good morning good afternoon good evening we are starting uh the episode number four of our mycotoxin info webinars and today there will be a presentation of the topic of mycotoxins and oxidative stress how they are influencing oxidative stress and which microtoxins are the most of the concern uh of course microtoxins causing oxidative stress and other things there will be a performance bottleneck today a presentation will be done by dr peters rai um i introduce myself because i am like a holder of of this event myself olga averkeva i am a phd in animal nutrition and taking a role of global category manager for microtoxin management in adcl in belgium our main speaker today will be peter surai dr peter surai started his career as a biochemist in ukraine and in 1993 he became professor of human physiology there in 1996 he moved to scotland and in 2000 he became a full professor of nutritional biochemistry at the scottish agricultural college since then he also had become a honorary professor at various universities in the uk hungary bulgaria ukraine and russia he has more than 800 research publications devoted to antioxidant he's really an expert in antioxidants for the last 20 years he has become lecturing all over the world visiting more than 70 countries around the globe his books devoted to anti-oxidants and become text books for many industry specialists and are translated in different languages during a presentation of peter if you find the interesting topic you can put your question in the chat area and these questions will be addressed to peter after the presentation we foresee around 15 minutes for the questions and answers session but don't worry if your question was not asked during this micro info webinar we will follow it up so we will answer this question later on uh during the question session uh my colleague julia dvorska will also help um to manage the question streams sometimes we receive a lot of questions and we try to pick up the most uh relevant or the most difficult ones that peter is able to answer them if you uh could not join or if you had some connection issues um you can always join our replay that you can find more information uh in the internet later on or we can send you a link for replay and will be individual link if you have issue with connection during the webinar you always can find our support and margot is here at your disposal to support you in terms of difficulties in connections so just to remind that we have five episodes and three of them they have done already and the fifth episode is the last one it's uh scheduled for may 19 and will be devoted to by an activation of mycotoxis by host organism by host cells and host microbiota enjoy your talk and please don't forget to to pull your questions for later on good morning good afternoon good evening ladies and gentlemen it's a great pleasure to me to join this webinar and talk today by my favorite subject mycotoxins and oxidative stress i have been working with my antioxidants for the last 40 years and about 20 years were also associated with mycotoxins and today it's a good opportunity to share with you a recent finding so usually i start with this slide just for you to understand that we are living in this dangerous world of free radicals and those radicals are produced in nature and we need some kind of umbrella which we called antioxidant protection we means our chicken our pigs our cows everybody need this kind of umbrella when we're talking about oxidative stress we need to talk about free radicals what are free radicals they are activated molecules of oxygens which actually can damage all types of biological molecules and there are internal and external sources of free radicals in biological systems and among internal sources of mitochondria are considered to be major source of free radicals mitochondria which are responsible for energy production but energy production never been hundred percent efficient and up to three percent of oxygen can escape from electron transport chain and they become free radicals there are also phagocyte cells in the body which are producing free radicals as a weapon to kill pathogens for them free radical production is a way to deal with pathogens there are also some other processes associated with free radical production as well as external sources of free radical production and if you look at this slide what i would like to show you here that we have antioxidant defense system in the body which is dealing with these reactive oxygen species or free radicals and here you can also see microtoxins as a one major source exogenous source of promotional free radical production and what also here important to mention that the physiological level of these free radicals are important for immune defense for cell signaling and many other functions in the body however the overproduction of free radicals it's a damaging effect and at this slide i would like to underline once more that free radicals damage lipids they damage dna they damage proteins in fact free radicals can damage all types of biological molecules and what is important to remember that the oxidative stress as a result of this balance between antioxidant protection and free radical production is a major cause or major molecular mechanisms or major stresses in poetry and animal uh production and if you look at this slide i just summarized main stresses in quality production including environmental stresses technological stresses nutritional stresses internal stresses and here mycotoxins are considered to be major