[Music] i would like to thank aliseo for inviting us you know to give this a short seminar and be part of this webinar series talking about methionine and the transition cows and our life you know just like brian said you know hope that you guys are safe and sound and keeping social distancing and being healthy and i hope you enjoyed this uh this presentation um okay so for this uh presentation i'm gonna go to this outline we're gonna talk first about the peripartial period of um of derek house you know major physiological and metabolic changes that occur during this time and how that can affect the overall um allow the cows to have a smooth transition um and then we're going to talk about how mentality can impact or have a beneficial effect on those if on those conditions they're less of desirable for transition cows and after that we're going to go over several effects that occur or that we have noticed based on liver function and how important is liver function for transition cows and later on we're going to talk about histo methylation and some of the newest data that we have collected here at south dakota and at the end i will give some summary and conclusions of this so first we know that transitionary cows has been well characterized there have been a lot of research on this area and we know that is around three to four weeks pre-partner to three to four weeks postpartum and it's very important this whole period because it's the most stressful period in the lactation cycle of their cows and this is illustrated by the greater incidence of diseases the cows may may undergo or may be more susceptible during this time than the first few days after calving and it's more prevalent for older cows as well now most of these is driven by the increase in energy or nutrients that need to be supplied to sustain the lactation so once a cow calf there's a huge amount of nutrients that are required for for for the cow to produce milk and this is compounded with the same at the same time with a known decrease in the feed intake for those cows in the days leading to uh to calving so both of these effects will lead to an eventual negative energy balance that is pretty well known and it's pretty common to most mammals so after cabin there is going to be this drop on energy balance and you most of the cows will go over a negative energy balance so because of this reason there is a common decrease in body condition score that cows will undergo after calving and we're going to take a closer look on that in this slide the there is a like i said the body condition will drop because the cow has to mobilize body reserves in the term in terms of fatty acids in that case and these fatty acids they're in the form of nifa will be mobilized from adipose tissue to the liver and also there is some of it will go to the mammary gland for meal fat now the problem that may occur here is that at some point there's going to be so much nephilim fatty acids that will filtrate the liver and the liver has to you know deal with that surge of nepha filtrating the liver so the liver what you will do is probably or more likely to um sterify those fatty acids there is some data for that episode for for that there is evidence for that um and there is going to be a potential accumulation of triglycerides that may lead to fatty liver now those triglycerides can be either stored in the liver or they can be packed and into blds and being sport out of the liver into the bloodstream in bldls okay and this bldl will carry those fatty acids or triglycerides to the mammary gland and other peripheral tissues so the animal can use utilize those fatty acids now on the other hand uh that these fatty acids in the in in the liver can be translocated into the mitochondria for being utilized for energy or to some extent at some point they're probably going to be so much fatty acids that there's going to be a partial oxidation of fatty acids leading to the production of ketone bodies and this may lead to ketosis as well now we talk about the decrease in feed intake around parturition so we know there's there's going to be this drop in intake and consequently you will there's going to be less synthesis of bfas as a consequence of that especially propylene that is utilized by the liver to produce glucose um but for the most part you know propylene is always going to be the major source for gluconeogenesis in the liver but the liver may rely on other sources for gluconeogenesis such as amino acids lactate and glycerol coming from the triglycerides now for we have observed a decrease in glucose so that's also common during the transition period and we have talked mostly on energy until this point but what about protein you know what's happening on the on the side of the protein during the transition period so we have this data from maryland where they where you're looking at here is bars based on body fat and protein content of cows going from minus two weeks and then it was measured also at five weeks and twelve weeks relative to perturbation and in yellow you see the fat content of those cows and in red the content of protein now it's quite evident that there is a decrease in the and the amount of body fat and also body protein uh on those cows going from uh gestation all the way into lactation so regardless of if there if there is a mobilization of fat in terms of nepha there is also going to be a mobilization you can expect that there is going to be a mobilization of protein that needs to be utilized by the cow during that time and this kind of goes back to the same idea there is a decreasing feed intake so the cow has to come up with with that deficit