The Dilemma of individual versus social housing of mice

Ajouté : 2026-06-03

[00:00:00]okay so uh good afternoon everybody welcome to this webinar um today uh i just want to make two points we will record this session for later viewing and if you have to leave early or you want to show it to somebody else then we will record it it will become available on our website and the other point that i want to make is that in the bar below there is q a session and there you can type any question that comes up during the webinar we will try to answer as many in the end depending of course of how many questions we get if there are too many to answer then we'll send an email anyway where we answer all the questions that came up during the during the webinar so today we have our speaker stefan from heininger stefan gradually graduated as a master in biology at the university of groningen he followed that up with a phd in behavioral neuroscience also at the university of groningen and his thesis is titled the driving force of metabolic programming and he will be submitting his thesis this month and he will probably do his defense later this year beginning of next year uh currently he is now working for knowledge as one of our marketing communication specialist and during this webinar he will talk about the individual versus social housing of mice and how the choice between these housing conditions can greatly impact your research so um stefan the floor is yours thank you very much renko for this for this nice introduction so good afternoon or good morning for the ones joining in from a u.s time zone um so indeed today i'm going to talk about this this dilemma of individually or socially housing mice because i came across this this problem actually while doing my my phd research so um to talk a little bit about who am i stephen from heidegger frank already gave a short introduction so i studied at the university of groningen and i'm submitting my thesis the driving force of metabolic programming so this title does not entail anything about housing conditions but i assure you that this was a very important part of finding um the appropriate models to work with so in 2010 i started at the university of cloning until 2015 as a as a bachelor and then master students and then from 2015 onwards i started working together with nutritional research in the netherlands working together on a phd project about about metabolic programming and then in july i um i started at naldis working as a as a marketing communications specialist so contributing to a lot of the neuroscience content that we communicate so the driving forces of metabolic programming we all know that obesity is a is a global epidemic so there's a lot of research about it it has a lot of media coverage this is not a new thing what we try to investigate is is obesity preventable is there a reason for obesity predisposition and how could we be able to prevent this for example early life nutritional interventions this is based on environmental factors and of which the first environmental factors actually take place in utero so these conditions in utero are obviously influenced by the health of of the mother of the diet of the mother and you can imagine that the brain and the body are developed in utero thus we need to provide the most optimal building blocks for a healthy development for later in life as well after birth we have nutrient transfer to breastfeeding and again the breastfeeding is also influenced by the diet of of the mother but obviously we also have a difference in bottle feeding versus breastfeeding after birth if we talk about nutritional quality and nutritional quantity so this environment during the nutritional environment the first thousand days of life has been adopted as well we have the term for this it's called early life nutritional programming and as i said this is purely about providing these these building blocks and using this early life as a window of opportunity to well to study this and this transition from early to adult life so um this was a bit of the the background on the topic of of my research but what i needed to do is i needed to search for an appropriate obesity model so this was one of the main goals investigate whether we can sort of model this obesity predisposition and then after it's prevented with dietary intervention so um we have a very clear obesity model of course which is the western style diet also known as sort of a mcdonald's diet if you use it in other terms but we also looked at individual housing as an obesity model and we incorporated a small little rear approach for this presentation obviously i'm going to focus on this individual housing paradigm and the problems that i for the problems the also the very interesting things that i ran into so mice as a model organism are obviously widely used in biomedical research because of their high similarity with with the human genome as expressed in this this this nice graphic uh you know easy to take care of you can breed them in large numbers relatively cheap and the most used strain in this case sometimes even called the wild type strings c57 black six mice mouse the very recognizable black black mouse we see we see a lot why did i specifically use this strain of mice and this is very important again for the background of my work if you talk about the early life nutritional programming so in metabolic studies in general it's a very established model they're very obviously prone and obviously we build up earlier studies that we've done in this in this context so during the first three weeks of life there is a post-natal leptin surge that takes place in these mice that play a role in the generation of hypothalamic feeding circuits so this is work that was done uh very important work by sebastian and you can see this in these in these graphics on the right here where on the on the top in these images of of graphic b these are wild type mice and you can see the formation of these of these fibers you can see in the in the in the hypothalamus with increasing h so p10 stands for postnatal day 10 horse nadal is 16 motion level 16 which is adult h and then on the bottom here we have the leptin deficient mice so the obobim is very popular model obesity model to use um so without this leptin surge