[Music] alrighty thank you very much for that uh introduction dr lukini so as he said my name is danielle coleman i'm a student at the university of illinois and today in my first presentation i'm going to be talking about the effects of rumen protective methionine supply to cows dream heat stress and how this can alter liver tissue protein abundance of mechanistic target of rapamycin or mtor signaling and so just to start off with here quick our overall takeaway message from this presentation is going to be that supplying room and protect methionine during periods of heat stress may help to maintain hepatic homeostasis in mtor insulin and antioxidant signaling and so to start off with a little bit of background here most of you probably know that methionine is often implicated as a limiting amino acid for our dairy cows and in several studies with our transition cows we've observed that supplementing room and protected methionine can help to increase dry matter intake milk and milk protein synthesis and those improvements particularly in milk protein have been linked to signaling changes in that mtor pathway however this hasn't been fully studied in the liver tissue additionally we know that methionine is going to serve as a functional nutrient and where we've observed increases in liver function and immune function in those transition cows and so those effects are going to be best exemplified through one carbon metabolic pathways and so this diagram here shows those one carbon pathways and we're primarily going to focus here today on the methionine cycle and the transferation pathway and the link to mtor and so just to walk you through quickly here methionine is going to be at the top and that can be synthesized here via homocysteine and can then be converted to s adenosil methionine and then escidenosyl methionine or what we sometimes call it sam can be used to allosterically activate down here cystothionine beta synthase this enzyme is the start of the transformation pathway and by increasing its activation we can increase the production of two antioxidants taurine and glutathione and that can help to reduce oxidative stress and therefore inflammation sam is also used up here to activate a protein called samtor when samtor is bound to sam we get activation of mtor here which can lead to greater protein synthesis however there has been very little work on the effects of supplementing room and protective methionine during heat stress and how this can alter liver metabolism in our dairy cattle we know that similar to the transition period heat stress is going to be associated with a reduction in milk pro milk production as well as intake leading to decreases in milk protein synthesis and we also know during this time that our amino acids are going to be reprioritized away from milk protein synthesis moreover this period of heat stress is going to be associated with increases in inflammation and oxidative stress as well as a reduction in immune function and so these are all going to be areas where room and protective methionine supply during heat stress could be beneficial and so our objective then with this portion of the study was to investigate the effects of rumen protected methionine supply during a heat stress challenge on the hepatic abundance of proteins involved in that mtor signaling as well as insulin signaling and antioxidant signaling and so to do this we use 32 cows in a crossover design at the university of illinois dairy farm and so we had two dietary treatments as well as two environmental treatments and so for our dietary treatments our first one was going to be our tmr in which we supplemented that rumen protective methionine at 0.105 percent of the dry matter and then our second is our control diet which did not contain any methionine supplemented and then our two environmental treatments our first one is going to be thermal neutral conditions which will be called tn on our figures and tables and then our second was heat stress conditions which we induced using electric heat blankets as you can see in this picture here on the left hand side of the slide and so to walk you through the study design real quick it was divided into two periods and then each period had two phases and during that when we changed to the periods our environmental treatments were inverted and so to explain that really quickly here we see in period one that group one was under thermal neutral conditions for the entire 18 days however group two was under thermal neutral conditions for the first nine days followed by nine days of heat stress and then when we go to period two we see that this is opposite group one was under thermal neutral conditions for the first nine days followed by nine days of heat stress while group two was under thermal neutral conditions the whole time and so we're going to consider this our sequence effect in our model another thing to note is that during phase one all cows were fed ad libitum however when we moved to phase two the thermal neutral cows were pair fed to their heat stress counterparts and this was to account for the decreases in dry matter intake that we knew heat stress would induce in our cows for this portion of the study we collected liver biopsies at the end of each phase two and then we took those to the laboratory and performed tissue explants where we incubated tissue in media for two hours and then use that to extract protein and perform western blot analysis for the statistical analysis we performed all of our analyses in sas using the mixed procedure our main effects were considered diet and environment and then we also looked at the interaction of those two and then to account for that crossover design we included the sequence and period in the model and so we'll move into the results here just to orient you to our graphs they're all going to be set up the same way we'll have our environmental conditions thermal neutral and heat stress on the x-axis and then our relative protein abundance on the y and then our blue bars are going to be that controlled diet while the orange bars are going to be the ruin protective methionine diet and so on this first graph we have the environmental by diet interaction for phosphorylated mtor and this is going to be our active form of mtor and we see that room protected methionine supply led to greater abundance of this pm tour under thermal neutral conditions suggesting an increase in protein synthesis here however under heat stress conditions we see a decrease in tour with room and protect and methionine compared to control so this suggests that potentially and we have a shift in protein synthesis when we supply roman protective methionine during heat stress and it's possible that methionine is being used for some of its other functions rather than protein synthesis here on this graph we have the abundance of phosphorylated eukaryotic initiation factor 2 alpha this is also a protein involved in that mtor signaling and we see a tendency here for an environment by diet interaction and so we see that the abundance here of peif2 alpha increased in our control cows during heat stress compared to thermal neutral conditions suggesting that heat stress may be altering the initiation of our hepatic protein synthesis here and then on this graph here we have some of our other proteins involved in that mtor signaling and we have our main effects of environment and diet shown and so what i want to focus on here is going to be the fact that we have an environmental effect here of heat stress on the ratio of phosphorylated 4ebp which is our eukaryotic binding protein to the total amount of that binding protein and we see that this ratio increased under heat stress compared to thermal neutral conditions again suggesting that we're changing some of our protein synthesis pathways when cows endure heat stress on this slide we have graphs for the abundance of total akt on this first graph and then the abundance of phosphorylated akt and so this is going to be a protein that is also involved in mtor signaling but insulin signaling as well and so in this first graph we see that the abundance of total akt was increased by heat stress in our control diet cows we see something