from summer so it's going to be using body reserves for that and what is also interesting from this data is that regardless of the concentration of crude protein in the diet there observed the same drop on body protein on those cows now in the same context there is data from some cornell these calculations from this this study from bell they uh calculate that around half a kilogram of metabolizable protein can be means or being a negative energy balance during the first week postpartum on transition cows so you can see that just as much as we can talk about negative energy balance there is also a negative metabolizable protein balance that very likely you know fresh cows will undergo during in that kind of scenario and in the same way as negative energy balance once the cows start having a more consistent and more significant feed intake it will come back up you know to positive values in metabolism or protein now this obviously this methodological protein is going to be utilized for amino protein obviously to some extent glucose synthesis and also the synthesis of other compounds so it's not just milk protein that we're talking about here but other compounds that are required for the cow and among those uh is adenosine methionine that is the major material donor in the body so methane in in choline in these compounds amidst the material donors can be um sources for the production of esoteric now uh glutathione and taurine also are also antioxidants there being that methionine can be a precursor for those compounds now we have um you know for data from nrcm before dnrc we know that methionine and lysine are the most limited amino acids in metabolizable protein in mint lactation cows so when we start doing these experiments several years ago we the main question was can we what kind of effect can we observe when we are feeding methanol and or balancing these amino acids um um from a middle lactation cow to a transition cow what will occur on those cows and that's kind of what we went on in in those data and i'm going to show you that in a little bit now the this is a data from denmark what they observe what you're looking at here is a fractional uptake or the influx of non-essential amino acids or essential amino acids in this case you see that most of these or probably all of their these amino acids here they have a linear effect meaning that there was a drop on the optic of this essential amino acids between cows going from minus 14 days pre-partner to 14 15 and 29 days so it's pretty evident a decline in the in the optic of these amino acids with the exception of metallic and histidine what we can observe a trend for a quadratic effect so what that means is that um at four days you know during the first week of lactation cows tend to increase the optic of this particular amino acid methionine histidine which is quite interesting you know there could be there is more than just um you know we normally sometimes we talk about gluconeogenesis but could be there are other needs you know for the other compounds that i just talked about that there's a need for these amino acids by the liver and it kind of goes back to the same point of within the first week you know that i show you that negative metabolizable protein that these at the same time that there are never levels you know very low levels of metabolizable protein in the cow the liver on those scalps require a greater amount of very specific amino acids um in in their system now this metabolizable protein i'm not going to go into much detail on this just to kind of recap you know what where is it coming from and all i saw protein essentially is rheumatoid degradable protein okay that is not being fermented or fermented to a lesser extent in the rumen and it will bypass or will go into lower tract and be absorbed there now we have also rheumatoid degradable protein that is essentially microbial protein that also will make up a great amount of this metabolizable protein now the point of this slide is to show you that or to make a point that is not only about metabolizable protein i know i show you that there is a decrease in the metabolizable protein balance during the early lactation in fresh cows but it's very likely it's not only about the amount of metabolism protein but also the profile of amino acids if you go back to that slide the from data from denmark you can see that the need for for certain organs like the liver they prefer very specific amino acids during the first you know weeks of lactation so keep that in mind that is not only about the amount of metabolism protein but the profile of amino acids now let's talk about methionine and the effects during the preparatory period of their cows this table i summarized the data from lactation on three experiments that were conducted at illinois and you can see that it was pretty consistent the amount of crude protein in the diet around 17 percent crude protein or dry matter the other important um point that i want to make here is the metabolism protein balance that it was negative for all the animals which is understandable because there is going to be a huge amount of demand on metabolism protein now you can see that there was less for about this tail in this case but this this you can consider that that was because she went to a lower amount lower numbers of daisy milk with those cows so by the time that she finished prod there was a better status in terms of metabolizable protein for those cows so um this is adjusted by intakes you know body weights of the animal and milk production so that's what it's been taking into account here now you also you can also see the lysine metallic