we see that this yeah there's there's actually a lack in the formation of these of of these fibers which you can quantitatively see in these graphs below you can see the fiber density and then the wild types at the young age and then at p16 wild types versus the ob ob mice and then again v60 so it's this this postnatal lepton surge is extremely critical in the formation of these feeding circuits so these are protect projections from the argument piece to the hypothalamus which um which are formed in humans this does not occur um you can see elevated leptin levels at the end of the prenatal period which then declined quickly after birth what that gives us is basically a postnatal model in mice in which we can study this hypothalamic feeding circuitry which relates to a prenatal period in humans so it creates an enormously uh interesting window of opportunity for us to do interventions and to do research into these into these feeding circuits so this was obviously also one of the main reasons why we use this model in my research however for in metabolic studies we often run into the problem with c57 black six mice that we have to house them individually we want to know food intake we want to know water intake we want to know uh energy expanded you want to collect feces these are all things you know for practical purposes but also if we want to automate specific measurements which can be challenging if we use for example socially house mice to be able to track these individuals in the cage so metabolic studies often opted for the individual housing of mice um this can however influence the outcome past the point of being of it being a relevant model and and this is why i talk about this dilemma individual for social housing in this webinar because i was really doing obesity research but i saw that the model itself was really affecting um affecting the outcome so even in terms of intrinsic behavior and primarily the metabolic and physiological parameters so let's talk about those uh for a minute here basically social isolation is considered the stimulus for condition which can lead to boredom and which can release to an increase in reward sensitivity so these animals become extremely sensitive to any form of reward you can think of the western style diets that i mentioned beginning as well increasing food intake furthermore because of this that this deprivation of social cues uh for example social facilitation there's less heating competition the social status is completely different this changes the energy use because um these individual animals do not have to expand the energy into you know social behaviors interacting with the cage mates etc and then it also changes the energy availability because of the lack of the competition there is um in in you know obtaining food in just general feeding behavior additionally um it is already known also that sustained social deprivation may provoke chronic emotional stress affecting neuroendocrine immune axes which can also lead to alterations in body weight and fat deposition so this this model itself which we you know also try to use as from pc model does have its implications downstream and this was reviewed in this review by skipping it all it's a very complex figure so you definitely don't have to understand it all but the general message here is that because of this individual housing you're actually messing around with a lot of factors that influence this metabolic profile also down to a behavioral level at the end but this is an interesting review to also uh look at and and the the main author's hipper nidalee skipper she was my supervisor at nutrition research so i worked very closely with her on this project we translated this into our um into one of my first chapters which we of my thesis which were already published in plus one uh which is titled individual housing of male black six mice after reading in and predisposes for obesity where we're going to show what i just said before the theoretical things about it we're going to show some of the results so here um you can see this paper so at the top you have the title and the author so you can look this up for yourself uh graph a shows adolescent growth this is from postnatal day 21 to postnatally 42 this is directly after reading and at the point of weaning of postnatal 21 these animals are put from their nest situation into either isolated states or individual housing or social housing which is pairwise in this case the two mice in the cage and see that these social animals gain more weight towards postnatal a42 which we use as a cutoff point for uh adulthood we transition the animals onto an adult diet at that stage and we consider them as young adults basically so there's a definite uh the difference between these socially and in which we house mice in terms of body weight accumulation which we in this stage they'll call growth then if we switch over to adulthood potion day 42 to 126 you can see that we also expose them to a western style diet but irrespective of this diet these individual animals from this point on actually gain more weight so this graph shows body weight gain so earlier in life adolescence it embarrassed growth but later in life you can see that they actually try to well that's the hypothesis try to catch up and add more weight compared to these social animals then if we look into these graphs we show a bit of information on body composition so we have lean body mass we have fat mouse and we have femur length femur length is a very robust parameter for overall development overall lean mass development also for growth length of the mouse we can see that the fat mass and the top graphs here are adolescents and if we um look at the previous graph where we see that yes the social animals hear way more than individual animals their fat mass is significantly less so uh we haven't we have a big difference in body weight but the body composition is completely switched around in this case really you know leaning towards a obisogenic phenotype which is still present in adulthood and possibly driven by this femur length difference also higher in social animals compared to individual animals in adolescence and in adulthood irrespective of the diet that they have given that they have been given so um so this was obviously