similar for phosphorylated akt which is going to be our active version of akt where the heat stress cows actually on the control diet had greater abundance than all other treatments and so this suggests that potentially there was a change in insulin signaling here but we do want to explore this pathway further this is going to be the start of kind of that insulin signaling so we do have plans to measure other things such as insulin receptor and some of the transporters and that to fully understand whether this is actually a change in insulin signaling and then lastly on this graph we have the environment by diet interaction for a protein called colon 3. this is going to be an inhibitor of a transcription factor nfe2l2 which transcribes many genes related to antioxidant signaling and so we see that the abundance of colon 3 was up regulated in our control cows during heat stress compared to our thermal neutral conditions however there was no difference due to ruben protected methionine under thermal neutral conditions but it did tend to be lower than those control cows during heat stress and so this suggests that potentially room and protective methionine is helping to maintain our transcription of antioxidant genes and helping to maintain our normal oxidative status during heat stress and so our overall preliminary conclusions from this work so far are that rumor protective methionine supplementation during heat stress may help cows to maintain hepatic homeostasis in mtor insulin and antioxidant signaling however we do have more work that we want to do and we do plan to do more measurement of proteins involved in mtor insulin and antioxidant signaling to really dig deeper into those pathways and then we also have plans to look at enzymes and metabolites involved in those one carbon metabolic pathways particularly at the enzyme level to understand if we're really changing antioxidant production with the taurine and glutathione here and so with that i just like to wrap up and thank um everybody who contributed to this project particularly my lab members in the nutraphysiogenomics lab as well as members of the dairy focus lab here at the university of illinois and then particularly i'd like to thank ediseo for their funding of this project and so with that um i'll take any questions um that you guys might have thank you danielle that was an excellent presentation and uh i hope the brains of the audience are not numb by now uh it is uh it is a hard hit starting the the friday morning uh but it was excellent and i really appreciate your clarity in in in explaining all these effects uh just for clarification to the audience uh th this is a part of an overall uh work uh there is a a a a published paper already publishing the results of this uh of these uh uh how the cows did uh and perform uh and and it is published on the journal of the science uh the references general theory science 2019 issue 103 page 22800 in case that you want to understand better the design and how the cows did daniel i do have a question for you um the uh uh uh you you do have uh these uh uh signalings uh that apparently uh do uh have an impact on preventing meal fat or meal protein or the overall metabolic status of the cows um do you um do you i mean and you look into obviously i mean methane do you think that there might be other nutrients that may help on that or or more specifically other amino acids that can help uh in this process yeah that's a a great question and something that we've been kind of exploring we think that there's potential for arginine to really have an effect here it's also linked to that mtor signaling as well as antioxidant status and so we've been doing some work over the last couple years myself included looking at arginine and as well as a potential interaction between arginine and methionine and so we actually have a couple abstracts um that were presented this year at adsa really trying to understand the length there we're seeing that potentially there is some benefit so far at the mammary gland level is what my work is showing um to supplementing both methionine and arginine excellent yeah i think this is a very hot and very interesting area of research i do have a couple of our questions here coming in as we speak uh one question is uh do you think if methionine is oversupply can negatively affect insulin dynamics or insulin resistance very interesting question um i'm not sure to be honest i would need to look into that a little more but i don't think there's been a whole lot of work so far looking at if methionine is over supplied in our dairy cows and particularly at the liver level you know i don't think we fully understand um these potential effects here yet at the liver level um we don't really know we don't really consider i should say hepatic insulin signaling as much as when we look more at like adipose tissue in that here in our dairy cows um so i do think that this does need more exploration and the effects that we're seeing here are potentially really interesting because we don't necessarily think about looking at hepatic insulin signaling so much so i think we would need to do some more work to fully understand if there would be any negative impacts there okay well uh home work to do uh it knows to be taken uh and then i will just ask you another question and then we'll move on with your second presentation danielle but i think this is also a good question in terms of uh uh is there any effect of uh heat stress on the activation of mtor or heat state or during heat stress can lead can be led to the reduced uh phosphorylated mtor so one of the hypotheses that we kind of have at the moment is that in this experiment we saw an increase in phosphorylated or active mtor with that heat stress and we think that that and the other changes that we saw potentially suggest at the liver level that we're increasing synthesis of some of our proteins and i think this could be linked to our acute face protein response here and we know that under heat stress production of acute phase proteins is going to increase and so this could be one of the mechanisms behind that change and so one thing that we're hoping to do to try to understand this response with heat stress a little more is that i do have the media that we incubated these explants in and so we hope to try and measure some of those proteins as well as some of our hormones and look at insulin and that in these in that supernatant fluid to try to understand further these effects that heat stress might be having on insulin signaling as well as the mtor pathway great thank you daniel for lack of time i know that we have more questions nice questions uh i will while you prepare for the next presentation i will try to answer the couple of our ones that are more so you go ahead and prepare for your next one uh uh danielle uh yeah uh now there are a couple of questions relating to uh to uh um main impacts on the cow's temperature and so on and so forth and those results are published in this journal of their science 10103 page two eight zero zero uh so daniel go ahead and go go for this with a second presentation please thank you very much thank you in this presentation i'll be talking about the same experiment um but i'll be talking about the effects on whole blood mrna abundance of genes related to the transsulfuration and antioxidant pathways and so our takeaway message here is going to be that supplying room and protected methionine can enhance the antioxidant responses and transferation pathway of whole blood however heat stress is going to have a negative effect on those pathways and responses and so again we are going to be talking about methionine which many of you know is implicated as a limiting amino acid for milk production and as i mentioned in the last presentation when we supply room and protective methionine to our transition dairy cows we have observed improvements in dry matter intake milk production as well as milk protein synthesis and on top of those production responses we do see effects more at a metabolic level we do know that methionine is a functional nutrient and it has been observed to promote liver function as well as immune function in our