[0:05]Dr. received his PhD at the University in Grenoble in France, and he is very well experienced and he has more than 16 years experience um in managing um R&D projects and he got a lot of strong experience in mice and rats, says the production technologies as well as Genesis and um nuclear transfer technologies and genetics.
[0:29]So I'm happy to have you here and I'm looking forward to your talk. So thank you so go ahead.
[0:37]Thank you. Thank you for this perfect uh introduction. Going to share my screen.
[0:54]Yep, do you see it? Nope, not yet. No, not yet. Okay. You should see it now. Yes, it's here.
[1:13]Okay. Uh last sorry.
[1:40]Okay, anyway. Um yeah, it seems to uh I can't oh yeah, that's works. Okay, fine. Perfect. Um first of all, I would like to thank the organizers for the invitation.
[1:54]It's a real pleasure to be with you to address such an important topic as the three Rs. So to start with, I would say, um a few, um a few words about Charles River and its positioning regarding the three Rs.
[2:12]Charles River is a leading global provider of essential products and services for the scientific community. Every year we do participate to the development of more than 80% of the drugs approved by the Food and Drug Administration.
[2:32]As you may know, the company has been existing for more than 75 years now and at and had always been working with animals. All along those years, the company has developed a strong three Rs policy with strong commitment to maintaining the highest standards in animal welfare.
[2:54]And thanks to that, all our facilities worldwide have earned accreditation for of their animal care and use programs from the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC) International, which is the highest accreditation you can get in the domain.
[3:22]And here we come to the extra bonus all we are using a Charles River. And for because uh we have added an all to the three Rs to end up with the four our four Rs mission.
[3:40]And uh for for us responsible science starts with uh this foundational fourth R. uh uh this fourth R stands for responsibility. Um and that has been developed specifically for us and to ensure that we are placing a lens of responsibility on everything we do, driving progress for patients and animals that really depend on our work.
[4:14]Today, I will make a focus on genetically engineered animal. So why is this? Because we do see that this type of uh uh models of animals are replacing standard wild type animal.
[4:30]And the revolution began in 1974 when Rudolf Jaenisch and Beatri Minx infected mouse embryos with SV40 virus and showed that the viral DNA was integrated in the germ line of the mouse. Since then, and thanks to several technical uh revolutions, including the recent uh emergence of CRISPR/Cas9 editing, genetically modified animals have gradually replaced standard research models in our facilities and when we can see that in our uh all of you uh in in your facilities.
[5:15]So different and complementary access of work are required to support the three Rs in animal research. The first one relates to the best practices for experiment designed and reporting.
[5:33]So different guidelines have been established by scientific community to support best practice. Those guidelines are distributed through different well acknowledged associations, you may know in the field of animal research.
[5:52]But I I would say this is uh the responsibility mostly the responsibility of the researchers and the PIs. On uh another side, recent advancements have uh enabled the emergence of new technologies and strategies which can positively impact the three Rs and the way we manage transgenic research models.
[6:24]And today, I would like to share with you our experience in developing and using state-of-the-art technologies to support the three Rs when using genetically engineered animal models. Uh since mice represents 80% uh of all animals used in research, I have selected illustrative examples of achievements in this specific species.
[6:50]The selected three Rs initiatives are powered by advances in different fields, including reproductive and genetic engineering technologies, genotyping, and digital tools.
[7:08]Let's begin with uh initiative uh linked to advances in reproductive and genetic engineering technologies.
[7:23]Most of uh mutations uh uh available uh in the mouse have been created in inbred strains or have been transferred to inbred strains through backcrossing methods. So one key development over the last 10 years did correspond to efficient sperm cryopreservation and in vitro fertilization technologies for inbred strains.
[7:51]Since uh since then, assisted reproduction technologies have taken more and more place in the lab for achieving key steps in the management of mouse transgenic colonies. And you know about those key steps, they include the production of live animals for experiments or for colony maintenance.