very interesting finding really proving that the body composition is is completely switched around in these two industry housing conditions we hypothesized in this in this slide you can see some data on on ucp1 we have prosthesized that there is an effect of thermogenesis um uct1 is uncoupling protein one and this is responsible for the heat generation in the brown adipose and this is considered as healthy fats babies have a lot of brown adipose tissue for example um so we thought these individual animals are also less capable of well keeping each other warm through you know hugging behavior and page mates so we did see some differences here but it tends to be a different pattern if we look at adolescents versus adult mice where in adolescent mice we indeed have a higher activity in the in the brown adipose tissue in individually housed animals so that would sort of confirm that theory but we don't see that in adulthood but then if we look at the activity in the white adipose tissue of uct1 which is also called browning of the white adipose tissue we see that the effect is largest there in adulthood where indeed these individual animals try to compensate a bit for that activity perhaps due to this lack of social thermal regulation [Music] so to summarize the main findings of this article uh individual housing impairs growth through adolescence but needs to increase body weight gain during adulthood regardless of diets leading to an obesity like phenotype possibly also due to the thermogenic strain because it's lack of this cage made based on the user b1 results we took these results and we went on with with our next study which is chapter three of my thesis where we use this model individual first social again um and then expose them to an early life diet where in this case we uh we did it got this this validation of this phenotype in terms of obesity but now we also added uh behavior to the equation um because we wanted to see is there behavior also changing because of this housing condition um we can see that in the top left we have a nice effect considering distance moved in an open field this is something we also know from literature that they get hyper that individually host animals get hyperactive in a in a normal arena so that's a nice confirmation but also if we look at an elevated plus maze where we study anxiety like behavior and you can see the adolescents adolescents individually housed animals have a higher open arm ratio as well as the adult animals which is interesting because we actually hypothesize that these individually housed animals because they are socially deprived would show higher stress levels but an open arm ratio on the elevated classmates indicates that they have lower anxiety like behavior so we were thinking about about how this would how this would come about why we were seeing this difference this different pattern and we took that a bit further and we looked into with my next chapter into how social housing actually affects specific behavioral um and behavioral outcomes so switching now focus a bit to the social housing condition and the reason why we did this is this center graph particularly is again the elevated plus maze but now we separated the socially housed animals into a group that was tested first and a group that was tested second and this is because we test the animals in a consecutive order so the results that we found before in socially housed animals we thought were actually influenced by the fact that they were separated from their cagemate mate so yes you can say that individual housing brings about uh probably behavioral consequences or because of the deprivation of social interaction however if you expose these social animals that are used to being housed together to a sudden environment where they are separated this can also induce stress and this was also affected by their by the hierarchical status within the cage so whether they were the dominant animal or the subordinate animal so we tested this by doing a tube test to see which one was done in the cage so so this was a very important finding for us as well which also led us to refine our methods of how we study or how we perform these behavioral tests in terms of habituation and also if you have social animals habituate them to a state first where they're used to being alone and then subject them to the testing arena itself um well yeah this is what i basically just explained so the main findings of these two chapters were the validation of the phenotype with individual housing we kept finding this phenotype so i won't show the graphs each time because we clearly see individually individual housing leads to increase body weight and fat mass and in terms of behavior the most important thing that you should take away from this is that you cannot compare individually housed mice versus socially host mice due to these large variations in behavioral response testing order affects this and the hierarchy in social cages and probably other factors as well so that is something that you should keep in mind by designing your study and thinking about how you actually when you're in the testing room how you perform the studies and to my surprise i was looking up some some literature about but it's really hard to find detailed articles of people looking into this so these are two examples that i highlight although they are in other strains of mice which i indicate on the slide i find it hard to really find um papers on on things like testing order and how this can obscure your results so but it's nonetheless extremely important to keep considering especially if we look at social mice mice are our social species they you know they state that they should be group housed as long as the groups are stable of course because it is vital to the welfare because multiple you know benefits which in an evolutionary level benefits to the survival of the individual lowering predation predation risk and reducing energy costs via the social thermal regulation which we talked about before and this is so so you know people in in terms of welfare leading towards the social housing but but this is where the dilemma that's where my title of my presentation comes into play a bit so but if you look at the social guidelines uh based um um this is the