transition cows and so those effects again are going to be best exemplified by our one carbon metabolic pathways and so we see those pathways here on this diagram again up here this time we have the methionine cycle with the transferation pathway being down here at the bottom and so we'll go through this quickly again we have methionine here which can be synthesized from homocysteine via two enzymes mtr and bhmt and then in these pathways methionine can then be metabolized to produce acidental methionine or sam which again is our methyl or universal methyl donor now sam again has a variety of functions but what we're looking at here is its ability to allosterically activate cystothinine beta synthase and thereby increase flux of this transferation pathway down here and so by increasing flux of that pathway we can increase production of both taurine and glutathione which are two of our major antioxidants and so in this way methionine can help to reduce oxidative stress and inflammation in our dairy cows however while we know ruin protected methionine can have these effects during the transition period there's been very little work with it related to heat stress and particularly in this case looking at the inflammation and oxidative stress that are associated with heat stress and as i mentioned in the first presentation we know that dream heat stress feed intake is going to be reduced leading to a decrease in milk production similar to what we see in the transition period and so because of this methionine could have a potential role in promoting production during heat stress as well as helping to reduce some of that inflammation and oxidative stress as well as improve immune function in our dairy cows and so therefore our objective with this portion of the experiment was to investigate the effects of rune protective methionine supply during a heat stress challenge on whole blood mrna abundance of genes related to immune and antioxidant responses as well as that transformation pathway and so this will be the same design as before and we use 32 cows in a crossover design our two dietary treatments our first one was our tmr with that rumen protected methionine at .105 percent of the dry matter and then our second was our control diet which was that tmr without room and protected methionine and then we also had our two environmental treatments our first one will be thermal neutral conditions which will be called tn on the tables and grafts and then we also had our heat stress conditions which we induced using electric heat blankets which you can see again here in this figure and then because this was a crossover design again we had two periods and two phases per period and so cows remained on the same diet for the entire experiment but what changed between the periods was their environmental conditions and so just to explain that again i'm using group one and two as an example we see that under thermal neutral conditions group one was exposed to those during period one but during period two they were exposed to heat stress for the second nine days we see group two is opposite of that where they experience heat stress in period one but did not experience it in period two and so we're going to consider this our sequence of environmental treatments and this will be included in our model additionally a thing to note is that during phase one all cows were fed at libidum however during phase two our thermal neutral cows were going to be pair fed to those heat stress counterparts in order to account for decreases in dry matter intake that we knew heat stress would be causing and so for this portion of the experiment we collected blood approximately four hours post feeding on day eight of each phase two we then took that blood to the lab and extracted rna and then we performed pcr for the statistical analysis we again used a mix model in sas for all of the analysis and our main effects were considered our diet and environmental effects as well as their interaction and then we also included sequence and period again to account for that crossover design so now we'll move into the results again we'll have our environmental treatments on the x-axis and our relative mrna abundance this time on the y and then our blue bars again are going to be our control diet and the orange bars will be the room and protective methionine diet and so on these first two graphs we have our interaction for two genes in the transformation pathway cbs again and then cysteine sulfinic acid decarboxylase which is also involved in the production of taurine in that pathway and so in both cases here we see that our rumen protective methionine cows had a greater abundance during thermal neutral conditions compared to heat stress conditions suggesting that there is some changes with rheumatoid methionine in the synthesis of taurine on this table here we have the main effects of environment and diet for some of the other genes in that transferation pathway and the ones i want to point out here are going to be the mrna abundance of cysteine dioxygenase 1 cystothionine gamma lys here as well as glutathione synthase and so for the first two cdo and cth we see a down regulation in their abundance with heat stress compared to those thermal neutral conditions and then we see a tendency for the same effect here with that gss and so this really points to the fact that heat stress is potentially going to be down regulating our transferation pathway and leading to less production of our antioxidants looking at genes involved in our antioxidant response on this graph we have superoxide dismutase one and we see here that our rumored protective methionine cows had a greater abundance of this enzyme during thermal neutral conditions compared to heat stress however there was no difference under heat stress here between the control and room and protective methionine diets then on this last graph here we have the interaction for a gene involved in immune responses toll-like receptor 2 and we see a tendency here for that environment by diet in interaction and we see that the mrna abundance of this tlr2 was increased in control cows during heat stress compared to thermal neutral conditions i'm really hinting at the fact that heat stress might be up regulating our immune responses dream in the whole blood and so this could really be contributing to increases potentially in inflammation as well as some dysregulation of the immune cell function and so overall so far these results highlight the negative effects of heat stress on the whole blood transfuration pathway and antioxidant responses they also highlight the fact that rune protective methionine may enhance the antioxidant responses in the transformation pathway of whole blood helping to potentially reduce oxidative stress however we don't see those effects of ruin protective methionine during heat stress and so we do plan to do some more work with these samples as well we plan to measure some of the cytokines in plasma from these cows at this same time point and then we also want to look at the mrna abundance of other genes involved in one carbon metabolism to really dig deeper and understand metabolism by methionine stream heat stress and so with that again i'd like to wrap up just by saying thank you to everybody that contributed to this project um especially the members of my lab group in the nutraphysiogenomics lab as well as the dairy focus lab at the university of illinois and then again a big thank you to adeseo for their funding of this project and um with that i'll take any questions related to this presentation as well thank you danielle for uh for again for another excellent presentation and uh and giving us all a big headache uh on a friday morning uh but uh i i just for the lack of time uh i do have one question for you and then we'll move on with the next presentations but just to put all these uh all this uh work down to earth um what would you suggest cows when whenever they hit stress obviously we know what happens and then they it takes a long time for them to recover uh particularly production with composition what would you suggest to a producer to use any nutritional strategy to help ameliorate this effect or help to cure