nutritional stresses which are causing a lot of problems and you already participated in three previous meetings related to mycotoxins where the negative consequences of mycotoxin contamination have been explained so when we're talking about mycotoxins we are talking about silent killers invisible thieves unavoidable contaminants natural toxicons all these names have been given to a group of fungal metabolites called mycotoxins and their negative effect on animal production are incalculable and if we look at this slide this is the most recent one 2020 which summarize the research in the area of mycotoxins you can see here that 22 percent of research related to aflatoxin 18 to dawn 16 percent zero unknown 15 percent if uh okra toxins and combined mycotoxins studies are related to 20 of total research and then here you can also see that the research related to mycotoxins in chicken in total comprise about 14 and take 23 percent so almost 40 percent of all research related to mycotoxins is associated with their effect on pigs and chicken and as i mentioned already that mycotoxins have decremental effect on poultry on pigs on cows on any farm animals and the major mycotoxins of concerns include aflatoxin occur toxin for monogenes t2 and dawn and zeralenon and today the major point of the presentation is to consider more in detail molecular mechanisms of microtoxin action with a specific references to oxidative stress and this paper came 2008 so it's already 13 years old at that time we suggested that actually microtoxins are in the world in creations oxidative stress and the inhibition of protein synthesis dna and rna synthesis and damages to dna this related to organ toxin tetrox toxin aflatoxin and other mycotoxins most of microtoxins impose lipid peroxidation and structure membrane damages then we have also induction of the program of cell death which is called apoptosis practically all mycotoxins are involved then we're talking about transcription factors and gene expression regulation where mycotoxins are involved and finally maintain our disturbances of redox balance in the gut and redux balance in cells are considered as a major consequences of mycotoxin toxicity so as a result we have immunosuppressant hepatotoxicity nephrotoxicity neurotoxicity genotoxicity and uh in this case we are talking about increased mortality poor feed conversion ratio poor growth rate feed refusal decrease fertility and hatch ability more recent paper 2018 confirms the same this is a review paper which again showing the tough latoxin for money xenochronotoxin xeralinone dawn nivalinol t2 toxin they impose oxidative stress they affect mitochondria the major source of free radical production they impose lipid peroxidation they impose apoptosis and they also also affect or stimulate inflammatory response therefore the preoccident properties of microtoxins are well established and this most recent review which is uh came uh this year actually uh showing that the major microtoxins including aflatox and ochre toxins and zeralinone fumanizing t2 ht2 and diva and dawn they are produced uh by uh various fungus and we are talking about aspergillus penicillium and fusarium as three major genera in the world in microtoxin production and you can see here in various food and feed materials those mycotoxins can be found and more specifically if we look at the molecular mechanism of action which was characterized in this most recent paper uh you can see that the major research in protein were done with broilers aflatoxin ochre toxin ht2 and t2 toxin were started and here you can see different doses of mycotoxins were used as an oxidative stress in this indications the melon dilde height final product of liquid peroxidation was used as well as a concentration of glutathione major antioxidants and total antioxidant potential and what they showed very clearly that most antioxidant enzyme activity were decreased due to mycotoxin contamination this means that the oxidative stress was imposed if we look at the same paper on the peak side again you can see here dawn zeralenona flatoxin for monizing they also were used in different doses similar oxidative stress parameters were started including mda glutathione total antioxidant activity and they were compromised and most importantly again antioxidant activity or or an antioxidant enzyme activity we're down like catalase superoxide dismutase glutathione peroxidase these are enzymes which are responsible for antioxidant protection and decreased their activity is related to oxidative stress and here is also interesting that for inflammatory cytokines are increased and what about ruminants yes look here goats sheep lamb cows again the same mycotoxins were tested various levels of mycotoxins and similar to monogastic animals again melon dilde height goes up total antioxidant activity goes down glutathione is a major cellular antioxidant goes down and similar to previous studies their antioxidant enzyme activities goes down so what is the conclusion the conclusion is that for all animal species including poetry pigs and ruminant animals mycotoxins are the problem mycotoxins imposed oxidative stress and this oxidative stress is mainly related to disturbances of antioxidant enzyme activity and this paper which came 2018 is very interesting in a way they look at the effect of mycotoxin sources on the mitochondria as i mentioned that mitochondria are major source of free radical production and if the mycotoxins can actually compromise function of mitochondria more free radicals will be