[8:15]The protection of genetics resources through cryopreservation. It also includes the adjustment of line hygienic status through pathogens removal, this is what we call rederivation. And of course, the generation of novel transgenic lines through model creation.
[8:41]Let's take first the example of the production of embryos. The standard way uh to uh to go to uh to produce embryos is to mate males with preconditioned female to generate and collect embryos at preimplantation stage.
[9:00]Those embryos are then processed uh for different type of services, like genetic engineering, cryopreservation or rederivation. The issue uh is that sexual reproduction is strongly influenced by different parameters, including mouse genetics and environmental factors, resulting in highly variable fertilization outcome and a significant fraction of mating failures.
[9:35]By consequence, it is very uh frequent to systematically uh either oversize, the number of animals used to ensure the successful achievement of the service, uh uh with a significative risk of animal resources uh wastage.
[9:55]So more than 10 years ago, we have introduced the IVF technology on our embryology platform and we are started to monitor its performance to replace mating.
[10:10]Here is a report of a seven years retrospective study we conducted comparing the efficiency of mating and IVF approaches in uh for the production of preimplantation embryos and live pups.
[10:27]And what we do see is that performance of uh uh you see on the right side of the slide, the performance of each method which is uh reported in the schematic. And on the basis of a pool of 100 osites collected from hormone primed females, our data show that IVF delivers on average 60% more embryos and 70% more live pups than the mating strategy.
[11:00]And the conclusion is quite clear is that IVF systematically outperforms mating for both the production of embryos and live animals.
[11:14]The outstanding uh efficiency of IVF is here expressed in that slide in term of number of saved animals per service. This number is indicated in green uh in in the table.
[11:32]How does this translate for one year of Charles River's activity in Europe? Thanks to IVF, we have saved more than 4,000 females per year. This great improvement in the three Rs also came with over advantages.
[11:53]A significant reduction in number of cages used and by extension all the associated workload and cost. So it's not only about saving animals, which is the first target of course, but it also helped in other domains.
[12:15]And by by extension, uh every process using sexual reproduction in startup can be dramatically improved if we use IVF instead. Here is another example.
[12:30]Conic inbred lines are massively used in research. Conic lines are created through the backcrossing method, which consist in repetitive mating steps aimed at transferring a mutation from one genetic background to another.
[12:49]The process is strongly uh accelerated when using polymorphic markers such as Snips. markers allow selection of mice most enriched in target genetic background. Those mice will be used for breeding to the next generation.
[13:08]And IVF gives sorry. IVF give the possibility uh uh to generate the next generation with a single male, thus reducing the total number of pups needed to screen from. And in addition, cohorts with IVF are synchronized in age and mating delays are dramatically reduced.
[13:34]So we get rid of all the issues we we have with standard mating and it is therefore possible to significantly uh accelerate the whole mutation transfer process. And you see uh an accelerated process takes 60 weeks and by this way, we can go under one year uh uh with 45 weeks.
[14:01]Modified versions of IVF can also strongly participate to the three Rs. Losing a transgenic model that has required a lot of animal resources, budget and time to be created is likely one of the worst event for animal welfare and for continuity of experimental studies.
[14:21]And this nightmare may come true if your line is showing very low reproduction capacity or if sperm cryostock is of poor quality. And so we have developed a laser assisted uh IVF to save endangered precious strains through the use of transgenic mates.
[14:41]The laser creates uh holes in the external protein envelope of the oocyte, which is called the zona pedum, thus facilitating the access of the oocyte to sperm cells.
[14:57]This approach has been successfully operated on our platform for several lines.
[15:07]I will now describe uh another technology we have been developing to support the process of model creation. Every uh every year, thousands of new uh uh mouse transgenic models are created worldwide. Transgenesis efficiency do rely heavily on the methodology applied to deliver key molecules such as proteins, DNA or RNA to the preimplantation embryos.
[15:36]Since the 80s, microinjection into mouse zygote has been the method of choice for this job. However, in the last uh two years, we have replaced microinjection by electroporation to generate mutations not requiring the transfer of large molecules.