percentage as percentage of metabolizable protein but i want to draw your attention here to the lysine to metallic ratio that we always talk about this you can see that by supplementing these uh these sources of metallic we were able to lower the the ratio of lysine to the tining from around 3.4 to 3.7 to um to 2.7 you know 2.9 so we were able to decrease that that ratio which is important now i just gonna summarize some of the effects on these three experiments and you can see that it was pretty consistent an increase in the in terms of meal gill by supplementing cows before calving with rumen protected methane we observed above three kilograms increase for the most part uh on milk yield and it was pretty consistent across all three experiments also um is for we have the same effect for intake around from 1.6 kilograms increase in intake on cows that were supplemented with methane during the fresh postpartum or first time now seem very similar to the previous data in terms of milk protein percentage we observed an increase in the meal protein percentage and was consistent with the other trials that were done in transition cows at illinois so because of this reason you know we it was pretty consistent and also we had the opportunity to go really deep into what was you know what what was the mechanism that the methanol was causing this effect on this cows and not just in terms of meal protein but also all the these other compounds that i talked to you about earlier so again in the same context you know in in at the farm you know in a foreign the their farmer would be able to observe an increasing meal deal and performance and perhaps also an improvement in health status in the in the herd but um this is less evident all these other effects you know that's why i always bring this iceberg analogy because we were you know we went really deep into what was these other um biological processes that methionine had an effect and we tried to prove that you know to kind of understand um how does methionine supplementation can allow these increa these increments in milky and performance so we observed that in terms of antioxidant effect liver function dna histomethylation and also a reduction in on immunosuppression that is pretty common on transition cows now for those that you know always don't think about the liver too much you know you should thank them is a pretty amazing organ and it has several um is involving too many too many things sometimes you know too many processes in in the body uh to store glycogen to make proteins uh to make cholesterol uh glucose in case of ruminants so there is a lot of um all different you know biological process that the liver has to deal with and i'm going to show you more of in the context of the transition how how important is this to take this into account now let's go back to that graph and let's focus on this effect where the nephas filtrate the liver and then tend to accumulate as triglycerides in the liver so what we did here is we kind of put together these three trials at illinois and we end up with around 100 transition cows in this kind of retrospective analysis and kind of meta-analysis where we took all the cows and we put it regardless of of treatment effects we divide them on the concentration of triglycerides in the liver okay and we um we run the analysis how much of that how much of that that impact can impact the meal yield so in this case we did observe a trend um um an actual interaction between fatty liver and group and daisy milk and it's pretty obvious you know that those cows they were in a severe um with a severe fatty liver um around 10 or more um they have a really hard time you know trying to get into into those first few weeks after cobbing and this exemplifier illustrated here with the actual means the those cows in a mild fatty liver they have around 42 kilograms of milk production all the way to severe fatty liver when cows they had around were producing around 37 kilograms of meal production so you can see that there is around five kilograms 10 11 um pounds of difference between cows they are in a mild fatty liver condition to cows they are in a very severe fatty liver effects so because of that you know we we're we start looking into how battalion can affect this you know what role plays mettani on on this um in trying to ameliorate this fatty liver condition so in this case um we can we can we know that methionine can supply or spare some of these matildas for the formation of phosphate phosphatidylcholine that is a major component of those bldl that i talked to you about earlier so these are um polyphoprotins or lipoproteins actually that allow the package of triglycerides and be able to export those out of the liver and most of these triglycerides are coming from those nephrons that i talked to you about the other point on methionine is also a precursor for these antioxidants glutathione and thorium and also metallic can make up you know as being an amino acid is required for the protein synthesis of important proteins such as apolipoproteins that these are also required for bldl synthesis or assembly and i'll show you more data on this then last one is production of albumin and there is a negative acute phase protein and i'm going to show you what that means and but albumin is a very important protein to be that is being synthesized by the liver and it's one of the major carriers in the blood for different compounds and other metabolites now let's focus on the other side of what the liver is doing with this amount of nifa there which uh their their their organ so the liver so this case nephi needs to be translocated into