it after it happens i think they're it would be very beneficial i think anything we can do to really improve those cows and help them recover faster it can really help one for one the cow to be healthier and have better wealth fare but it can also help the producers economically and i think that methionine in particular could be really important there and i think that's another step to potentially look at in the future is to really understand is this having effects after heat stress helping those cows recover excellent well thank you danielle for taking the time uh and and doing these presentations uh for lack of time or in in respecting the time for the other presenters uh we're already five minutes uh behind i will introduce the next presenter uh and the next presenter is uh jorge carnero uh he is down south very much down south uh in brazil uh he is a student of dr rodrigo almeida and uh i i will just uh uh let uh jorge to uh move on with his presentation thank you thank you daniel thank you brian and all the dead sale group for the the opportunity and my my presentation is working yeah now it is a jorge yes okay well um my name is georgina hickey carnero and i am a page student a student in the university of south of brazil and my advisor is dr rodrigo meda and we are going to talk a little bit about our projects that has the name effect effect of rooming protect methionine supplementation and its relationship with lipids metabolism and high producing dairy cows with different dietary energy concentration when you look at the previous studies with a high methylene concentration the diet in additional uh to the constant effect in in milk protein content with methionine we realized that in some studies there were a milk fat increase also in particular in post-partum trials beside that we observe in some studies that the cows became a little bit more efficient to carry over the consume energy to the milk for example and in recently some some studies has shown a post-absorptive effect of methane in milk fat synthesis in memory epithelial cells several pathways has been suggested to explain this response but the main point seems to be linked with srvp ebp1 for example and its respective controls like crct2 and another protein contrast in this contest our our project has the the main point to evaluate the possible effect of a high methylene intake in the lipid metabolism of high producing their cows at the peak and mid of lactation into different concentration of energy in the diet as a second point we would study a possible effect on the ability to export lipids to whole body with a high methane intake and also a possible effects on the novel synthesis of fatty acids in the mammary gland to do this trial we used 112 hosting cows with 49.4 kilograms of milk and 146 days milk and average we blocked the cows by lactation number mucus and daisy milk the experimental periods lasted 38 days and we used the first three days as a covariate the tmr was orphaned once a day with or without the high energy treatment on the the wagon on the tmr and the methionine treatments was offered twice a days on top dress mux samples was collected weekly and in the last nine consecutive milkings we use it for compositional milk fatty acid profile and to future gene expression all the data was analyzed and mixed proceed of the sauce with repeated measures the experimental diets was based in a typical diet of south brazil based in corn and soy and so the first treatment was a control diet the second treatment was a supply of methionine then uses martimint then and our point was to met 2.4 percent of metallic and metabolizable protein the third treatment was a high energy treatment and we used it uh hydrogenated fat to do this and the fourth treatment was a combine of high energy and methionine we used this product a hydrogenated hydrogenated fat to increase the energy in the the high energy treatment because we don't want to change the fermentable carbohydrate in the rooming and also we we would to get the to the cows uh a fat that has a similar profile of fat and the body fat and it has the uh smaller risk to change the hydrogenated fat in the rooming in this diagram we can see uh when each procedures was done during the the trial and here is the private results in the private results we can see that high energy get more milk but without any increase in in content of the milk in fat or protein but methionine was able to increase milk fat content and milk protein content with interaction between energy concentration diet in protein content and protein yield but we we observe are reducing in lactose content when we increase the supply of methane and when we get more energy for this cause the lactose increase again the energy correct milk was not different between high energy methane and high energy plus methane and we observe a little increase in mercury and nitrogen with high energy treatments when you look to the the behavior during the time the milk protein content and milk fat content has a constant increase during during the period and the treatment with methionine was more constant in energy correct milk for example when we look the the muco energy output we can observe that the high energy methionine and high energy plan plus methionine treatment had no difference was the same and this can just suggest and for us that maybe the cows just change the the place when they put the energy in the end of the day as a conclusion the greater supply of methionine show potential to change lipid and protein metabolism interaction between energy concentration concentration and methane on total milk protein as well additive effect on meal protein production and we need more analysis to the fatty acid profile of milky and the possible pathways of the novo synthesis in the mammary gland to be better understanding the mechanisms involved between methionine and lipids metabolisms well thank you for the time for the the opportunity and i hope then i can then i could answer some some questions thank you thank you georgie for uh for the uh your good presentation uh excellent results uh very interesting very interesting the data just have one question um did the cows uh uh fed just the lumen protected methionine by themselves uh they uh did they uh uh lose body weight yeah no we i don't showed this data but no the by the way was not affected be the treatments okay thank you um so so um well the diet the basal diet had a a a good dose of fat uh it's uh you know it was a 4.3 uh the control diet compared to the uh they died with the hydrogenated fat um so um do you think that uh if the the fat content of the basal dye would have been less uh would you have seen these effects well and i think that the point is the this fat was helping the cows to increase again the the mucus for example when observed when we observed the treatment with methane the milk mucus was a little bit lower than the than other treatments and i guess that the cows expend more energy for example to synthesize fat and protein and this and the the cause then has methionine and energy and get and work with this better i i guess i mean i don't know if i we got less fat if the cause was had the same results i guess maybe not okay okay thank you um and then i see that uh did you um you show you are showing here uh and i'm going to uh kind of uh set up uh andrew's uh talk uh you did set up here the uh the grams of methionine uh metabolizable energy uh oh you have there for the lysine as well okay i got it okay okay yeah okay okay well thank you uh thank you for your uh your uh uh excellent presentation uh georgie uh and uh thank you rodrigo also uh for your work i'm looking forward for the following up results on this trial uh the next presenter it's going to be andrew uh lapierre uh he is a ph candidate uh at cornell university under the supervision of dr uh my mike barnumber so i will um let andrew uh uh go on with his presentation uh i think uh george's presentation was a nice setup for uh for uh for andrews thank you good morning andrew yes good morning good morning uh thank you daniel and thank you uh jorge for the for the great presentation um so today i'd like to talk a little bit about some of the