[15:58]So in this slide, this slide illustrates the advantages of the electroporation method versus the microinjection approach for the generation of a knockout model by CRISPR/Cas9 system.
[16:16]In that case, the excision of the target sequence only requires the transfection of the Cas9 protein or MRNA and one or two RNA guides. Our data demonstrate that electroporation needs on average half the number of embryos needed by microinjection to generate the knockout model.
[16:43]Similar results uh are obtained uh for the generation of knockin models when DNA insertion does not exceed 1Kb. And beside the obvious, I would say, uh uh three Rs advantage of the electroporation approach, one should mention that electroporation is much more easy to operate than microinjection.
[17:11]So that means that the experimental outcome is less dependent on the skill of the operator of your technician. And in addition, the process uh takes only one fourth of the time of that of microinjection.
[17:31]Electroporation can also be successfully applied to the recombinase systems. And beside CRISPR/Cas9, recombinase systems constitute a very different a very efficient way to introduce target mutations in mouse genome.
[17:49]And the most popular recombinase system is the Cre/lox system. The Cre recombinase is able to recognize and attach specific short DNA sequences called loxP sites on the target DNA. And depending on the number, the orientation and the localization uh of the loxP sites, the recombinase will operate various types of DNA rearrangements.
[18:18]Typical rearrangements uh include excision events, sequence inversion and translocation.
[18:29]So the schematic in this slide describes the very standard process used to obtain a knockout through a target sequence excision in all the cells of the mouse using the Cre/lox system. So that approach very standard approach use mating, and in the first step of the process, the flox line is mated with a Cre expressing line to obtain the knockout uh allele.
[19:01]So this is what you see in this first. In second step, the resulting heterozygous knockout line is bred with wild type animals to remove the Cre transgene from the line in order to avoid its potential impact on the final phenotype.
[19:20]And then in a third step, the pure excised heterozygous line is then expanded by mating again with wild type animals. Once you have enough heterozygous animals, you can bring you can breed them together and to obtain the homozygous colony.
[19:40]And now is described in the schematic below, the same process using IVF and electroporation method. So the the philosophy of this approach is to produce a large number of embryos through IVF, and those embryos contained uh the floxed allele.
[20:06]Uh the embryos, those embryos are submitted to electroporation in the presence of the Cre protein or Cre mRNA. Embryos are then transferred to foster moles to generate the to obtain live pups to and obtain heterozygous for the knockout line.
[20:26]And the good thing is with IVF, you can scale scale your IVF. So up to obtain in within the same step, the addition, so the excision of the flox allele and the expansion at the same time of the heterozygous knockout line.
[20:45]So the resulting heterozygous are then directly can then be directly interpret for the generation of the homozygous uh colony. And with this strategy, uh breeding steps uh two breeding steps have been eliminated, thus allowing to save dozen of animals uh for uh within the approach, including we save Cre expressing animals, wild type animals used for breeding and also wild type litters which are not of interest.
[21:23]And this approach can be translated to over recombinase system over than the Cre/lox. It can also include the flip FRT system with the same efficiency or over recombinase system. There is no need to import and breed the recombinase expressing line.
[21:43]Thus you reduce strongly cost and workload. And in addition, the absence of mating with an external line eliminates eliminates completely the risk of introducing foreign alleles into your original mouse model.
[22:01]Uh the approach is therefore ideal when looking for generating uh for generating ubiquitous genomic uh mutations.
[22:13]So something else to say is that leveraging on technologies is key, of course, but not sufficient to sustain a robust and consistent three Rs policy. This is why at that stage of the presentation, I will I will take a step aside to discuss further why responsible science starts always with the fourth R of responsibility.
[22:42]Let's take the example of our embryology platform. As highlighted in this schematic, technologies constitute only a fraction of the parameters affecting the global efficiency of our processes.