the mitochondria and there it can be better going over under better oxidation and produce energy that is highly needed during this time as we we look at the energy balance and all these other effects but at some point it is going to be so much nifa and depending on the cow you know the the genetics of the cow and also the amount of body condition score that the cow is putting up during the late gestation and the dry off then the dry period um you might have a huge or lower surge on nephi's concentration in the blood so the point here is that it's going to be so much that the mitochondria will start doing a partial oxidation of those fatty acids and that's where you start seeing a lot more production of ketone bodies and that's why where all ketone ketosis is derived from so in that context what you're looking at here is pretty much kind of like the membranes of of the mitochondria in the cell if you will this uh a liver cell or a hepatocyte as we call it and this is the cytoplasm or the cytosol inside the on the hepatocyte of the liver cell and fatty acids coming here they're usually activated with a koi and they have to be translocated inside of the mitochondria so this is the side of the mitochondria so to do that the fatty acids needs to be ligated with carnitine and carnitine can allow those fatty acids to be translocated inside of the mitochondria now inside the mitochondria the carnitine is going to be dissociated from the fatty acid and the fatty acid is going to be reactivated with a koi so it can go on and do and be you know oxidized but that carnitine has to go back outside um to the cytoplasma and pick up another fatty acid so that's kind of like the someone you will see this as that carnitine shuttle pretty much you know allowing all those fatty acids to be move across into the mitochondria so they can be oxidized so in that sense um if you stay with me for a little bit this is this is lysine and lysine we know there can be three methylated to form trimethyl lysine and these material donors you know if you are supplemented uh material donors such as methionine it's very likely that you're going to have a greater pool on the field donors so you may have we can easily associate this effect of a tree methylation or lysine um in in in calcium supplemented with methionine and what is this important uh tree materializing is the right limiting step for the formation and it's upstream for the formation of or the synthesis rather of l-carnitine okay so this is important if you have a product that or amino acid they can also make up this important compound carnitine that allow you to or allow the cow to mobilize those fatty acids inside of the mitochondria to be oxidized and rather than stay there and be stored astral glycerides it's something that is helpful so on the other side you know this can lead also to oxidative stress but we're going to talk about the antioxidant effects of methionine in a little bit so going back to the same you know carnitine story um we actually measure carnitine or free carnitine in the liver and we observe an increase of carnitine in those cows that were supplemented with methylene this was metasmart and sm is smart mean and i'm going to keep the same legend for for all the other graphs so by the time of 21 days postpartum it was not as much of an effect but it's pretty evident that as certain days you will see this an increase in carnating at least that's what we saw and also it kind of coincides you know when the other data that i've been showing you in terms of you know negative metabolizable protein uh the uptake of amino acids like metallic and histidine in the liver so it kind of goes back around to the same you know early lactation first week the first days after calvin so this kind of you know popping up these things that pop up in this data will show us that it's really important to really um can allow the cow or provide a nutritional management the the cow you know set the cow to a good start you know into the lactation um if you wait too long price is is not ideal now the other effect that we want to go walk you guys through is the mobilization or sporting those packing those triglycerides into bldl so for that um we have observed and a trend in our experiment a trend for an increase in the concentration of apple b 100 in the blood of cows that were supplemented with methionine so apple b 100 is this um protein in the cell and in the membrane of this bldl they are required you know once the the bldf go outside and you kind of have to um reach other peripheral tissues so apple b100 is kind of the other tissues are able to recognize ah okay this is a build yell so it has fatty acids or cholesterol so let's take this guy in and and we we need this guy you know for for for getting those fatty acids so that's kind of the other peripheral tissues once the bltl is outside of the of the liver can recognize this particle but but it's required this apple v100 now in a later experiment they'll actually observe an increase in a significant or statistical increase in apple b100 and it was nicely coupled with an increase in bldl concentration in the blood as well so there is some evidence that um tell us that methionine can have an effect on these polyphone proteins or the whole lipoprotein secretion in the liver now um i always like to put this light is a little but just to make a case you know or stress the importance or liver function we have you know to the studies you know when we start and and we we recognize very rapidly the importance of liver function and how these uh you know effect usually you cannot measure at the farm can allow the cow to have a smooth