work that we did uh in the last few months uh with the addiso group uh discussing uh digestible amino acids uh in in dairy dairy cattle their efficiency of use and what happens at it to that efficiency of use when we vary uh dietary carbohydrates in the form of starch [Music] so to start my work is interested in the interrelation between the supply of amino acids particularly essential amino acids and the metabolizable energy in which those dairy cattle uh receive uh within their diet and so that really uh stands on the shoulders of a previous phd student who did his work with the uh updated version of the cncps uh and evaluating what happens when we do relate uh supply of amino acids relative to metabolizable energy and so through some data set corroborations and generating some results he ended up finding some what we deem optimum supplies of uh essential amino acids relative to metabolizable energy and this is a common practice that we see uh with some of our folks over in the swine industry relating um standard ileal digestible amino acids relative to the energy that those cells are being fed uh and so this isn't necessarily novel but we are looking to relate this relative to energy and so with that in mind that whole principle allows us to look at some amino acid balancing relative to that grams of essential amino acid relative to energy uh which would permit us to feed lower mp diets while maintaining lactation performance and we've done this in several studies already through help without a sale so it is possible we are able to shave down some nitrogen that's being fed and more precisely implement maybe some rp products that allow us to uh dial in our methionine and lysine contents within those diets but to further expand upon that there has been some literature that would suggest that if we were to alter the supply of any gluconeogenic nutrients whether that be propionate through vfa production or actual intestinal glucose presence we do see alterations in milk protein output relatively speaking through the through up regulation of igf-1 and the somatotropic gas access pathway so there might be some opportunity there if we were to influence the supply of gluconeogenic precursors to influence our efficiency of use for those essential amino acids some studies have been done by increasing gluconeogenic supply but that's typically been done through abomasal infusion and when it's not done throughout nasal infusion the range in which the inclusion for dietary starch or non-structural carbohydrates is typically out of the range in which we would see in our north american diets so that in mind our goal was to actually vary non-structural carbohydrate levels within a typical range that we would see in north american diets and then evaluate what happens when we test that those levels of amino acids relative to metabolizable energy so we conducted a study from december to april of this past year was conducted as a penn study to mimic more of what we would see out in the industry with four different diets it's a two by two factorial design and we fed it to three pens per treatment and within those pens there were 16 cows so a total of 192 cows with a 25 75 split of permipurous and multi-brisk cows and we did want to make sure we were balanced within pens so cows were blocked by parity body weights and previous milk production as you can see that there the treatment outlines are as follow um we had two levels of dietary starch inclusion as well as two levels of supply for essential amino acids relative to uh metabolizable energy so you can see in the left hand side we have our ls100 and our ls105 both of which are being fed or formulated for 23 percent starch on a dry matter basis and then the 100 would designate the 100 grams optimum which is what you saw in the first slide trying to target those ratios there and then the 105 would be a 5 increase of those ratios to see what would happen if we were to titrate in a little more supply if we would see some greater protein output in those cattle on the right hand side we have a similar uh designation uh just that we have an increased formulation of dietary starch at 29 dry matter with the hs 100 and the hs105 we did want to make sure we're testing amino acids here so we did want to be isochloric for all four diets varying only in dietary carbohydrates and amino acid supply and we did formulate these diets using version 7 of the cncps we had a 10-day covariate for all pens they were all fed the hs 100 diet to get baseline levels uh for milk production as well as blood metabolites and um total tract digestibility and then the pens were assigned to one of those four treatments for 50 days we took daily dry matter intakes and milk yields weekly body measurements milk composition uh plasma samples every other week for pun and plasma levels of amino acids as well as uh feed ingredient tmr and refusal samples on a weekly basis uh again we used 192 cows 12 pens 4 dietary treatments and given some of the constraints that we had at the farm we had to uh split this up into two longitudinal enrollment periods for these uh for these cattle oh and then we also had three fecal spot samples for total tract digestibilities for those four diets all stats were analyzed using stats our statistical software we had descriptive feed and tmr samples in animal performance under the notion that uh our block or pen within enrollment was used and then the main effects of starch level amino acid level in time and we use interactions of those three when appropriate and then we included the random effect of cow within pen and then whenever available we use covariates and because this was a large longitudinal study we use repeated measurements with two key adjustments for those treatment differences a breakdown of the four diets you can see here on a dry matter basis and what i really would like to point out is that in order to have differences in uh starch levels and non-structural carbohydrates we did include steam flake corn and cornmeal at higher inclusion rates for that hs100 and hs105 diets but to maintain isocaloric nature for all four treatments we did have to include a high palm energy booster into the mix uh for the ls and ls105 we did utilize some urea to maintain uh room and end balance as well as the inclusion of some smartamine m and ml uh to really dial in uh the methionine and lysine levels for these diets from an observed chemical composition for these four diets you'll notice that crude protein was different as expected for the 100 and the 105 groups ticking up for the 105 groups relative to the 100 groups unfortunately looking at the starch levels we did see a relatively close 23 percent uh dietary starch of the ls 100 and 105 group but we didn't quite get to the 29 percent for the hs 100 and 105 but with that in mind we kept moving forward our mp supply here for essential amino acids looks pretty similar to what we would expect when we uh have the 100 supply relative to the 105 so particularly looking at the lysine and methionine here we did see uh the trends going up as we move from 100 to 105. our me as you can see on the bottom was nearly identical for all four treatments and then when we relate methionine lysine relative to the metabolizable energy you can observe those numbers there looking at some of the results uh for our dry matter intake we did see some differences uh relatively speaking between these high starch and the low starch diets with the low starch diets having lower levels of dry matter intake we do believe that's because of some differences in forage inclusion as well as the different types of fiber that we were feeding these cattle so the hs 100 and 105 groups did have increases in soy hulls and other uh more highly digestible levels of fiber which would have allowed for greater or quicker passage and digestion so that their room and fill wasn't always met looking at energy corrected milk the hs105 group was the highest out of the three but what we did notice was that the energy corrected milk for the amino acid level at 105 was higher relative to the 100 group we do believe that's because of different reasons one of them being that the true protein yield was greater when