[22:58]Each cause or reason for imperfection within this system is a source of variation and low performance. And we, at Charles River, are placing a lens of responsibility on everything we do.
[23:13]So we do consider that beside beside the technologies, we do consider that the training, the continuous assessment and the rewarding of our staff is of equivalent importance than the type of technologies we are using.
[23:30]And also choosing relevant equipment and maintaining its performance in the long run is also key. So our responsibility, I would say, essentially resides in creating the appropriate environment to sustain continuous improvement in all dimensions of our activity.
[23:54]And any failure or under performance in this system would lead to experiment repeats and wastage of animal resources.
[24:08]So once we have said that, we come back now on the technology side and the role of innovative approaches in supporting the three Rs. Let's talk about genotyping.
[24:22]Genotyping is uh as you know, is allowing to determine the genetics of an animal and constitutes therefore a crucial step in colony in colony management.
[24:37]Genotyping usually requires to biopsy a live animal using a surgical procedure. In this part of the presentation, I will describe alternative genotyping ways either to reduce the number of animals involved into the genetic testing process or to minimize animal discomfort during the sampling.
[25:02]First approach relates to the genetic testing of sperm and embryos. I'm going to illustrate the strategy with three examples of application. The first one is the validation of cryostocks.
[25:21]And the rest of the examples will relate to the assessment of experimental procedures and the optimization of model creation process using mouse embryonic stem cells, which is something which has been already approach in the previous talks.
[25:40]So as you know, cryopreservation is key to secure genetic resources. Once constituted, the quality of the cryostock should be carefully evaluated since the line may may be thereafter discontinued.
[25:58]So you have to be sure about what you have in hand and what is in liquid nitrogen. Quality controls should normally include a functional validation and a genetic validation. The functional validation is aimed at indicating if the line can be revitalized.
[26:21]And and and the genetic validation is aimed at indicated at indicating if the cryostock is showing the expected genetics. Indeed, uh we can face errors during the genotyping process of the animals.
[26:41]And we can face errors during the freezing process itself. This is why we need to verify both. The quality control strategy should be based on the best compromise between predictive ability of the test and the number of used animals.
[27:01]This schematic shows how quality controls are usually performed. Floxed embryos uh or embryos generated by IVF using frozen sperm are transferred into one or two foster females to obtain pups. Genetic testing of the pups is then conducted.
[27:28]Um so in one step, generating live animals, you do your genotyping and you verify you can obtain live pups. Then if you do this type of QC, generated pups and foster females are usually after the QC.
[27:48]And this approach also needs to maintain a regular and significant production of pseudo pregnant females using a dedicated colony. So you need quite a lot of animals to uh to go through this approach.
[28:07]In this slide is described the optimized three Rs version of the process for functional validation. So the functional validation here relies on the assessment of the in vitro development of the embryos generated after flowing or flowing the embryos or obtaining them through sperm and IVF.
[28:41]And our data shows that there is a very strong correlation between the ability to of the embryos to develop in vitro and their capacity to develop to turn so in vivo. So there's no need to replace the embryos uh if your test is performed in the right way uh to know if you will obtain pups uh a turn.
[29:09]And then the genetic testing because we need a genetic testing is performed directly on cultured embryos issued from the functional assessment test, and the embryos should contains enough cells. So we do use mola and blastosis stage to get enough DNA to run our uh PCR.
[29:32]In the case of sperm cryostock, then you can also do your genotyping directly on sperm cells. And this approach by going only in vitro avoids using six to about six to twelve animals on average per cryostock QC.
[29:56]And we do perform a lot of cryopreservation on our platform. My last example of the great possibilities opened by sperm genotyping is the optimization of the model creation process when we are using mouse embryonic stem cells.
[31:39]So despite the emergence of the CRISPR technology, ES uh cell-based genetic engineering is still required to generate complex mutations, and those complex mutations usually involve large genomic uh sequences replacement.
