Embryo transfer

The text on this page is taken from an informal compilation of opinions of contributors to the online VOLE List. As such, they are not peer reviewed and may contain differences of opinion. Those wishing to contact the list may contact Adrian Smith.

I would like to know your recommendations for anaesthesia (both inhalative and injectable) and analgesics protocol for surgical embryo transfers in mice. Also I would like to know about your experience with non-surgical ET methods in mice as well.

This paper is very informative: Koutrili et al. (2014): Effects of using the analgesic tramadol in mice undergoing embryo transfer surgery. Lab Anim (NY). 2014 Apr 21;43(5):167-72.

For surgical embryo transfers, we use a mixture of buprenorphine and butorphanol as pre-emptive analgesia, prior to induction and maintenance of general anaesthesia with isoflurane. We have not found that post-operative analgesia makes any clinically observable difference to the mice and currently do not use it.

Non-surgical embryo transfers (NSETs) are also carried out under general anaesthesia using isoflurane, but without either pre or post-procedure analgesia, which we consider to be unnecessary. We originally used TCET devices to transfer manipulated blastocysts into the uterus of 2.5 dpc pseudo-pregnant CD1 females, but due to an inconsistent supply (and continually changing design) of the commercially available TCET, we started to produce our own in-house versions about 2 years ago.  These are constructed by gluing a fine piece of capillary tubing to a pipette tip and are then sterilised in an Ethylene Oxide chamber before use.  A small piece of plastic drinking straw is used to create a speculum, and the preloaded device gently threaded through the cervix of an anesthetised mouse.

The Advance Science Training Centre at MRC Harwell hopes soon to be able to deliver hands-on practical courses in a number of surgical techniques in mice, which will include surgical embryo transfer and NSET.

Why do you mix buprenorphine and butorphanol for pre-emptive analgesia?

I knew this was going to be a talking point. They used to give buprenorphine once the mouse was anaesthetised, rather than 30 minutes beforehand, and as the technique only takes about 10 minutes, I reasoned that the buprenorphine wouldn't have kicked in it before the mouse woke up. I reasoned that with a shorter lead-in time, butorphanol might give short-term analgesic cover up to the time that the buprenorphine took over. I expect that there are many flaws in this, as with most of my "cunning plans". Happy to be corrected.

Yes you are quite right that with a 10 min procedure, the buprenorphine will barely have kicked in once the mouse has recovered. I’ve given buprenorphine an hour or so after reversing fentanyl mixes with butorphanol, but I am not sure what will happen with simultaneous administration of a mu antagonist/kappa agonist and a partial mu agonist (of course if we decide butorphanol is a partial mu agonist too then there is no problem!).

I am very concerned by your statement 'We have not found  that post-operative analgesia makes any clinically observable difference  to the mice and currently do not use it.' How was this clinical assessment done to make sure that the animals do not experience pain? It is, after all, a surgical procedure. I would highly recommend to reconsider the post-operative analgesia, please see also: Fleischmann et al. (2017): Voluntary intake of paracetamol-enriched drinking water and its influence on the success of embryo transfer in mice.

Many thanks for that. We have not tried paracetamol in drinking water post ET, and I will discuss this with the scientists involved. Does everyone else give post-op analgesia after ET and for how long?

Sorry to stir things further, but the paper cited shows only that paracetamol was consumed, and potentially analgesic plasma concentrations achieved, and there was no effect on litter sizes, but their was no measurement of post-procedural analgesic efficacy. The papers by Flecknell and Miller and Richardson are suggested dose rates based on cross-species extrapolation. Our several attempts to provide post-surgical analgesia in both rats and mice with paracetamol have not been particularly encouraging. I’d only consider use of paracetamol post surgery if opioids and NSAIDs were contraindicated.

Yes, I was concerned that the 2 drugs were potentially antagonistic, but at the risk of causing more opprobrium, when we introduced this a few years ago, we couldn't see any behavioural differences post surgery between the mice that had buprenorphine alone, those that had buprenorphine and butorphanol (given at the same time but in different sites), and those that had no analgesia at all. As you can imagine, this didn't help my case for the use of analgesia, so we now give it on the principle of giving the mice "the benefit of the doubt". What we can say (as with the paracetomol) is that it apparently does no harm to the recipients or the embryos. In other surgical procedures we use carprophen post-operatively, but we have always worked on the understanding that NSAIDs are contra-indicated in ET. Again, I would be very interested to hear from anyone who uses these routinely as analgesics for ET.

Schlapp G, Goyeneche L, Fernández G, Menchaca A, Crispo M. Administration of the nonsteroidal anti-inflammatory drug tolfenamic acid at embryo transfer improves maintenance of pregnancy and embryo survival in recipient mice. J Assist Reprod Genet. 2015;32(2):271-275. doi:10.1007/s10815-014-0378-x: The use of tolfenamic acid at the time of embryo transfer improves both pregnancy rate and number of live pups in recipient mice, with optimal effects observed with flunixin meglumine. We suggest that the use of tolfenamic acid has beneficial effects on the maintenance of pregnancy and embryo survival in recipient mice, which should be taken into account for further studies in other mammalian females.

And this paper comparing adding an NSAID to an opioid:

Parker JM, Austin J, Wilkerson J, Carbone L. Effects of multimodal analgesia on the success of mouse embryo transfer surgery. J Am Assoc Lab Anim Sci. 2011;50(4):466-470.

At a practical level, I can say that we routinely use meloxicam in wet mash after ET surgery (with buprenorphine given s.c. at induction) to cover overnight. We previously used carprofen in medigel - but can’t get it now, so we changed. The mice happily self-medicate and the technicians were immediately won over when the first transfers had substantially higher numbers of pups. We've never done a comparison - but there is definitely no evidence of reduced success or fewer pups. We also use the NSAID wet mash in our stock mice if they have a bad parturition and they happily self medicate.

The prostaglandin discussion is, in my opinion, one of those "known facts" that needs challenged. I remember having this argument about our experimental sheep in my previous job: "you can’t give an NSAID after a bad lambing because it will stop the animal from cleansing due to the prostaglandin effects". What about the effects on normal processes from the pain? Again, best proof was just to show it (as I had used it routinely in practice for bad lambings/calvings and caesarians) - we gave one ewe who had had a bad time a dose of Finadyne and she dutifully cleansed about 30 minutes later. The potential effects of pain are frequently in my opinion ignored. Only an anecdote - but just to add to the real science.

The effects on NSAIDs on the risk of retained foetal membranes (RFM) is something I feel I can actually comment on with some degree of authority (or at least in dairy cows!) There is reasonable evidence that meloxicam does not affect the risk of RFMs, there is some question mark over flunixin (but the evidence is older). Routine use of meloxicam at all calvings (birth events) had no effect on measured production outputs:

Effects of meloxicam on RFMs in cows

Review of routine use of NSAIDs at calving

Effects of meloxicam on dairy cattle following assisted calving