produced and here again you can see aflatoxin dawn for monozine ochre toxin t2 toxin zeralenone and they actually were tested in various modal systems and it was very clearly showed mitochondrial dysfunction due to mycotoxin contamination so this is a one of the possible mechanisms of toxic effect on mycotoxins affecting mitochondria by affecting mitochondria free radical production is increased antioxidant defenses are compromised and we are talking about oxidative stress and most recent papers actually have more and more attention paid to two transcription factors these are specific factors which activate genes so one transcription factor called nrf2 this transcription factor is responsive for synthesis or activation of synthesis more than 200 of different molecules most of them are antioxidants and aflatoxin dawn or toxins if they affect the expression of this transcription fact and this effect is those and exposure dependent in the ways that low doses and short exposure usually activate nrf2 which means at the beginning the body can try to protect itself from a toxic effect of mitochondria microtoxins while higher doses and longer exposure inhibited and they have to expression and another transcription factor called nf kappa b and this factor is very important because it is responsive for synthesis of pro-inflammatory cytokines and pro-inflammatory cytokines impose inflammation in their gut in other tissues and you can see here data on on the effect of dawn and other mycotoxins zeroly known on the expression of this nf kappa b look at this paper in particular it's 2010 paper and what is shown here that an f kappa b expression increased almost six times uh uh so in this case dawn can substantially increase the expression of this transcription factor more pre-inflammatory cytokines are synthesized and this explains why dawn is so damaging on the god now effect of mycotoxins on an f kappa b expression is condition dependent depending on type of microtoxin those duration of exposure they can activate or inhibit an f kappa b and most importantly there is a balance between these two transcription factors and this balance is very much determine redox balance in the cell therefore what are consequences of redox balance disturbances by microtoxins i mentioned already lipid and protein oxidation by the way more and more attention today is paid to protein oxidation because lipid oxidation that something described many years ago but in recent years more attention is paid to protein oxidation as a major cause of negative effect of mycotoxins apoptosis which is a program cell there's leaky gut and microbiota disturbances immunosuppressions damages to healthy tissues including liver decreased productive and reproductive performance and this a few papers just to confirm the negative effect of mycotoxins on apoptosis so mycotoxins in including fumanizines t2 toxin and other fusarium microtoxins they easily impose a program cell theirs which is called apoptosis and here one of the paper and you can see here the tito toxin increased the activity of caspase-2 this is a specific enzyme responsible for activation of apoptosis you can see increase in four times eight times 12 times so the activation of apoptosis is very high one more point and again one of the recent paper considering compromise intestinal barrier induced by microtoxins so microtoxins can actually compromise microbiota in the gut compromise chemical barrier physical barrier and immunological body in the gut and that's why microtoxins very often are associated with various diseases because the protective effect of the god is compromised so when we're talking about healthy gut we are talking about digestion and absorption protective barrier immunity and balance microbiota you can see here normally lies which are responsible for absorption of nutrients and these are damaged relies which could be due to mycotoxin do not expect good absorption and good conversion feed conversion ratio in this case so free radical since they got produced by mitochondria phagocytes and various enzymes and today the actual topic redux balance is a god is a very fashionable one you can see plenty of reviews in a recent couple of years and redox balance and microbiota another topic helping to explain how mycotoxin can affect the health in particular mycotoxin can compromise microbiota and in general terms if we look here the syndrome of malabsorption that something which is uh considered to be a common for aflatoxic causes t2 toxic causes or toxic causes and this is a syndrome malabsorption is a result of damages to their god so what we have here we have mycotoxins in the feed stimulation of lipid and protein oxidation intestine oxidative stress and damages to parasites nutrient absorption decrease antioxidant system is compromised at the same time mycotoxins are absorbed delivered to target tissues and metabolized and oxidative stress is imposed so we have detrimental consequences for health and generally speaking antioxidants actually can help to deal with this problem and they can help with gut protection with liver protection and immunity maintenance therefore the whole idea is that previous first part of my presentation proves the point that micro toxins in the diet can actually impose oxidative stress which means there is a disbalance with a risk between a free radical production and antioxidant differences on one side mycotoxins increase free radical production for example by damaging mitochondria on the other side they decrease antioxidant differences due to