[32:00]And in this process, which is described in this slide, the chimeric males obtained after injection of recombinant embryonic stem cells into mouse blastosis, uh those chimeric males are bred with wild type females to transfer the mutation to the next generation, this is what we call germ line transmission.
[32:26]However, in within this process and as mentioned in the previous talk, only a fraction of the generated chimeric males are usually able to go germ line.
[32:41]This means that a large fraction of the generated F1 pups may be one type and will be euthanized. On the other hand, use less breeding pairs are also set up because there's quite uh you can face some sterility or infertility with the chimeric males you obtain.
[32:56]And my last example of the great possibilities opened by sperm genotyping is the optimization of the model creation process when we are using mouse embryonic stem cells. So despite the emergence of the CRISPR technology, ES uh cell-based genetic engineering is still required to generate complex mutations, and those complex mutations usually involve large genomic uh sequences replacement.
[33:07]And in this process, which is described in this slide, at the chimeric males obtained after injection of recombinant embryonic stem cells into mouse blastocyst. uh those chimeric males are bred with wild type females to transfer the mutation to the next generation, this is what we call germline transmission.
[33:26]However, in within this process and as mentioned in the previous talk, only a fraction of the generated chimeric males are usually able to go germline.
[33:41]This means that a large fraction of the generated F1 pups may be one type and will be euthanized. On the other hand, use less breeding pairs are also set up because there's quite uh you can face some sterility or infertility with the chimeric males you obtain.
[34:10]So the approach using genotyping, sperm genotyping, combine is combined with some over assisted reproduction technologies and uh it can dramatically improve the global process. And it is described here, what we do, we do collect sperm from the chimeric males and we do cryopreserve the sperm.
[34:33]Then a straw from each cryostock is used to perform automated sperm parameters analysis and uh is also used for genetic testing using QPCR. And from this analysis, we can identify males of interest, showing both uh a good potential fertility uh capacity.
[34:57]And also a good ratio of sperm containing the expected mutation. So and then we do select the best sperm, showing the best performance in both sperm parameters and ratio of uh the mutation.
[35:46]And we have selected this male. We have run an IVF and we have obtained a very high ratio of heterozygous pups in the F1 generation.
[36:14]So and and beside the reduction uh in the number of animals used, um the approach also demonstrate clear advantage regarding the project timelines. Indeed, uh the expansion of the F1 heterozygous colony can be done at the same time as the obtention of the germ line transmission.
[36:44]So once you you are pretty sure that you will obtain a large number of heterozygous pups, so it helps to set up thereafter your colony. And finally, if needed, you if you need some excision of unwanted DNA constructs, uh such as selection markers like neomycin uh resistance uh cassette.
[37:16]Then you can also achieve this within the same IVF session through electroporation of Cre or flip recombinase as we have seen in one of my previous uh slides.
[37:34]So we have seen through the different selected examples that sperm and embryo genotyping can really constitute a plus when managing transgenic mouse colonies. And at Charles River, we have developed efficient methods for the genetic testing of sperm and embryos.
[37:51]Analysis can be performed at the level of a single blastocyst with amplicon size up to 300 base pair. And for sperm, a single straw is sufficient to perform genotyping with amplicon size up to 1.5Kb.
[38:11]In the case, animals can't be replaced in the genotyping process. We have focused our efforts in reducing the discomfort of the sampling method. Indeed, for many years, mice have been sampled through surgery by taking either a piece of ear or tail.
[38:30]Um some already some already established non-invasive genotyping methods have used oral swabs or hair, fecal pellet, anal swabs and tears. So there have been different approaches. At Charles River, we have previously established the value of oral swab genotyping in rabbits.
[38:51]And recently we have adapt the sampling method for mice. The method involves mouth swabbing of the inner cheek of the mouse using a cotton stick, followed by appropriate lysis procedure of the harvested buckle epithelial cells.
[39:19]This method shows similar efficiency as ear biopsy for every kind of PCR analysis. Samples can be stored and shipped in varying conditions, making it very easy to use.