we look at the high starch diets whereas when we look at the fat yield it's greater for the low starch diets now we do we do think the greater protein yield is because of the starch effect however the low starch diets having the greater fat yield may be attributed to the greater supplementation of pre-formed fatty acids in the form of that high pump of fat so you can lactose yield was a little bit different but that would be indicative of the changes in milk yield uh and then the comp milk composition uh for protein fat and lactose does follow suit for those yields body weight and condition score didn't change for any of the four treatments relative to the initial or the final body weight as well look at efficiency results so milk yield and energy corrected milk relative to dry matter we do actually see greater efficiency in the low starch groups relative to energy corrected milk again we do think that is partially an artifact of of of the hot energy booster that we did feed to these cattle whereas when we do see the hs100 and 105 they're not far off but they are statistically different than the low starch group from a nitrogen efficiency standpoint however we do see that um the lower levels of amino acids were actually more efficient with their nitrogen so the ls 100 and more importantly the uh hs100 had greater nitrogen efficiencies both around uh 33 mun levels again you can see there lowest on the ls 100 and highest on the 105 level we do think that's because the ls-105 didn't quite have the fermentable energy it needed to utilize the extra amino acids that we fed those cattle so in conclusion uh increased levels of dietary starch had greater milk protein percentage and yield however the lower starch diets had greater milk fat percentage and yield so that would be attributed to why we see those differences in energy-corrected milk production for the ls-105 and the hs105 group again cattle fed a lower level of dietary starch consumed less dry matter but that was likely due to the greater inclusion of forage in the diet and it did appear that cattle on the lower starch diet reached physical fill based on fiber levels but we are looking at that further feed efficiency was improved for cattle and low starch diets but again we partially attribute that to the inclusion of high palm fat but i think most importantly and the really goes with the hypothesis of the study is that the optimum amino acid per mcal of metabolizable energy so that 100 percent had the greater nitrogen efficiency with the high starch 100 diet having the greatest nitrogen efficiency of all so there is more work that needs to be done uh juggling everything with colbit and stuff here on campus we need further feed chemistry and laboratory analysis to help clarify these results uh further so with that i'd like to acknowledge my pi mike vandenberg and everybody around us in the cornell team the cornell room knit center team for their help with the experiment and of course addiso for the financial and intellectual expertise so with that i'll take any questions thank you andrew for your your excellent presentation um i i have okay i have one question what time did you take the blood samples yes good question uh blood samples were taken uh for every uh time point they were taken um eight to ten hours post initial feeding so feed was usually dropped between six and seven in the morning and then when they returned from from milking about an hour after that we took samples from them uh in the afternoon so it would be about two to three pm in the afternoon okay uh do you think uh does it matter uh if you're in your gluconeogenic diet uh does it matter if it was glucose or propionate yeah i think it's uh i think it depends on which form that you're that that you are feeding um particularly in this study we were really going after appropriate response um to try and drive uh not only in insulin or igf-1 response but also microbial yield to really help with that protein output i do think they have similar pathways in which they work uh being pro proprionate versus glucose um but i think for this study we were really interested in in dialing in that that appropriate response yeah one more i have two more questions uh um in the india high starch diet obviously it was lower fat do you think that uh because the those cows produce less meal fat do you think that that was could have been because of a lack of long change fatty acids uh uh precursors to express the the meal fat of those cows yeah so this um you know the supplementation of a high palm fat was to help with obviously with the isocaloric natures but as it has become increasingly clear the supplementation of the type of fatty acids that you're giving these cattle will have an effect on on how they partition particular nutrients so we have been working with a collaborator here on campus um dave barbano on taking some of the milk that we sampled from these cattle and really looking at the fatty acid profile um from these cows and really seeing is if there is a response in the amount of preformed or de novo or mixed fatty acids and then how that may or may not have effect affected the efficiency of use of these amino acids that we gave these cattle wonderful uh and last question just for lack of time but one down to earth uh which one of these four diets would you feed to those cows so i think given everything we've got here we really i given circumstantially the way milk protein is these days i would really try to dial in on that hs100 diet um because that really we're trying to feed for that optimum we're really trying to hone in on uh microbial yields and really try to get a propionate response for these for these cattle so that hs 100 diet is is likely where i would go there may be some opportunity to maybe push it towards that 105 but again that's i think that's dependent upon feed availability and financial resources for those for those diets and by 100 you mean not only lysine and methionine but all the amino acids correct yeah so that's thank you that's an important distinction um yes we focus on lysine and methionine because they are most limiting but again with with this with these studies um this one that i just presented as well some others that we've done in the past we're really trying to dial in all essential amino acids um so not just titrating in different levels of lysine and methionine although having products rumor protected products does certainly help get us to where we really need to be for those levels of methionine and lysine yeah i asked that question because i know how uh how uh how much hard how hard you have to work to make those damage so yes there's a lot of a lot of uh moving parts there yes yeah well thank you andrew for your presentation and uh for the lack of time i i need to introduce you guys to the next and last presenter for today's uh webinars uh i mean for today's round table uh the next presenter is uh dr kirby carpeting uh i don't think he needs much more introduction but he is obviously a professor of nutritional physiology department in the department of animal science animal sciences at penn state university uh with that i will let you uh going into kevin's presentation thank you thank you for for having me um uh in talk to here to talk about our uh experiment where we're extending past some of our previous work that we had done with with hmtba um so bioetragenation do smoke fat depression probably doesn't we don't need to talk about that very much but it's really common cause reduced milk fat um it doesn't cause every change in milk fat that we see on a herd but it is a common cause methionine is supplemented in different ways so we have hmtba that's a rumen available form hmbi that's partially room and available and then we have rumen protected encapsulated synthetic methionine so we've previously shown in a number of experiments that hmtba supplementation reduces the shift to the alternate biodrogenation pathway and reduces that induction of milk fat depression we're feeding low fiber and high starch and unsaturated fat diets just quickly wanted to show a little bit of that data so this is our our first experiment with hmtba where we had low and high producing cows and