decreased activity when torques and enzymes and this is related to few transcription factors including nrf2 which is responsive for synthesis more than 200 of different antioxidants and an f kappa b the specific uh transcription factor responsible for inflammation and let's now on the second part of research attention let's look at nutritional antioxidants and protect as protective strategy against microtoxins we need to remember that mycotoxins are very damaging even if you use some microtoxin binders you are never be able to bind hundred percent of mycotoxins those who which are escaping binding they are coming to the liver to be detoxified and the detoxification in the liver causing oxidative stress and damages to the liver so we need liver protection on the other side dawn is detoxified in the gut and gut microbiota also can be uh damaged due to mycotoxin contamination and we need to maintain healthy god so when we're talking about antioxidant protection we need to remember that all antioxidants in the body are working together as a team we call vitamin e headquarter one two oxen defense carotenoids communicate in services of antioxidant defense flavonoids and polyphenolic antioxidant police we are talking about vitamin c and special forces selenium chief executive antioxidant defense and here we are talking about ministry of defense which is gene expression and synthesis of various antioxidant enzymes so when we're talking about nutritional modulation of antioxidant system we can consider selenium vitamin d vitamin c polyphenolics and other nutrients which actually can affect antioxidant systems and by improving antioxidant defenses they can help to deal with with mycotoxin contamination so i will start from selenium because it's my favorite subject and i have been working with selenium for the last 20 years today we know that selenium is a part of cell of 25 celino proteins those celino proteins are synthesized in the body in response to stress and at this slide you can clearly see that more than half so from those 25 celina proteins are involved in uh uh in their antioxidant defense and signaling and now we are talking about dietary selenium which can affect antioxidant defenses it's our review in 2019 and in this case we are talking about breeder egg embryo deodorant postnatal cheeks they are protected due to dietary suppleme selenium supplementation if we look at the balance between an f kappa b and nrf2 in the gut you can see that mycotoxins pathogens and various stresses can impose oxidative stress and dysbiosis and in this case an f kappa b and nrf2 can be affected in this case you can see the nf kappa b is increased so the synthesis of brain inflammatory cytokinesis increased and the nf nrf2 is decreased which means that antioxidant defenses are decreased so if we look further we will be looking at inflammation apoptosis immunosuppression gut structure damages now what's happened if we have optimal selenium status so optimal selenium status will help salino protein expression so there will be 25 celino proteins synthesized then we have redox balance regulation uh improvement of antioxidant protection and this antioxidant protection will affect or prevent this oxidative stress imposed by microtoxins then we have effect on uh prevention of activation nf kappa b which means prevention of uh inflammation and improvement of antioxidant defenses and we also have maintenance of commensural microbiota as a result we have activation of nrf2 activation of vita genes and decreased activity on f copper b and finally maintaining redox balance gut structure immunocompetence and gut health so here this selenium is taken as a just as an example so in general terms the antioxidants in the god can help can help to maintain these transcription factors in optimal activity can help to maintain integrity of the god and this will help with first antioxidant defenses and secondly prevent the actually penetration of pathogens through the god and here the review of 2018 and using this review we can show how the system works so we have oxidative stress imposed by mycotoxins the disruption of epithelial barrier is taking place bacteria penetrates this barrier as a result the dendritic cells and microphages will be activated microphages started to produce reactive oxygen oxygen species which free radicals and they disturb redox balance as a result we you can see mitochondrial dysfunction which means that free radical production will be increased even more and what we have finally we have leaky gut leaky gut means that the barrier is disrupted and the situation is very difficult and in this case we can talk for example about syndrome or malabsorption so on the right side look what selenium can do selenium can affect the prostaglandin synthesis and activation of various transcription factors which actually prevent activation of this nf capability which is responsive for inflammation so the pro pro-inflammatory cytokines goes down anti-inflammatory goes up we are talking about additional synthesis of selena proteins we are talking about additional synthesis of antioxidant enzymes like superoxide dismutase and catalase and what is happening having here we are talking about redox balance reestablishment and inflammation resolution so this means that inflammation is taken under the control and in this case we are talking about maintenance of the gut structure and antioxidant defenses in the gut will keep healthy god and prevent those damages caused by mycotoxins so now when we're talking about selenium we need