Hi Marco,

How old are your frogs? How many times have you performed surgeries on them to collect oocytes? In my experience is it always good to start with new frogs from time to time. Also, for a reason that still remains obscure to me, we have observed seasonal changes in the quality of oocytes, even though the frogs are kept under controlled light cycle and temperature. 

I can't recall the name of the supplier we were using back in France, I think it was in "the Dombes", you may be able to find the info on the net. We also bought them once directly from South Africa as they were supposed to be of better quality, but it wasn't worth the troubles... 

Hi Marco,

How old are your frogs? How many times have you performed surgeries on them to collect oocytes? In my experience is it always good to start with new frogs from time to time. Also, for a reason that still remains obscure to me, we have observed seasonal changes in the quality of oocytes, even though the frogs are kept under controlled light cycle and temperature. 

I can't recall the name of the supplier we were using back in France, I think it was in "the Dombes", you may be able to find the info on the net. We also bought them once directly from South Africa as they were supposed to be of better quality, but it wasn't worth the troubles... 

Hi Marco,

we are using NASCO frogs for many years though we are using them for having embryos, but only good oocytes give good eggs and thus embryos. We tried Xenopus Express in France ([email protected]) once, though with moderate success, never better than NASCO.

It might be a problem with the water hardness (too low) and food or the female haven't been used for a longer period (old oocytes) as well as an infection.

Contact the Hubtecht Institute in Utrecht (Netherlands). They have expereience in cultivating Xenopus:

[email protected]

+31 (0)30 212 18 00

+31 (0)30 251 64 64

Hi Marco,

as Norbert already mentioned, it´s good to start with new frogs from time to time. But sometimes, initial oocyte quality is not good from new frogs. Then, it´s a good idea to keep the frogs for a few month without surgery. Some people even treat new frogs with hormons to induce shedding of eggs in order to get rid of "old" oocytes.

Good luck!

Jürgen

We use Nasco frogs and found them generally to be the best, compared to Xenopus express (France) and another German company, which had to be closed down. Temperature and water quality are our major concerns, water temp. above 21 °C for longer periods is bad for oocyte quality, the same is true for water clearing after feeding and removal of the feces. We have bad experience when feeding the frogs with meat (cow heart) and changed to more vegetarian diet ("frog brittle"). Between surgeries, we give the animals at least 3 months to recover.

Hi Marco,

we also use the frogs from Nasco. We tried Xenopusexpress, but we not so satisfied. Nasco usually always were good frogs. Nasco also always breeds them by themselves and has no wild catches which may be send to you by other suppliers sometimes without even mentioning this. The microbiological status may not be so clear in this case. The first idea is also: how about the age of the frogs? How often have they been stimulated or operated? If you remove immature oocytes the number of narcosis may accumulate with time. If the frogs are squeezed for mature eggs than the period between the injection may be too short or too long. Especially when the frogs are not used regularly the egg quality is reduced and the frogs should be pretreated once before putting them back into a normal injection cycle. How about water quality and water temperature? Are ther differences between the tanks? Our observation was an influence of the season as well with usually having very good eggs in spring and autumn and poorer quality (or better: more variable) in summer and winter. This can be due to the different temperature of the water inflow. Maybe these frogs are at the end or the beginning of the water system and get more cold water and therefore have more variations in temperature going up and down.

Other idea: position in the system with the frogs being on top of the system and therefore closer to the ceiling: do they have a little bit higher temperature there and more light?

What kind of infections were tested by your vet? Virus infections may have a even higher influence on egg quality than bacterial infections and are difficult to detect. Any parasites tested? Most infections are subclinical in frogs and never show in any behavioral or physiological changes.

Hi Marco,

We are working more than 20 years with Xenopus oocytes and have had similar issues. It can go perfectly well for years in a row until suddenly oocytes become bad and stay bad for a long time for no obvious reason. At the moment we are also buying our frogs from Nasco and they are perfect for over two years now.

Once when we had a bad period, we changed antibioticum from gentamycin to neomycin and that helped. Another time when it was bad, we increased the calcium concentration of the Barth's solution to 2 mM calcium and that time that helped. So these are two things you can try.

I cannot recommend breeding frogs yourself. Once we have bought very young frogs which were as small as the fingernail of your thumb and it took about 2 years before they had enough oocytes. In addition more than half of them appeared to be male. It's too time consuming.

We have bought frogs from Xenopus I in France in the past, but I don't think they exist anymore.