[39:35]And by consequence, oral swabs can be used for routine genotyping. And in addition, oral swabbing is perfectly relevant when a second invasive biopsy is not possible, or when the animals are old, or are identified with ear tags or tattoos that avoid taking some pieces of any any biopsy.
[40:03]In all those conditions, oral swabbing can make a difference and can be and so that can can be of help. As for over techniques, a good training with the method is key for achieving good results.
[40:22]Of course, this is why Charles River is providing clear and precise instructions for operating oral swabbing. We now leave genotyping methods aside to discuss about the input of digital tools on the three Rs.
[40:38]With two examples, the first one is about data collection and record keeping. And this is one of the major goal in animal colony management. It is really to keep track of the historic of the line.
[40:56]One should use a very robust system that assigns each animal a unique identification number. And by recording every single detail, the system should allow early detection of problems and trends.
[41:17]Such a system do strongly participate to increase the breeding efficiency and to reduce the number of animals used and of course, all the associated cost.
[41:31]And at Charles River, we have developed a proprietary system that some of you may know, more than 10 years ago now. And this system is called Internet Colony Management system, ICM. This custom designed software solution is a tool that is used for both project and animal colony management.
[41:59]So we use them for our standard colony production and also transgenic colony production. ICM is a two-way system, allowing our customers and our project managers to pilot mouse colonies in real time.
[42:15]So it ensures that accuracy of the data and and the project goal alignment between us and our customers. And this system allows tracking of hundreds of parameters, including animal ID.
[42:31]Animal location, genotype, genetic background, all the sampling we have done on animals, pedigree and and more and more parameters. ICM enables controls over colony size, avoiding duplication and oversizing animal population. So we do save a lot of animals with such a robust system.
[42:51]And finally, the tool shows extensive filtering and sorting data capabilities. So you are able to retrieve the data that are um can be used for analysis of your uh colonies, breeding uh approaches.
[43:06]So you can adjust uh your uh procedures. In the same way, Charles River has been developing the use of innovative identification system for mice.
[43:24]And our genetically engineered models and services teams have begun to use mini colored mouse ear tags with scannable barcodes for lab animal identification. So in this system, we are using tags which are very small, 3 millimeter in length and can be easily decontaminated by standard methods, including autoclavin.
[43:45]So very simple to use uh within our breeding systems. And multiple color options are available. And it really helps to quickly identify cage mates and dosing groups, for example, so through the observation of the mice, you can also get information uh seeing the colors of the tags uh you you have been using, beside, of course, the possibility to scan those tags and obtain all the information contained in the tags relating to uh each uh animals.
[44:17]The approach provides with millions of unique IDs with zero error when you scan when you scan them. And the process is fully integrated with our Internet Colony Management uh tool uh that uh enhance also the power of uh our uh ICM uh system.
[44:34]The system uh of this what we call rapid ID tags is minimally uh invasive. And this is why it also improves animal welfare. And by strongly reducing the risk of errors, it also participates to reduce research failure and animal wastage. So you don't face any issue selecting the wrong animal for your experiments.
[45:00]And as observed with over three Rs supporting solutions, and as we have seen for in my previous slides, this one is also coming with many over advantages regarding the workload and cost reduction. So this is mainly due to the facility of use of the system and it's because also it's very reliable and accurate.
[45:06]So this presentation has now come to an end. I will just try to summarize the some key points uh in my conclusion.
[45:22]So, um we have seen that genetically engineering animal models have really become uh in the last few years key tools in fundamental research as well as in the drug and discovery process. And those genetically modified animals are gradually replacing standard wild type animals.
[45:47]And each animal user and its related institutions, we all own a responsibility uh towards the management of animals involved in our activities. And at Charles River, uh we have a long and proud history of investing and embracing the companiments of the three Rs.
[46:11]And we are trying our best to demonstrate responsibility with regards to the management of our genetically engineered rodent colonies. And during this webinar, I have been giving you many examples of our embryology and transgenic technologies, but also digital tools can lead to dramatic improvements of the three Rs.