transition or at least give them you know the tools that they can you know sustain um a harmful inflammation or uh some or uh oxidative stress and to some extent there are some cows that you know you will see this very skinny cow in early lactation but you just find you know she can deal with that you know amount of nifa reaching the liver and just process those fatty acids and she will be okay so some of those cows have those genetics and are able to do that but at the same time you should be able to provide you know management you know by not allowing those cows to get too fat during the late gestation or late lactation or during the dry period or at least to have an optimal body condition score at the time that they cap and also you know by nutrition effects that we have been talking about here now i like to put this slide and this is the inflammatory response this is very busy you know and i'm just i don't want to go over to a specifics on this but the the main point of this slide is that um even even though we don't we don't know but the tissues in the body they talk to each other they have ways that they can recognize that there is something else going on or there is a an infection or a local inflammatory response and they can communicate with other tissues or organs in the body and they do that by secreting this kind of cytokines in the bloodstream and these will travel you know in the case of the brain is gonna these signals are gonna go to the brain and you know tell the brain to produce more adeno adrenocorticotropic hormone in this case and this will travel further into the adrenal glands and at the end it's going to produce cortisol so essentially these are signals and these signals are telling in the liver that this other you know the mammary gland or the ures there is a local inflammation or there is some infection or some pathology so the liver has to respond to that has to do something and the liver will respond that depending on the level you know of the signal will respond by changing its pattern or its behavior if you will of the secretion of certain proteins and we call these acute phase proteins so when the liver recognizes this this signaling that there is a that there is an inflammation it's going to start producing positive acute phase proteins these are serum amyloid a haptoglobin and cerebral plasmin and there there are other positive acute phase proteins but uh during this presentation we're just gonna focus on this and also at the same time uh the liver is gonna start secreting or synthesizing less of other proteins and these are negative acute phase proteins albumin and alpolypo proteins that are polyco proteins we just talked about apple b100 now in other terms this the same slide but it's just like a you know the way to to think about this is that just these tissues they may or may not have a pathogen you know or they have a pattern in potential pattern infection but they will send a signal all the way to the liver so the liver has to deal with and producing more of these acute phase proteins positive acoustic proteins so this help you know those tissues or move on into a different signals to allow the cow to respond to those potential infections now if you put this into perspective you know at the same time the the liver will receive this inflammatory response from the mammary gland or the euros at the same time you have to keep in context that there is a surge of nifa going into the liver at the same time and what the liver has to deal with you know changing this okay we have to synthesize less of this protein and more of this and there is more nifa coming up and we have to make more glucose from propionate at the same time and also cholesterol and produce secrete bl at the same time so there is you you have to get this into context how much or how easy it can be for the liver to get overwhelmed during those first you know few weeks postpartum so this is quite important to recognize and to recognize that the importance of liver liver function in that context now let's talk about negative acute phase proteins like albumin so what you're looking at here is albumin concentration in the in the blood from cows they were in a controlled diet or in a metasmart or smarming and you can see that there is a drop in the concentration of albumin in the blood on those cows that were in a control diet especially around the first and second lactation it's second week of lactation and in the in in contrast those scouts that were supplemented with methane that you can see there they kind of remain stable and at some point they even increase uh by you know second or third week postpartum now this red line is just to highlight this 35 grams per liter is not not a cut off or anything just to highlight to to this other data from italy where they actually run a retrospective analysis and this is the 35 grams per liter and what they did was um they separate cows based on their liver function and um they put a cows into an um upper mid upper lower mid lower and lower uh concentrations or or liver function and the point here is primarily is to you know so you guys can see that there when front account that is have a good liver function to a a really lower liver function you will see that there is about 6.5 kilograms difference in yield yield we talked about 12 you know 11 12 pounds of increasing meal production when the when you when these cows have had a good liver function so it's easy you see that we can associate liver function with meal performance and health overall in cows and this goes back to the same idea that is so important for for transition cows now let's look at a a