guide for the care of laboratory animals the national research council they state that uh housing should be based on natural living conditions of species so um and also that this home cage should be an environment that provides for the animals physiological ecological needs again going back to that statement that i made about being a gregarious species so they would better cope with stressful procedures and your experimental results will be more reliable but we run into this dilemma and this is very nicely explained in this article actually that i found to group or not to group and this paper basically states a few things here that they do focus on male mice i have tested both sexes in my experiments although i do not show data on this um but male mice naturally do not share they do live in groups however they are very territorial and aggression towards each other can be a serious problem um which affects the welfare of these animals um and and this can even more negatively impact your animals in terms of stress if you look at social defeat for example social defect it's an extremely stressful model and this can happen in a hometown situation if you have multiple male mice that are you know aggressing towards each other and there's one consecutive mouse or more circuit mouse that keeps keeps losing in the social defeat paradigm so but they also state this paper individual housing may not be an appropriate solution giving the welfare implications associated with no social contacts um this is exactly why i find this presentation the dilemma of individual for social housing what's what what should walk for them what is then the most uh optimal solution because there are a lot of factors that influence behavior we have handling for example tail or tube handling which can which can affect behavior so i tested both of these methods in my research as well to see if they indeed get less stressful if you if you handle them with a with a tunnel or a tube habituation is an important thing that we talked about also considering separation from the cage mate that we can also choose to separate these social animals before subjecting them to a test which they have to perform on an individual level even the researcher itself can influence this vector so this is always a topic that keeps coming back try to control the variables try to make it a level playing field as much as possible um if we focus a bit more on the separation from encagement because this was something that was very interesting for me to investigate we know that social isolation induces like behavior in terms of individual housing how i did it i did it for long term so are these animals better coping with this isolation compared to the always socially housed animals this could be the case if you look at this study however they saw that they have an isolated group which is the so the first one is a control group and the third bar is an isolate group they check this over a longer period of time so they indeed see so if we look at time in the center zone of an open field that also long-term isolation can lead to increased anxiety like behavior furthermore on a neural level we see that it can um not only induce inside like behavior but decrease bdnf levels as well um greatly affecting the plasticity in these animals thus also if we you know take apart the the metabolic side of the story it could also affect cognition now what's my answer on should we house mice individually or socially because the discussion goes from from one point to another and i've also noticed that when giving similar presentations um yes social housing is more naturally relevant and yes individual housing does cause the presence like phenotype leading to body weight and increase social housing increases some logistical challenges in terms of identification of the mouse we have a hierarchy we have testing order effects for which you then all have to try and control for with habituation methods um however yeah individual housing is inevitable in some cases uh think about can you cannulas implants uh we talked about fighting behavior and also metabolic ages um in which we want to know uh the exact energy expenditure of one mouse instead of you know an entire cage my approach to this is actually pragmatism pragmatism is key in in choosing your and choosing your preferred way of housing because that's what it comes down to there is no there's no perfect way but it should be tailored to what you need for your research so use concepts from other studies or previous study center studies and um and learn from um from those previous experience like i showed you in this presentation and make sure that then you take those things into account when going for your model of choice and also a very point consider what you actually need from your data for example food intake i've thought about this point for long for a long time in my studies i wanted to know food intake quantity of my animals but was it um really of such an added value that i needed it from individual mice or could i just take the cages as a statistical unit and the latter is exactly what i did in my previous study and i was also privileged to be able to use a high-end because of how i designed my studies so i didn't have any any large interventions or anything it was only dietary intervention so i could you know have a high high number of animals so i could still retain the statistical power when only opting for using cages statistical units um but still it's always good to to stand still with what do i actually need is is this you know can i um in some different form of analyzing this or still by grouping this data get the information that i'm actually looking for to be able to accurately test my hypothesis um and then you know considering all these other effects like testing artifacts counter balancing habituating testing during rest and active phase make sure you you check everything beforehand that everything is is the same and no errors can be made so i had a lot of students working um working with me a lot of interns on these projects and things like rests and active phase testing was a constant discussion because of the times at which they wanted to they wanted to work or do the do the project and it was my job to really keep in check like okay we have