more.  This enabled accurately sizing follicles, followed by IVP from the released COCs, plus a multitude of follicle stage and outcome matched material was collected.  The results were very clear: aspiration of the larger follicles produced more blastocyst stage embryos, but importantly, the presence of the dominant follicle considerably suppressed blastocyst development.   This was further supported by investigating the level of apoptosis in small follicles, demonstrating that the presence of a dominant follicle increased cell apoptosis in such (subordinate) follicles.  As follicle aspiration triggers a new follicle wave to be initiated, perhaps a 10-day gap should be considered as optimal!!  These cows were 2-wave cycling cows, of which the majority of cows are, but there are also 3-wave cycling cows that would push this timing out a little.  However, I’d be glad to hear if someone uses such timing and if they believe it has a positive impact!

What does “serum-free” medium really mean?

While scanning the cattle IVF literature, you will likely see the references that a medium, especially culture medium, is “serum-free”.  As we have already discussed in previous blogs, addition of serum is a ‘two-edged sword’.  On the one-hand it can promote development, particularly if embryo growth is slightly retarded and morula formation is hindered. But on the other-hand, serum addition at levels of 5% or more will increase triglyceride content that negatively impacts cryo-survival, especially with ‘slow-freezing’ protocols.  Furthermore, serum addition is a causal factor for ‘Large Offspring Syndrome’.

The solution seems obvious – remove the serum and replace with a purified version of bovine serum albumin.  And for further development promotion, perhaps supplement with one or even a cocktail of growth promoting substances.  There is nothing ‘wrong’ with this approach.  But do we need a protein source at all?  The answer is “yes” but it appears to be stage-dependent.  There is clear evidence from literature that polyvinyl alcohol (PVA, or similarly, PVP) supplementation instead of serum can support development to the 16-cell stage, but further development requires a protein source.  Many years ago, one of Australia’s leading embryo researchers, Peter Kaye (now retired), demonstrated that albumin is essential for, and literally, being ‘gobbled’ up by post-compaction mouse embryos via pinocytotic vesicles, possibly as an additional source of amino acids.  I conducted some follow-up work, and showed similarly the uptake of albumin and labelled caseins into the trophectoderm of cattle embryos.

However, be wary of the quality and source of serum albumin.  There are different ways in which albumin can be extracted, the simplest being a crude ethanol-precipitation of serum, known as Cohn’s Fraction V.  It is not much better than straight serum.  Serum substitutes are a mix of albumin, and other factors, most common is the use of “ITS” (Insulin, Transferrin and Selenium).  A problem is that you may not know the origin of the albumin.  There are also many growth promoting cocktails now on the market, particularly developed for stem cell production and differentiation.  We advocate chromatography-purified albumin, sourced from a disease-free country like New Zealand.  Also, look to see what the fatty acid content is.  Again, low- or fatty acid-free is preferable, especially if attempting slow-freezing of cattle IVF embryos.

To have your questions answered contact the team at admin@artlabsolutions.com. We look forward to hearing from you.

The risks we take – an IVF story.

Most who work in the cattle IVF field as a commercial enterprise are, in some way or other, challenged every day.  However, some situations are so challenging, they are worthy of a story to be told.

An Australian Wagyu breeder enlisted a team consisting of an experienced cattle ART vet, IVF and genetics leaders, including ourselves and SpeedBreed, Gnarwarre (near Geelong, Victoria, Australia), to deliver as many calves as possible from just two straws of semen.  The genetics within those two straws was of such high value that each straw cost tens-of-thousands of Australian dollars.  And to complicate things further, we had no idea how the semen would perform under IVF conditions!  This was a BIG investment by the breeder – it had to work.  The first decision that carried a risk was to cut one straw in half, use one half for the first day’s collection and the other for a 2nd day’s collection.

Although the OPU went well on the first day of collection (29 donor cows, providing 284 good quality COCs), and the transport maturation looked fine, there were fewer cleaved embryos than expected following IVF (51%). It gave, what we would categorise as a poor, but by no means uncommon result from untested semen.  As a result, good quality embryo number was also lower than expected (56, 38% of cleaved), but this is also a characteristic of poor fertilisation.  We suspected the issue was a not-optimized IVF step.  The sperm motility was fine, but we know some bulls that have good AI fertility may be poor at IVF.  We agreed to increase the heparin level, to twice our normal dose.  This was a further risk, but we needed to boost IVF rates.  It was worth it, as the cleavage rate was 75% of COCs (304 from the same 29 cows), and good quality embryo production from cleaved was just below 60% (135 embryos).  For OPU-derived oocytes, this is at the upper end of the spectrum.

We are now awaiting the pregnancy outcomes, but this story is about the need to adjust practices, evaluating the risks and making decisions that really impact IVF success.

 

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. 

 

THE BUZZ ON GROWTH FACTORS IN EMBRYO CULTURE MEDIUM.

Back in the ‘80’s -‘90’s when cattle IVF began to be seriously used for breeding, and when the “large calf syndrome” was recognised as a consequence of embryo culture, the use of growth factors in medium was snubbed, as it was thought that such factors caused the syndrome.  The real truth is that some cytokines and growth factors are very supportive of embryo development and quality.  They decrease embryo ‘stress’, and reduce the incidence of cellular apoptosis (regulated cell death). 