positive acute phase protein cellular plasmin and serum amyloid a in this case is a little bit counterintuitive because what you want is to have lower or at least a greater concentration of these biomarkers will will mean that there is a greater inflammatory response and in this case the control cows have greater concentration of cerebral plasmin and serum amyloid a in contrast to those cow that were supplemented with metasmar and smartmen and it's pretty consistent on this on both of these uh biomarkers now this is another graph from methyl from the metallian cycle and i'm not going to go into detail on this but just to show you that at some point in this this is adenosine methionine methionine and at some point in the methane cycle you're gonna have a formation of homocysteine there is a precursor for cysteine and um and this antioxidant taurine and glutathione and these are related to liver function as well so in this case uh what you're looking at here is auric so orac is oxygen radicals absorbance capacity so in a way is the antioxidant capacity of the animal measured in the blood essentially so what we're looking at here is we we observed is was an increase in the concentration or the capacity for cows that were supplemented with methionine with a greater antioxidant capacity to that extent then on the in the case of glutathione this is glutathione in the liver so this is liver tissue and this is blood blood blood samples so just similar to oracle glutathione there was an increase in glutathione concentration in the liver of those cows that were supplemented with methane and what is interesting is that we can break down this effect and we can observe that there was most of that effect was due to this increase in the glutathione concentration at minus 10 days before cabin on those cows and furthermore on these and we have observed the same pattern on these other experiments that were run at illinois when you supply methane in you know 10 or 11 days before this you make this liver biopsy you will see also also an effect for an increase in glutathione concentration on those cows so it has been pretty consistent across these experiments and we kind of call it a primary effect and one of the things that i always like to talk about this because i think it's quite important that you you allow the cow to have this increasing glutathione because to some extent in other models like rodents and humans glutathione is called as or is being suggested there is a storage for amino acids in the liver so in a way you know you can see that there is this is kind of like putting uh amino acids in the form of glutathione um in the bank so the cow is kind of like you know storing some of this glutathione when you're feeling that supplemental methionine before calving so the excess of methane if there is an excess is and it's pretty evident that the the liver is going to start accumulating this with the time what's important on this is that if the cow has this greater concentration of glutathione before calving is the cow already have this powerful antioxidant already in the system so you don't need to depend on the feed intake or the cow because that's gonna be really a real issue uh you don't know how much that cow is gonna be able to to eat during those days so you cannot um rely on that but rather having already an antioxidant ready for being utilized by the liver is a plus and you know i think that's one of the one of the the the underlying effects that we're seeing in this data also the fact that you can have this glutathione be breakdown into three amino acids and one of them in timing can be a really helpful you know for the um for the animal as well now let's talk about uh histomethylation there is more recent data that we're collecting on this so uh this is the central dogma of molecular value so in theory or not interior but we know that their dna contained information for all the proteins in the body we always hear about people saying about this you know but there is an intermediate there is rna so this information of dna has to be transcribed or put in another form so these other proteins can read this information and make up you know proteins at the end so there's a middle step in of this and most of the you know dairy farmers and and technicians at their farms are more associated with dna with this uh with genomic testing when you take the ear notch or any other sample from animals and you can kind of have an idea of the of the of the of the genome of your or your herd or any other any snips you know and any differentiation in your in your heart um but for those that you know we work with this regulation between dna and proteins we study a lot what regulates this flow of information and i always like to put this this analogy with tiramisu and i probably need to change to another analogy on this but um the main point of this is that if you you know humor me for a little bit the dna if is written in a spanish um language it has to be transcribed into an rna or an english is the same recipe the same ingredients but it differentiates in the language so these other proteins cannot read spanish for instance so they need this information in english so they can make up you know this final product that is so in this case it could be casing or copper casing so all this you know is a flux flux of information from dna all the way to make up these proteins so there are a lot of a lot of things that can regulate this flow of information one of them uh we're gonna talk about that in a little bit so going back to methionine this is um a result from a microarray that we did in liver tissue from cows going to