to remember that there are different sources of selenium and there is an inorganic selenium which is its first generation of supplements which came in many years ago and in this case the this sodium selenite is used in many commercial premises but the efficiency is low uh second generation of supplements include serena yeast and pure selena methionine and they have some problems like selena methionine consent proportion in selena yeast only 60 70 percent and we have also some problem with stability issues pure selena methane so finally the such generation of supplements came on their market which is hydroxy selena methionine helping solving selenium problems and helping to maintain optimal optimal antioxidant defenses and this can help with mycotoxins okay that was a selenium however the most important antioxidant and most study antioxidant in biological systems is vitamin e so vitamin e is antioxidant which is located in cellular membranes and vitamin e is preventing damages to those membranes and if you can look at the our review 2019 you can see that vitamin e has protective effect for breeders and cockrells for semen for egg yolk for embryo and for newly hatched chick and you can see here vitamin e increase antioxidant defenses not only by increasing vitamin e itself but also by activating superoxidase materials glutathione peroxidase and catalase you remember from my previous slide the disease enzyme activity was actually decreased due to mycotoxin contamination so in this case by using vitamin e we can reestablish antioxidant defenses in stress conditions do we have any evidence related to microtoxins yes if you look at this publication 2008 it's a effect of t2 toxin and dawn on dna damages so you can see here dawn and t2 toxin impose dna damages and by adding vitamin e you can decrease those damages and if we look more specifically in the review paper 2018 which actually look at the protective effect of vitamin e again is mycotoxins we are talking about alpha toxin dawn humanizing or heratops and t2 toxins zeralinone in all those questions cases of vitamin a has a protective effect again protecting against oxidative stress protective against membrane damages against are actually this activation of antioxidant enzymes what about vitamin c another antioxidant again you can see in the same reviews they showed that aflatoxin dawn occurred tito toxin and xeralinone they also they are negative consequences also can be mitigated by using a vitamin c and again you can see increase in glutathione concentration glutathione antioxidants and superoxide dismutase activity those enzymes which are inactivated by microtoxins decreased mda level which means decreased lipid peroxidation and again in previous my slides you saw that mycotoxins actually negatively affect uh lipid peroxidation increase lipid peroxidation the vitamin c c can help you and finally the most exciting part it's a polyphenolics the polyphenolics there are more than eight thousand compounds in nature uh you can see there are flowers flavanoids flavanols flavonones prontocyanins and other group of these compounds and in general terms polyphenolics received a lot of attention in last 20 years and they were considered as important antioxidants and more and more data accumulated that the action of this polyphenolics is related to those transcription factors i mentioned already so do we have any evidence that that they have protective effect in microtoxic causes so if you look at this big study which is 2019 and you can see here that the activity of catalase glutathione terexitase and superoxide dismutase in duodenum in the gut was compromised you can see a significant decrease and by alien grapeseed extract which is a source of polyphenolics and this was re-established which means that by using polyphenolics we can maintain antioxidant defenses at theodinum the same is true for the liver you can see here again in the liver the protective effect of polyphenolics is obvious another paper 2019 the effect of polyphenols and vitamin e on antioxidant status and meat quality of broiled chickens fed naturally contaminated with ochre toxin a again you can see here the same parameters antioxidant enzymes total antioxidant potential in the blood in the liver in breast muscles and again occur toxin compromise those parameters and polyphenolics and vitamin e actually having protective effect and in some cases the protective effect can be in combination in higher than in individual antioxidants this means that by using these polyphenolics in the diet of chickens it's possible to maintain antioxidant defenses in the gut that's the place where actually damage is most important as i showed you previous slide for example leaky god at the same time uh is a god is responsible for duncan detoxification and again to maintain gut integrity to maintain the microbiota it's a very important task and this kind of compounds dietary compounds can help and finally one more compound called silimarine which is a plant extract which is used more than two thousand years in human medicine and this is our big review 2015 which actually described antioxidant properties of silimarine and silimarine is very popular in a way showing antioxidant protection and in the same at the same time uh you you can you can see here that uh silimarine actually affect the toxicity of aflatoxin so you can see here the aflatoxin they were used in chickens and you can see body weight 20 day 28 and day 35 you can see significant decrease and by using silimarin it was recovered the same