Furthermore, cytokine and growth factor mixes can counter the need for serum in culture medium.  But there is a catch – some factors will only be effective if they are from the species of origin.  For example, GM-CSF, a cytokine found in the reproductive tract, needs to be of cattle origin when used for embryo culture.  Furthermore, different suppliers provide factors that differ in their level of ‘activity’.  Most cytokines and growth factors have an optimal concentration for a biological impact – too low or too high concentrations leads to largely no effect, but with some factors, can be detrimental.

We believe growth factors are helpful at the right source and concentration during embryo culture, particularly under serum-free conditions.  But we don’t provide the growth factors themselves, especially not as a specific medium additive (it’s an Australian regulatory issue).  Adding growth factors to our media needs to be done in consultation with us, and we cannot accept any liability for any advice given.  But if you want to learn more about cytokines and growth factors, we are happy to provide you with information that can help you make an informed decision.

Contact admin@artlabsolutions.com for more information.

Read our Founder’s Story…

Written by Professor Jeremy G. Thompson himself, he shares insights into his passion for the ruminant IVF industry, his extensive research history and where cattle IVF is headed for the future. 

“My passion for ruminant, especially cattle, IVF was born from two things: Being a city lad but with many relatives on my mother’s side were farming, I loved working on farms, the beauty of the country and the enjoyment of working with animals; and appreciating that the research work could lead to real benefits to industries important to the Australian and New Zealand economies.  Besides, unlike human IVF, when I began studying ruminant IVF, it didn’t work at all.  So there was the challenge there that remains, of improving it.

I had just finished my B.Sc. (Honours) on the ‘reproductive activity’ of an Australian native mouse (!) in 1980 and was looking for something to do when I was approached by the legendary Prof. Bob Seamark asking if I wanted a temporary job as a goat embryologist working on a farm/breeding centre in the Adelaide.  They were long days and not much sleep, as I was embryologist and farm-hand all in one. It was a life-changing experience.  It led me to the Vet School at University of Queensland to do my PhD on IVF in sheep, which then led me to a short-post doc at Murdoch Uni vet school in Western Australia, with Prof. Ray Wales.  The project was looking at creating transgenic sheep with different wool properties. Alas, the funding was halted prematurely.  At that time IVF in humans was in its formative days and I was exposed to clinical IVF for a very brief period.  Looking for where to go next, I was approached to be recruited to the then Ministry of Agriculture and Fisheries (MAF) research centre, Ruakura, on the outskirts of Hamilton, New Zealand, filling the shoes by the departure of another doyen of the field, H. Robin Tervit, who had been attracted into a commercial breeding company.  These were heady days when multiple ovulation and embryo transfer (MOET) technology had matured in sheep and cattle to the point where breeding using MOET was having a major impact on changing the genetics of flocks and herds.  The NZ government had wanted to introduce new breeds of sheep into the country by MOET via embryo cryopreservation.  Robin had gone to Scandinavia and collected and froze the embryos, but then left MAF.  I was recruited to continue the program, a huge responsibility for a young post-doc.  Fortunately, before too long, Robin returned and we formed a great research team and friendship that continues today.  This where the transition from sheep to cattle IVF also began for me, as dairying is such a large industry in NZ.

After 13 years there, with one year in York (UK) on sabbatical with Prof. Henry Leese, with whom I learnt so much about metabolism of the embryo, it was time to return to Australia with my young family.  I was approached by both Profs. Rob Norman and Sarah Robertson from The University of Adelaide in 1999, to manage the clinical laboratories that served the clinical infertility group, ‘Repromed’.  I had only been an observer of human IVF, but this plunged me directly into clinical practice.  Although I learnt a lot and still engage with clinical IVF, I found that I missed the research and the work on cattle IVF too much.  Fortunately, I convinced Michelle Lane to come to Adelaide, where she took over the running of the clinical IVF programs, and following successful applications for research fellowship and grants, I returned to being a full time researcher in 2004, where I remained until this year.

My time at Adelaide has been special, mainly because of the cutting edge work conducted there in gamete and embryo biology with great researchers such as Prof. Rob Gilchrist and more recently with Dr. Kylie Dunning.   I was blessed with being involved in large block grants throughout my time in Adelaide, and with entities such as The Robinson Research Institute, ARC Centre of Excellence for Nanoscale Biophotonics and more recently the great collaboration with the Davies Research Centre. 

Ruminant ART research in Australia is hampered by the fact that few veterinary and/or animal science schools teach it.  At present, with current technologies, it has limited opportunity for new career pathways.  Over the past 2 decades, incremental improvements have occurred that are making IVF more attractive, but still requires a revolution in technological impact to make it highly attractive for industry.  I am convinced that, not just in Australia, globally we need IVF in ruminants to be simpler, yet more efficient and with less cost.  If we succeed, it will provide more meat of higher quality, but with fewer animals and will be more ethically produced.  There is a future for cattle IVF, because we are so close to solving the previously intractable problems of reduced efficiency and higher costs.  Automation of all aspects of the lab production will be feasible in the next few years, it just needs investment first and training opportunities as well.”

If you would like to know more about Jeremy’s history, contact him directly at jeremy@artlabsolutions.com