to do this in a very similar way and also not doing too many animals on one day that the first animals tested at the beginning of the day and the last animals so but these are all things that you've probably heard about but it accumulates it accumulates all these factors that you have to take into account so it's always good to make a list so how can all this help actually in this in this whole topic and we're talking about this entire topic of my of my of my webinar here um and i get the question a lot and obviously my pg defense coming up if i would do it again what would i change what would it do differently or what would i do in the future and um [Music] i would set up my research for continuous monitoring with no spinal diaper the reason for this is that i really miss a full metabolic behavioral and activity profile on these effects of social versus individual housing um these these things like humming behavior social behavior but that's primarily eating drinking and activity patterns is something that i just need to increase my resolution on i have these small snippets within their developments which simply do not give me enough insight in how these metabolic profiles are being built up if we look at an illustration of this this is how i depict the entire timeline of the development of one of my mice for example and the snippets what i'm talking about is for example here we have some behavioral tests and here i measure for a few days uh energy expenditure for example in an indirect calorimetry setup but but with using knowledge phenotyper i could actually really that's what i mean with increasing the resolution really increase these um well these these time frames in which i measure things so gathering a large amount of information actually from a very powerful very powerful setup so that would be my answer to if i would do this again how would i approach this i've come to the end of my part so for any questions you can always email me you can also contact me at this email address stefan.fan hiding at moulds.now and i would like to thank you for for joining in and i would be happy to answer some of your questions so i give referral back to you yeah so thank you stefan for this excellent webinar um we have a few questions that came in and one of them is also in line with the question that i wanted to ask but this one is from uh margarita sorry if i pronounced it wrong so what she wants to know is please indicate the group size which is crucial for interpreting the graphs in the first part of your talk and yes my question which is a little bit related to that is um [Music] like when is something considered the social housing is that two animals is that 10 animals or [Music] so yeah the term the term that i use social housing in this case could be a bit misleading because i try to keep it simple in my case because i wanted to test a condition of one mouse versus two mice in the cage because if you add even more um if we talk about you know high rocker structures it gets even more complex so um it's really a terminology thing um i should it's more appropriate to call it pairwise housing in my case um but you consider it social housing in in my case more than more than one miles per gauge okay and the second question is also from the same person and that is uh in the first part of your talk what was the gender of the animals um and if you have any notes on the susceptibility of female mice in social isolation yeah so the gender i talk about i talk about male mice in my results in this case primarily and this is because in the first few experiments we only applied for the mice and then in my final experiment i i wanted to look at the sex differences as well but i didn't include these behavioral profiles in my my thesis because i focus on the metabolism so any note so so all the results you saw were up real nice but any notes on female mice is what is interesting is that they do show um a different response to the social isolation basically the obesity like phenotype that you see in these male individually housed mice the females are less susceptible to this and i i don't have enough information to explain as to as to why and i know that also the social structures and socially house females are also different but they do not gain as much body weight and fat when individually housed from weaning as the males do so um if they are less susceptible to stress i am that that i do not have the answer to but they do definitely do have different feeding time okay and uh there's another question from julia odermatt did you observe a difference in the behavior of the mice when they were handled by the female or the male researcher oh yeah that would be a very interesting thing to um to look at actually we did have uh different gender researchers but yeah so this is something that we have quantified basically so this is exactly what i talk about when i'm increasing resolution and this is not only done with phenotype of course but um all these things that i talk about like how you handle your mice and and how you control for specific things are often overlooked so i think we can gain a lot of information from uh well exactly your question do these mice respond differently so um yeah that would be that would be my answer it's a very nice suggestion but it's a lot of work you know quantifying this i sometimes i you know i work with large groups a lot of animals so logistics is obviously an issue there okay and then we have another question from stephanie marcea can a reversed cycle also affect society um i guess with the reverse cycle we're talking about the light dark cycle yeah it's not uh mentioned like that so maybe we can answer that via email that might be a better way just to get the understanding of what exactly is meant is it for a reverse cycle for the animal for the researcher um yeah i i think i i suspect that it's about a reversed uh light dark cycle and it can definitely affect anxiety but this is this is also why i tested the mice in a in a light condition because if i i have bad experience with testing in a dark condition with with entering the room for example if we look at anxiety tests there's a high chance of sort of polluting your testing area with the light distribution and i'm not a fan of working on the red light conditions