is true for feed conversion ratio again you can see control group aflatoxin be a contaminated group and protective effect of silimarine and again silimarine is the most effective compound to maintain antioxidant protection in the gut to activate a transcription factor nf2 which is responsible for synthesis of antioxidants enzyme and down regulating nrf nf kappa b transcription factor responsible for the uh inflammation uh one more paper again quite quite recent one uh aflatoxin and taking ducklings effect treatments with lycopene and silimarine you can see the protective effect and finally what about what about other botanicals i came to the end yes the other botanicals can also help to maintain antioxidants defenses including rosemary boulder and others and one more point probably the last one bht bht is commonly used as a feed additive to deal with a lipid peroxidation and in this case the phd can also help to deal with mycotoxins so conclusions microtoxins are major nutritional stress factors in protein production ketotoxin don xeralinone orchard toxin aflatoxin and phenol medicine can impose oxidative stress oxidative stress is major mechanism mycotoxin action and as a result of oxidative stress we have got integrity damages got microbiota damages immune system damages and various organs can be damaged as well this could lead to decreased production and reproductive performance and what antioxidants can do until dietary antioxidants can help to maintain antioxidant protection in the gut antioxidant protection in the tissues and the dietary supplementation of dietary antioxidants like selenium some photogenics or botanicals can help to maintain antioxidant protection and can help to deal with mycotoxin toxicity and at my last slide and i thank you very much for your attention thank you very much peter it was very exciting very interesting presentation personally i also learned a lot and i like to follow up with some things we have several questions and the first one is that what is the mycotoxin of most concern regarding oxidative stress in general terms when we're talking about oxidative stress the mycotoxins practically all mycotoxins impose oxidative stress the most dangerous one is t2 toxin the most toxic one at the level of oxidative stress ochre toxin dawn zeralandon and aflatoxin these toxins are major concern of a poultry pig and dairy industry and this exactly uh mycotoxin impose oxidative stress and that's why the general point of my presentation was to show you that all these mycotoxins can impose oxidative stress i did not talk about combination of microtoxins because i did not have enough time to explain but the combination of microtoxin can give even more important negative impact on antioxidant defenses and yeah i just would like to follow up this question with my question regarding the levels of mycotoxins uh that for instance european community accepts quite low levels compared to other countries for mycotoxins like t2 for instance do you think that oxidative stress can be also caused by levels which are below a limit allowed in feed yeah the problem is that mycotoxins never come singly so if you have any feed you would not if you found for example aflatoxin it does not mean that the feed does not contain uh t2 toxin or dawn and so on so forth so in most of it there is a range of microtoxin as a combination of microtoxin is more toxic than the individual microtoxins and therefore the level of microtoxins which would be even lower of their legal limit in combination can still have that mental effect on their on the redox balance in the cell on the antioxidant system on those transcription factors i mentioned and they can actually have detrimental effect first on the gut and secondly on the liver and if we're talking about occur toxin it will be kidney so what we're talking about we are talking about the independently of the level we have we need to remember that this is a problem and we need to find a solution for solving this problem thank you and may i ask the next question is also about the uh species are ruminants equally affected as swine and poultry regarding oxidative stress any difference between species well i i would say that with monogastic it's much easier to study because the problem is that the aluminium bacteria can affect mycotoxins themselves however as i showed you with this uh picture from the last review uh there was a proof that yes mycotoxins also have detrimental effect on antioxidant system in cows in calves in gods in ruminants in general therefore what i would say that mycotoxins are also toxic for ruminants however they as a scientist i would say it's much more difficult to study microtoxins in ruminants especially how we would uh use for example different antioxidants in the diet to deal with those mycotoxins because rumen sometimes can actually destroy antioxidants for example if we use sodium selenide as an example okay sodium selenite is not coming through the room and it's precipitated there therefore when we're talking about women we need to pay more attention and the form of antioxidants we use and how long we use what doses we use but generally their idea still the same thank you another question is what should we test in animals to see the anti-oxidative status well that's a difficult one because you see when we look at the antioxidant status which is used in human medicine it's very simple there is a enzymatic activities and there is a range of activities which are considered physiological okay so you have a table you open the table