then we fed diets that were low medium or high risk for milk fat depression so basically what's interesting with milk fat depression is that we know what our risk factors are uh we know high producing cows are at higher risk and low producing cows we know that as we increase starch and increase unsaturated fat or a number of other risk factors we increase the chance of dropping into milk fat depression so we're able to experimentally uh create situations where we have higher risk so in this experiment the the blue is control red line is hmtba you can see the low producing cows didn't have much trouble that they maintained pretty normal milk fat as we went from that low medium to high risk diet our high producing cows on the other hand as we went to a medium and high risk diet the control cows dropped rapidly in milk fat so this is a 0.7 unit difference in milk fat during high risk phase while our hmtba cows are able to maintain uh higher milk fat when we look at our transcend isomer we can see that our control cows in blue in the high group had a really big increase in transcend as we went to that medium and high risk risk diet so this really shows that there's a ruminal effect of hmtba we have a recent paper out collaboration with dr peta at upenn where we show that there's a microbial population change so we're maintaining increased diversity in the microbial population when we have hmtba and there's a lot of thinking that diversity is really important to the microbial population and maintaining normal normal fermentation there must be what some of those populations that we're helping sustain in in face of this high starch and high unsaturated fat they're important for bio hydrogenation so our objective and current experiment was to compare hmtba to other sources of supplemental methionine when feeding diets with increased risk for milk fat depression we had 10 permaparas and 26 multiparous cows this is done in kaelin gates uh and we had a five by five latin square um so the the the experiments get really difficult for me to follow once i get beyond three treatments as i have to really really concentrate remembering what all all of the treatments are i i always tease lou armitano he he designs these five by two by two experiments and makes makes my head explode we have a little bit of that uh but we really wanted to get out these differences in our different sources of methionine so we have control which is this their corn carrier we have hmtba which is targeting 30 grams of uh hmtba hmbi 43 grams of on hmbi from metasmart hmtba plus hmbi so we have 12 grams of hmtva and 26 grams of hmbi and then we have rheumatoid protective methionine which is 26.5 grams of smartamine m uh and then to make this even more complicated we have different dietary phases because basically what comes down to that if you expect to uh uh your treatments having a difference during milk fat depression you have to induce milk fat depression to see that difference right so if we go back to that that previous data showed that if we just did that experiment on safe diets like a lot of our experiments are done we don't uh give the opportunity for a product to actually show what it can do in in uh in a risky situation or when that cow is starting to get challenged uh so day one to 20 we have low risk for milk fat depression this really allows diet adaptation also uh day 21 to 27 we go to moderate risk milk for milk fat depression so we're dropping forage ndf um in increasing soybean oil in the diet so this is mimicking rapidly available unsaturated fat day 28 to 30 we go to a high risk of milk fat depression so we decrease forage ndf even more and increase soybean oil and then we make observations across these phases uh of course the the cows don't always read your experimental plan and hypothesis and sometimes uh they they don't react exactly as you would like uh go we started this experiment i believe it was in late november or early december we had to start with our new core new crop corn silage so it was not um as long through the fermentation process uh it was happened to be drier corn silage and we had a harder time inducing milk fat depression so in period one and period two uh we went into when we went into the moderate and high risk situations we didn't see very much of a decrease in milk fat so we made a little bit of an adjustment to these moderate and high risk diets we made them a bit more challenging and we did get more of a decrease in period three and period four but then period five we actually had those cows are getting further out in days and milk and then we didn't have as large a response during the induction in in the moderate and high risk phases in period five so a little bit when you look at the data uh there's there there's a little bit of a difference in the overall magnitude of those inductions during those periods um we did test for that interaction and i'll i'll come back to that in a second so our our model we have a random effective calm period fixed effect of treatment milk fat depression risk during the period are parity in their interactions repeated observations of day and subject of cow by by period so now you can understand why why uh it's really complicated to try to keep all this this data in your mind at once luckily we had no two or three-way interactions of treatment and parity so i'm not going to show any of the parity data uh today because there there weren't any interactions there we also tested for this block effect so pure block of uh period one and period two and then period three first to five where we changed the magnitude of those inductions and we did not have treatment by block interaction so that allows us to look at all the data together so i would say is that even though there's a slight difference in the magnitude of of that decrease in milk fat that we're getting between these periods overall it did not change uh what the treatments did right so so we're we're able to look at those those main main effects but uh we're modeling out those those impacts um milk yield was increased by supplementation only on on day 13 uh so to just kind of orient you to my meforma and my data and upper right hand side we have the overall effective treatment day and that treatment by day interaction so we have treatment by day interaction for milk yield and then the first line in the data is the overall effect across uh all of our experimental days and then we look at the data by uh by day and then our p-values we compare uh hmtba column is control versus hmtba and uh same thing for all the others they're we're comparing each of the treatments uh back to the control to say are they having an in impact um so if we looked at that overall effect there's a tendency for hmtba to increase milk yield uh but this impact really was coming from um from day 13. so on day 13 we have a significant increase in milk yield for hmbi in the room and protect methionine we have a tendency for hmtba and hm2ba plus hmbi then there's a tendency for or actually i think this is significant and just under p .05 uh for hmtb on day 20. this has confused me a little bit on why we saw this on uh day 13 and and not on the other days you know at first i was kind of thinking maybe this is just just something that popped up as as uh being on that day but remember this is a five by five line square so this day 13 is replicated over five different periods so this isn't that that it happened to be a change in the silage or something like that a day effect that way it is replicated and i'll show later we also have some changes in milk protein on day 13. so i think what we might be seeing here is uh some sort of a transient adaptation that's occurring uh to these diets during during that that phase i think that's that's uh interesting response uh milk fat was our primary interest so we have an overall effect of treatment effective day no treatment by day interaction it is down to 0.16 but it's not significant so if we look at our overall effect controls 3.62 across our entire experiment on hmtba is is increasing um hmtba plus hmbi is significantly higher and rheumatoid and methionine is also significantly higher uh the hmbi is is not it's numerically higher