One good alternative is to buy  ready to inject oocytes from Ecocyte, a company in Germany. You can order defolliculated oocytes from them and they arrive within 24 hours after harvesting. We have very good experiences with oocytes from them.

I hope that one of the suggestions will solve your problems.

Hi Marco, 

Unlike most of the other people who responded we actually switched from Nasco to using Xenopus Express (USA) and have seen some improvement in our oocyte quality.  The seasonal oocyte quality issues (oocyte quality is worse in the summer) were a problem for us and in order to get around that we switched from laboratory strains to the wild caught southern hemisphere frogs that Xenopus Express provides.  We also eliminated survival surgery entirely after a series of experiments using translation of an injected RNA as a measure of ooctye heath indicated a decrease in oocyte quality with multiple surgeries from the same frog.  

For albino oocytes we tend to use younger stages and we specifically request that the frogs be ovulated before they get sent to us from Xenopus Express.  This does result in the death of a small percentage of the frogs after arrival but the ones that survive usually have large amounts of healthy oocytes 1-2 months after the frogs are received.  

As others have also suggested the water hardness has been reported to affect oocyte quality as well as standing vs circulating water.  We also performed some preliminary experiments with normal vs ciclid salts and standing vs recirculating  water but the results were inconclusive.  

Also, there will be variation in oocyte quality just generally speaking I wouldn't go changing things up if this is something that happens rarely.  We only changed things when we went months with oocyte quality issues. 

Good luck!

Hi Marco- We have used frogs from Nasco, Xenopus I (USA) and the Xenopus National Resource at Wood's Hole labs.  We've had great oocytes and poor oocytes from all providers over the last 30 years.  One thing that really affects oocyte quality is stress- that can come in the form of problems during shipping, recovery or changes in environment.  For example, construction of a new building next to our animal facility corresponded with poor oocyte and egg quality for many months ( I'll note that the labs using mice had similar stress problems with their colonies). 

In our experience, if oocyte quality from a frog is poor, it's best to euthanize the frog- they are unlikely to get better with time.  I would also let Nasco know that your new batch of frogs had problems, that could help sort out it the problem was at Nasco or stress during shipping.

Good Luck

Dan

Hope to be able to send you some information after the Easter holidays, i.e. 29 March.

Bad batches of oocytes may be obtained from frogs kept for a long time without spawning. Over-aged oocytes degenerate spontaneously. One possibility is to inject HCG, induce spawning and then wait at least 3 months to allow for new oocytes to grow to full size. Indeed, stage 5 oocytes are usually in good shape also in such ovaries and may be injected easily.

In case of an urgency, you may also centrifuge the oocytes as used to visualize the position of the nucleus for nuclear injection. Fragile oocytes lyse at centrifugation or turn white and may be eliminated.

Hope it helps

Duri

Thank you very much for all your replies! You gave me several good points to follow up on, and I will do so in the next couple of days. I'm especially thankful for the thoughts on breeding, I will most likely (and gladly) abandon this idea again.

Maybe of interest, I also found this 70 page pdf guidance quite helpful (link below).

greetings, 

Marco

http://science.rspca.org.uk/sciencegroup/researchanimals/implementing3rs/housingandcareaquaticspecies

Beside ordering frogs from Nasco we also used to breed froggs by ourselves. It works well but takes some time. So this is an option when you have enough place and time to do so. And always a nice job for young students or trainees :-) Usually they also have very good eggs. But nevertheless we always do refreshment of blood from time to time and order new frogs.

In our laboratory, we have a good experience with the Diacleanshop company. Here is a link: http://www.diacleanshop.com/epages/Diacleanshop.sf/de_DE/?ObjectID=6212031&ViewAction=FacetedSearchProducts&SearchString=xenopus. We use oocytes for the functional studies regarding human uric acid transporters.

Best regards

Vladimir Krylov 

We euthanise our frogs and remove the ovary, so they never get multiple surgeries. We do find that frogs which have previously been super ovulated are much less likely to give good oocytes. Moreover, we tend to get very good oocytes from lab reared frogs (at about 2 yrs old). The oocytes form these tend to be of higher quality that those from Nasco frogs. There was another place from which we obtained some frogs, and those tended to have very bad oocytes, so I would say that the supplier does make a difference.

I’m afraid I cant be of much help with European suppliers as, for some years now, we have only used Nasco.

If you would like to know more about in house breeding then please don’t hesitate to contact me directly (Dave Simpson: [email protected]).

best wishes,

David Simpson,

Gurdon Institute,

Tennis Court Road,

Cambridge,

England,

CB2 1QN

+44 1223 334 087