say hey this activity is lower than this data and this means is activity low it could be oxidative stress uh at the same time melon daily height as a final product of lipid peroxidation most commonly used how is the sensitivity of this test is not very high specificity is also not very high it's easy to use but information which you have is not good enough therefore the most likely scenario you need to use several tests simultaneously there is no symbol simple test the one which can give you the whole answer for example the total antioxidant potential it's very good one you can you need only 20 microliter of blood you put in the tube in five minutes you have a result however if you look at the total antioxidant potential uh if it goes down it's bad but you are not answering the question which antioxidants are involved then you need to look at the antioxidant activity like antioxidant enzyme activity superoxide dismutase glutathione peroxidase catalase and again the major problem is for poetry and peak and ruminants there are no stabilized data or data and table which are accepted by everybody therefore what you need to do to look at the normal animals which are without contamination of the mycotoxins for example and your experimental animals contaminated if you see that in experimental animals the activity on antioxidant enzymes goes down lipid peroxidation goes up this would give you the indications that oxidative stresses place and just related to that question is there any uh difference between species in terms of uh checking the antioxidant state status or the parameters to be checked or you can take blood of ruminant swine and poultry and you can check the same type of compounds yeah you can check the same type of compound however the the data which would be related to physiological label you have to establish yourself for every species and this more importantly those data will depend on the diet will depend on the age will depend on other stresses because when we're talking about mycotoxin stress i did not mention my presentation but you should remember that at the same time there are environmental stresses like heat stress there are ammonia for example in chicken house which is also stress so in this case when we're talking about nutritional stress yes mycotoxins are major nutritional stress but they are not working uh you know separately they are in combination with environmental stresses with technological stresses vaccination for example so those three which is a post-vaccinal stress is a common strain therefore those stresses are working together and when you have a combination of stasis plus combination of microtoxins yeah so every time you have a different situation and you have to establish for example it's a lab whatever the lab is that's a like as a big pig farm for example yes they can take a blood from piglets they can blood they can take blood from cells they can take blood from boss and analyze for example total antioxidant potential yeah there is no problem their essay will be still the same however what is a norm what is a pathology you have to establish okay okay thank you next questions are about the antioxidants uh with the limited amount of money to spend on the diet what nutritional strategy selenium or vitamin e or c would choose to be or anything else would be chosen to be most effective against free radicals well first of all uh we need to realize that for exams a diet is usually supplemented with spremix okay and premixed contain vitamin e uh for example if you look at the vitamin level in the premix for breeders it will be 100 ppm okay if we look it's a growing chicken it will be around from 30 to 50 ppm uh if we look at the piglet diet okay 100 ppm can do the job as well so if everything is right i mean the diet right the premixes right is that 100 ppm is sufficient to deal with oxidative stress in most cases years but when you have high mycotoxin contamination you might go from 100 to 150 okay then we look at the selenium study okay selenium is also part of premixes and most of premixes selenium is supplemented at the level 0.3 ppm the question is is 0.3 ppm is optimal well well there is no answer for that one because the 0.3 ppm in most countries a legal limit you cannot give more legally okay and this this means that as a nutritionist you are restricted how much and what you can add however if you look at the botanicals there are no limit what you can use on them but at the same time you need to decide how effective they will be because as i mentioned to you all antioxidants are working together as a team now you have to understand who is doing what in this team and is it increasing the activity of selena proteins is enough to deal with everything no the question is the nutritionist has to design an optimal diet taking into account all available information about stresses about the level of mycotoxins for example in the diet about the weather for example is a heat stress outside and the ventilation system is not able to deal with that heat stairs therefore the nutritionist has to decide what to do there is no limit for vitamin e you can go up but it's an expensive place as for vitamin c well vitamin c is not a part of premixes because vitamin c is synthesized in most animals and that's why the additional level of vitamin c usually added in specific anti-stress premixes like in heat stress or maybe with mycotoxin stress again vitamin c is good on one side but vitamin c uh when it's reacting with iron for example it's actually reduced iron from iron three