[46:53]Performance, and we are developing we are optimizing those technologies all the time and trying to introduce new technologies to support the three Rs, but it's also clear that from the selected examples that those innovative strategies and tools, while they are supporting the three Rs efficiently, they are also strongly participating to securing research projects by and also reducing delays, they are reducing staff workload and they are reducing internal cost. So when you are working for the three Rs, you are also working to improve all the other parameters of your activity.
[47:39]And we thus do believe that managing effectively genetically engineered animals and and more generally animal resources through the length of the four Rs is the right thing to do. Thank you for your attention.
[48:07]You sure. A very interesting talk. Um I would just like give a little comment. I like the mouse picture on slide one. Um very nice. However you did that.
[48:21]Um we do have uh one specific question coming in from the audience um and I would just forward it. The question is about if you see any change in the fertility um I of transgenic lights after rederivation.
[48:37]No. We do not see uh this. The only thing you normally you should even depending on the type of rederivation you are performing. There are many cases and everything is based on the genetics of your mouse model.
[49:02]And you should be fully aware of the genetics you starting from. meaning if you are using only males for rederivation, you usually usually take osite from wild type female for your rederivation.
[49:13]And doing that, you are most of the time eliminating spontaneous mutation from genetic drift. So it can only improve the fertility of your of your mouse model. But if you do that and if you do not select carefully the genetic background of the of the females producing the oocytes, you can also induce a kind of genetic mix in your model and maybe decrease uh um the uh the fertility of your uh of your model after rederivation. So you need you need to be very careful uh on the type of animals and genetic background of animals you are using for rederivation.
[50:02]But if you use males and females, there's no there's no way you can see any decrease in fertility especially when you remove pathogens. Most of the time the pathogens have more tendency to uh uh decrease fertility than to uh improve uh fertility.
[50:26]Okay. Um so when I was looking There is an audience the only question so far. It was just a comment that it was great, very interesting with the swab genotyping. Um so Orly is raising his hand. Orly please speak up.
[50:42]Yeah, thank you for very interesting uh presentation. I have a question about the the IVF the IVF to produce embryos. Do you see differences or you tested between inbred, outbred or hybrid lines?
[50:54]In efficiency, you mean? Yeah, for with respect to the IVF procedure instead of breeding. Very interesting, thank you.
[51:00]I bread and all bread are more efficient than inbred for sure, more robust. no problem with that. Uh but now we we and as we are doing in routine, we are doing IVF every day.
[51:15]At least four days a week. Uh so we have great uh very well trained technicians and everything is also about training and expertise and experience and and doing it uh on a regular manner.
[51:33]Uh we do obtain very good fertilization rates with uh common inbred strains, I would say uh C57 Black Six, Bold Sea, 129, there's no no issue with that. But on average, uh hybrid and outbred uh provides with better results for sure.
[51:51]Very interesting, thank you. Thank you very much, Jean for your presentation.
[52:03]And uh and yes to to follow up with Oris's question. I would say also between inbred strains, we we find uh differences like uh Bob's sea. Not very nice results compared to a black six, for example. Maybe you have or also an FVB good results, I would say. And um for Jan, I would like to ask you, so if I understood correctly, it is feasible to perform uh genotyping in a sample in a sample of sperm cryopreserved sperm, right?
[52:42]That is correct. Yeah. Okay. And um would it be feasible to perform also a Snip test?
[52:50]We have not tried. Um it's just a question of getting the right amount of DNA, I guess. We have not tried but this is something which can be tested. Yeah, why not?
[53:03]Uh because we would like to submit a sample. So Absolutely. Uh it's just a question of uh processing the sperm in the right way for the PCR.
[53:18]So you can maybe ask uh Yes, yes, we are already in contact. Uh so if he can adapt his uh procedure because I know in some companies everything is automated and not very flexible.
[53:31]Uh but I don't see any to be honest, any block uh for doing this.
[53:38]Thank you.