minus -10 721 days and as you can see the pattern of expression on those genes is quite different between cows in a control diet and a methionine diet and we observe around 2600 genes that were differentially expressed in in interaction so it is pretty obvious that when you feed methionine you will have some effect in the transcription so if you go back to that you know there's going to be an effect on the level of transcription for that recipe for for to make cap tiramisu if you will um now because of the same reason that we have seen this you know uh differential expression on this in the liver when you uh supplement the timing we kind of have start putting together a model for how methane may regulate gene expression in transitionary cows or in their cows in general and this might help us you know for later on if we wanted to use the same methodology to actually come up with a theory of regulation for other other nutrients but the point here is that you know we know that methanol can have an increase in saline symmeting there's a major material donor as i mentioned before and we have tested in dna ventilation we know there is an effect on dna methylation when you supplement cows with methane now we don't know in terms of transcription factors if they are um activated by the timing which one it is we don't know until this point there are some there could be some other intermediate methodologies besides s-administering methionine that can have an effect on the gene expression and also cell membrane transporters or trans transceptors that can have an effect on gene expression and now at south dakota we have worked more on histo methylation so now if you go back to the same graph what we have been focused on in in terms of histomethylation is or or epigenetics is kind of like the regulation of information that is available or not to be transcribed or to be makeup as um as a protein as a final product so like i said we have focus here on histo methylation and if you go back you know and and start thinking about the dna how is the structure and it's very packed in the nuclear or the nucleus and it has to be like that because it cannot be floating around um and how all these sections are floating in the in the nucleus so it has to be very compacted and to some extent and to be in a more structural uh organized manner so the dna does that by pulling around into these proteins histone proteins in the nuclei and these histone proteins it can be depending on how tight they are it can be really packed and and really tight um the dna enough that can inactivate or close up some sections of the dna but in another sections it can be very loose you know it can allow this information or this recipe for for you know if you go back to the analogy of the tiramisu you can allow to see that recipe of the tiramisu and is a more active or open uh section so depending on how the dna is organized or spooled around in with the dna these two conditions can happen now let's go back to the tiarasu again and just to give an idea that if you go to the to um to a restaurant and the menu that you can get a menu and the menu will symbolize or will be an analogy for the the genome so it's all the information in in the menu is all the information for proteins and known proteins in your body but what happens that once the dna starts pulling around and to have this section their close-up it could be as if you were not able to see or order some specific dishes from that menu that they just give to you so in that case one would not be able to see you know if you want to order jeremy zoo or any other side dish here lobster whatever you do prefer you're not gonna be able to see that in the menu so it's kind of like the same thing or idea going on here that depending on how the structure of the arrangement or the dna it is it can have a huge impact in the amount of information that can be utilized or seen by the by the by the in the cell now what we have done here is to um to utilize this modified histone and we kind of have insert that type of recipe or or that gene or genomic genetic material inside of the bovine cells and so to make them bright and we can kind of trace back which ones the number of cells that we have and how bright they are and with this fluorescent microscope and we have focused on very specific uh methylation sections the k27 and k9 doing this um research and the other important point is that we have moved from a qualitative microscopy to a more quantitative microscopy so what i'm saying by that is that we're not as much interested on uh you know pretty pictures you know and they're nice to look really nice but what we're interested more is to get data and to get a number and with this cell profile software we can actually run in a batch all those images and it will tell us how many cells we have how much is the intensity of those cells and we can kind of actually start doing some mathematical or actually have a mean and actually prove that there is some effects in terms of histomethylation and that's what we have done here or at least fernando was the one fernanda rosa a former student have done here in this experiment where she increased the concentration of methane from zero all the way to 500 micromolar and you can see that it's quite interesting because at 125 there was that greater relative histomethylation it was not a 500 so this tells us that there is not a dose-dependent matter at least until this point so what what we're trying to say is that at some point the cell will know that there you have you got to stop doing histo methylation so there is likely to be other regulatory mechanisms that we don't