to iron two and impose oxidative stress so the situation is not straightforward it's clearly needs more research to understand how you will combine all those antioxidants to have the maximum protection and stress conditions and the question very close uh is it mixed of antioxidant or botanicals are more effective than single silver well again there is no simple answer on that one uh because uh when you mix it does not mean automatically that you if you have one plus one plus one is it three is it four or is it two there's a question because for example when you're working with botanicals you need to be very careful because uh uh botanicals uh they have plenty of those polyphenolics okay as i mentioned there are more than eight thousand different compounds we know not much about them we we look at like uh quercetin or some other single compounds which most most study but we don't know about others and we are not controlling others that the problem is because we can control the total amount of polyphenolics yes you can have the chemical analysis and say the total polyphenolic this number is that number good enough for you you don't know what are they you you know that for example maybe two or three of them you might do the concentration chemically analyzing and you will see the concentration what about others there is no answer therefore uh research should be done or researchers should spend more time to understand how those botanicals works for example the absorption of there is very low and and and they still can have a tremendous effect in the god okay if they are not absorbed it does not mean they do not affect the god right and they even it's sometimes good that they are not absolved because they are moving in the large intestine because the most antioxidants are absorbed in the small intestine like vitamin e for example so vitamin e is absorbing the small intestine and not coming to the large intestine while polyphenolics they are not absorbed well so they go through up to large intestine with a major microbiota reside so the microbiota can be affected by those botanicals and actually the relationship between botanicals and microbiota that's the current topic of research most exciting part of research because today microbiota is considered as a separate organ in itself because in the in the body we have more microbial cells than cells of the body itself so we are talking about trillion microbials living there and in this case those microbial can do good job bad job and this job will depend on dietary supplementation as well thanks a lot peter the question is about the species what are the most sensitive animals to antioxidant stress well uh with oxidative stress of course uh the uh most sensitive will be early life animals uh for example newly hatched sheep the chick during the first week of the development uh if you look at the piglet new orleans born piglet or piglet just wind so the winning for piglet it's a very high stress already okay you should realize that when we have piglet let's say for four weeks for four weeks on milk so the digestive system of piglet is adapted to the meal consumption and suddenly on day 28 no more milk dry feed this is a very very high stress and in this case the digestive system very often compromised and there is a diarrhea and many other problems appear so if you have oxidative stress at the same time because the body is in compromised position already if you have stress like mycotoxins or heat stress or any other stress the the sensitivity or consequences will be much higher if you look at the breeder side okay we can say they are more resistant but remember that there is a progress there is a science called maternal programming which means that what you affect how you affect breeders will have consequences later on for example if we look at the human side we could say that few weeks of women women pregnancy determine the health of future baby until end of their life so which means a baby born and then in 70 years later there will be still consequences what mother was consumed the same is true for for piglet for hosanna for example okay we we give for example oxidized fat for sow okay we might not see immediate effect however those changes at the gene expression level and gene expression means that some genes can be switched on switch off and we don't know if those genes which were off will be on later on therefore when we're talking about breeder diet or sow diet or cow diet pregnant cow diet we need to remember that we need to do maximum to prevent any stress at this period of time otherwise you will solve one problem like in including my uh antibiotics for example okay you include antibiotics you saw the problem of microbial contamination but you affect those genes i mentioned already and you can have uh consequences in terms of for example meal production of your future cow i think we are running out of time we have several questions but also several comments i would like to address to you peter so there is a personal uh message to you that it's a great job uh very well done so from the participants and there is also one comment that it's a miracle that we are still alive because so much strength yeah so we are alive because we have good diet and good sense of humor yeah so we will follow up other questions we still have several and we contact uh participants with email okay i will be happy to answer those thank you very very much peter okay thank you thank you it was a pleasure thank you thank you bye bye thank you very much thank you bye bye