because you know it's difficult to see but uh yeah the opinions are are are really spread out about about that what is the best way to test in light or dark so that's rest for active phase for the mice respectively so um yeah but i didn't test that okay and then we have a last question which came in by the chat so i would like to ask mark to also put it in q a so it's safe after the meeting i cannot do it for you and his question is shouldn't the first and second tested buyers disappear with handling the mice giving you handle them one after each other um [Music] yeah the handling is really important here um and i think the differences here is not that we handle them differently but it's the time that they have to wait in in sort of their home cage before their cage bait is done with the test and i think this separation induces a stress in both the first and the second tested mouse of course um because you know one of your cage mates just just disappeared so you can imagine that this could be a stressful situation for for these animals in terms of handling could this be avoided uh that's our question by sort of control handling these mice uh that is that is not exactly what we did in our last experiment but we tried to approach that by first separating them into a different gauge it did have home cage betting and then afterwards subjected them to the test and we did see well we actually didn't see this testing order effect anymore so um but we didn't have a control condition for that considering like [Music] that we do a habituation i got don't do a habituation i just incorporated it in my design altogether okay and then we have a very nice question from david and uh he's not familiar with the plastic cup handling so if you could explain a little bit more how it works and um yeah so um that's a very good question and it's actually very relevant topic so it's not a plastic cup it's more of a tunnel or a tube which i place in the cage as cage enrichment actually so extra cage enrichments aside from any housing or bedding material and the my get very used to to this tube also because it's in the home cage obviously but the the tail suspension or picking up by the tail is actually considered stressful for the mice as well you know all their weight is hanging on this point they can handle it um but in in practice you you know if you even if you miss grab or or you know if the mice runs away you try to grab it really quickly it can induce it can do stress so um what i did is i use this tube and i just when it's in the cage when i go weigh them or something i just grab the tube and they have to get used to this but after a while they just walk into this tube you carry it over to your your weighing station for example they walk out of the tube again you weigh them and then you know the same thing in reverse um so you can find it under research or if you google for or pubmed for you know tunnel handling or tube handling of mice so there's a lot of research popping up on how this benefits the stress levels or anxiety levels of your of your mice so it's definitely worth to check that out if you look if you're looking at refining the handling of your mice okay so i just want to stress that all the questions that are being answered we will answer them later on in an email to everybody but i think we have a little bit more time to do just one or two extra if that's okay with you uh okay so russia has a question um also concerning the light dark cycle did you use a reverse light dark cycle or just normal one just a normal like dark cycle so during well our light hours it was also light in the rooms and this was purely for logistics because we find it very difficult to keep working in these dark conditions since we also had a lot of mice to take care of and a lot of uh handling to do so um this is a discussion that always pops up in our department as well like do you do the testing in the dark phase or do you have a reverse light dark cycle um yeah what i said because of really logistical reasons we opted for normal light dark cycles starting at around the gate in the morning the lights went on okay so uh there's another question now from uh jeffrey alexander gimenez um and it's regarding uh the anxiety levels do you think the anxiety level uh we find in mice that are singly housed equivalent to the level of anxiety that we find in a submissive mouse that is paired with a dominant mass so if the anxiety level of a of an individual mouse you mean is equivalent to a subordinate mouse yeah um [Music] i wouldn't know actually i could go back to one of my slides about this um where i uh where i looked at these differences and we can try to find an answer on this so um what i found in general is that socially housed mice showed higher insights like behavior because of a lower open arm ratio and um what i saw is that this only gets if we include this testing order it only gets stronger if you add this hierarchy factor so in subordinate animals so i would guess that subordinate animals are more susceptible to this separation anxiety separation from cage mates and um where in my first experiment in individually housed animals we saw a lower anxiety like with a lower anxiety like behavior i think is really um related to this to the separation and um so i i think yeah i'm not sure if they compare if you look at these levels they do seem to be around the same same level here but here we didn't find this individual for sparewise difference so that was really the previous experiment so that's the only thing i could say about it for now that i do think there's a higher vulnerability to support us to experience a structure like this so thank you very much for the people who attended i just want to stress that we recorded this webinar and if you want to look at a certain slide again or the entire presentation then you can go to the website in a few days and there you can see the entire webinar again for the people who answered questions and for everybody attending we will send them like a more detailed answer in the coming days also so i would like to thank everybody for attending and especially steven for presenting today and then that's the end of the webinar thank you all for attending and for the nice questions thank you arenko for hosting no problem