know yet uh we have measured some of the histone uh material transfers as well and because of the time and we'll not going to be able to shoulder that data but there is an effect on that as well but we can tell you that um at the berkeleys there seems to be an effect by methionine in histomethylation as we were expecting that now we have couple these effects or these data from histomethylation with gene expression and we measured these three uh gene genes for these enzymes along the metamin cycle and just to give you an idea we observe a significant or a trend uh pretty consistent for a linear increase in the uh in the transcription of those of these four genes along the metallic cycle when we increase the concentration of methionine and this goes uh in line with other gene expression data that we have done in liver in vivo in those transition cal studies now another important point of this is that when we observe this increase in relative histomethylation we also observe an increase in total protein synthesis in the same cells in the same batch so this tells that there we got something going on here that there is there is to some extent there is an effect on histo methylation that may allow us to understand um or at least to be an indication that most of these effects that we're seeing you know greater performance greater middle protein percentage some of that may be reflected from a change in the structure or the dna to what extent we still don't know but at least this is a good or encouraging data that tells that there is something else something going on here on this and there is also other data backing up this idea this is um just to give uh information on this l-chromatin is the same uh as when i was talking about when the dna is open and there's space for gene to be accessible so is more active in in theory when you have a l-chromatin state in the dna and when you have a eternal chromatin is a more close-up and more packed together type of dna structure however what's interesting is that this experiment or this data from massimo bionas where they did microarray from cows in minus 30 days all the way to the end of the subsequent lactation in mammady tissue on those cows they observe for those genes in the microwave they were associated with a chromatin meaning that the dna is supposed to be more accessible to be a decrease once the cows calve you know and goes into the lactation there is a decrease in the chromatin status based on those transatomic data and it comes back up until the end of the lactation so this is kind of you know there are some hints and instead knows that there is something to it there is something the by the dna to be more inactive and i prefer to say in the case of at least the mammary land that it might be a case that there is more focus type of dna structure you know you close up other genes that are not necessary uh just to make milk milk synthesis probably just kind of open up all the regions for casein and kappa casein and leave other regions that are not as important for meal production so it kind of is a more targeted and focused um structure of the dna and that's kind of how we're looking at this at this point and this pcg is kind of like a gene um general repression actually so this data is pretty line up in the spring consistent with the other data that i just show you now the final point of this is that you know to what extent dietary methylene might be affecting this uh because to close up this or to have a heterochromatin status you need methylation you need material donors such as methane but at this point we can just speculate and and this at least very interesting data they might tell us you know that there is another um effect or another reason or role for these material donors to be you know readily available on those uh on transitionary cow diets now with that i want to close up and to give a summary so we have observed that you know with this supplementation on the timing during the transition period was pretty consistent across those three experiments in increasing performance also we have seen metabolism improvements especially in the form of um of these acute phase proteins and the other proteins that i show you in the blood and also there was a huge impact in the gene expression in the in the liver so all that you know can tell us to you know allows is a strong signal or indication that methionine have a profound effect in the in the in the liver function um based on the data that we collected and this you know goes back to i'll say the same you know stress enough liver function is important for transition their cows so the more liver function you have the lower uh you consider the stress you you you do you have and also you allow those cows to deal with harmful harmful inflammation as well and this was clearly reflected on the performance at the end of those cows now at this point you know histomethylation looks like a the data we're we're getting is pretty interesting and encouraging and to kind of complete that methane model and and and and it's pretty the whole thing driving this is that we observed this huge impact on the regulation and to kind of be able to understand how does metallic can have this impact on performance at the end and with that i can entertain any questions i would like to uh thank also the experimental station or south dakota they have helped us with some of these uh collecting this data and also to my students because i really right now i don't work anymore it is all them they do the work in the lab all the time and also you know just remember to keep social distancing so thank you very much for your attention you