but it's not reaching reaching significance if you look at our our decrease on you know day 20 these cows are three seven seven we're taking them down to a 3.54 so we're getting a small decrease in milk fat percent and our uh responses are mostly at that medium and high risk phase and that's what we would expect um that are products able to have an effect when we start uh into those challenging situations milk fat yield is a similar um story that we have an increase with hmtba hmtba plus hmbi protect methionine here on the yield that increase in milk yield on day 13 is driving a significant increase in milk fat yield on day 13 and then we also have an increase in milk fat yield on that medium and high risk diets on day 27 and 30 that's being driven by that that change in milk fat percent so looking at trans 10 18 1 this is our best indication of that alternate biological nation pathway we have no effect of treatment effective day and no treatment by day interaction that's significant but when we look at the data with our pre-plank contrast we do have a tendency for hmtba to be lower than control um and the response is mostly on this day 27. so we are increasing um trans-10 from 0.45 which i consider normal 0.4 to 0.6 is about where we we normally see herds uh so we're we're increasing that two to three fold um so getting up to 1.24 so we're getting a a modest amount of milk fat trans 10 type milk fat depression um our our hmtba and is significantly decreasing on that day 27 but not as large of an effect in transtend intermediate as we had seen in in our previous experiments de novo fatty acids so normally we see de novos decrease during diet new smoke fat depression we do have a day effects we are decreasing these denilos but we're not having any effect uh on from any of our methionine supplements and this is a a little bit unexpected because in our previous data um we would uh we would see an increase we saw an increase in de novo um so it it seems like the changes in milk fat that we're having in this experiment are not simply from uh alleviating a classical diet induced or biogenation induced milk fat depression looking at milk protein data so there's an effective treatment and day i end the treatment by day interaction room protective methionine is increasing milk protein percent there's a tendency for hmtba plus hmbi um and as i mentioned before this effect is most significant on day 13 so all of our methionine supplements uh increase milk protein not a lot but they increase milk protein on day day 13. rheumatoid protective methionine was increased or tended to be increased on on all days milk protein yield we have a tendency for an increase in both protein yield for room protective methionine again that day 13 everybody's increasing milk protein yield being driven by that increase in milk yield um in in rheumatic methionine is more consistent in that increase so summary in conclusion our challenge resulted in only a modest decrease in milk fat so we're decreasing to about a 3.5 percent but trans 10 18 1 was nearly tripled hmtba a mixture of hmtba and hmbi and rheumatoid 9 resulted in increased milk fat concentration and yield the mechanism appears to be independent of trans 10 under these circumstances and may be due to acetate supply or post-absorptive mechanisms so uh you know transtend isomers are not the only thing that impact milk fat yield uh there's a number of other other potentials and we're really excited about the the opportunity for uh methionine to work post-absorptively to increase milk fat you know there's great mechanisms at work that have been really well investigated in liver fatty acid transport and and metabolism during the transition period and we think some of those same mechanisms could be important to supporting milk fat synthesis during during normal lactation uh so like to uh thank you for your attention project was supported by addison and penn state i also need to recognize that the support we get from usda and other other sponsors in the lab so happy to take any any questions thank you uh kevin for uh for your excellent presentation uh uh yeah i mean this is a really uh uh full circle uh that we went from the beginning to the end now and we're back into inside the uh the cell of the cow for what you're trying to say now in terms of uh the impact of uh try to elucidate what is affecting this meal fat depression uh we always uh or you know we're always they were thinking this is just just this uh lumen effect but obviously uh something else is going on uh now uh uh you did mention that there were some uh uh you did study some microbial uh uh effects uh can you expand a little bit on that or do you have anything to make more comments about it yeah so uh so so the microbial effects were not in this experiment they they were in a another experiment with with hmtba um you know so so we originally had gone in to characterize uh what what we call our our favorite 12 microbes so there would be your your common culturable microbes like strep office and fibrobacter that everybody recognizes and we we can see those change during milk fat depression when we induce milk fat depression but we were not able to see an effective of hmtba on those what we did find with real-time pcr is an increase in protozoa abundance and then we we went on in and worked with dipty to do the microbiome analysis um in talking about complex data you're you're you're looking at everything that's there right um and then you you see a lot of different microbial species um you know there there's there's a couple microbial species that um microbiologists look and see their favorite guys changing and and kind of get excited about the possibility of this one or that one i i will say that what we see is really a subtle change it's not it's not um a sledgehammer effect uh but i think this actually interesting that that hmtba um seems to be maybe targeting what are some important important populations i i i think we're you know with that you have a characterization so you're saying these guys are changing at the same time that you're seeing your production change but it's really hard to say which ones of those are the important ones that are changing um but but the biggest kind of global thing is that that increased diversity that there's a couple different metrics of diversity and and you seem to be maintaining uh more diversity now why that is i don't think we really know um but but it there is there is a positive effect there oh and and so going on the other side now uh there is another question is okay so how does rooming protected methane now prevents meal fat depression what would be how you explain that yeah so so that's where i'm really excited about possibility for um post-absorptive uh facts um you know i i think we could think about uh you know we there there's well-documented effects on liver metabolism so there would be the potential that um you are assisting in in fatty acid packaging and transport that would be making those fatty acids available to the mammary gland um i think there would be all sorts of of what i think of as sparing effects so if you're changing liver metabolism in a certain way uh you change then change the nutrients that are available to the mammary gland because they they have or have not been used by by the liver um you know i there there could possibly be some direct effects within the mamrie gland um i i don't think those are as as well documented as as what we we understand in the liver yeah and i think uh you know this is again goes back to uh you know the availability of these uh fatty acid precursors and what you're trying to and what you were saying they are the disappearing effect it's a is you know it's a is really interesting uh area of work that we need to uh to uh focus in but uh um i i i really thank you everybody uh for uh for sticking in uh into this i don't want to go much longer i really thank you kevin for your excellent presentation and every all of the other presenters just as a reminder uh these presentations have been and are uh recorded uh you could have access in feed channel uh dot online and with that i i hope to see you guys next week thank you very much have a good day