[Music] so we're gonna spend the next 40 minutes or so talking about methyl donors from transition into a lactation and methyl group or methyl donor is basically chemically an alkyl derivative which is basically a carbon that's not completely occupied all four valences with hydrogen's would be basically ch3 and we like to thank that carbon is an energy source but in this particular case methyl groups can have numerous cellular functions such as methylation of genes DNA what we call DNA methylation they play a role in phospholipid synthesis and they play a role in protein synthesis because they can affect the transcription of genes and therefore the message that's used for synthesizing amino acids and they have a dietary origin and they come from three main molecules most likely methionine quantitatively it's probably the major methyl donor but there's also folic acid or folate an intermediate of folate metabolism can donate methyl groups and another one would be an intermediate of choline metabolism so choline once undergoes oxidation can produce bake baiting and baiting is one of the methyl donors present in in cells so we have a multitude of compounds of different origins that can be methyl groups and provide methyl groups through transmetallation reaction so one of the effects of methylation is that it can affect genes it can transfer methyl groups to the DNA and the site of methylation is usually a cytosine which once and their goals methylation you become 5-methylcytosine in this reaction is carried out by an enzyme that's called dnmt our DNA methyltransferase and it requires a donor which can be one of those compounds that i mentioned before in this particular example here I'm using s adenosylmethionine as the methyl group donor which you can see it's been transferred to cytosine to form 5 methylcytosine so why is this important because during this process of methylation of DNA that alters how genes are behaved or how the chromatin behaves so here I have a diagram of a piece of chromatin that contains multiple genes in the chromosome of a cell and if this chromatin is loose and usually not methylated he can undergo transcription to generate the message for subsequent protein synthesis but if it turns out that some of those genes become methylated such as in the example that I provided before the addition of methyl groups prevent a normal gene transcription and as a general rule it's not always the case but as a general rule methylation works by silencing genes because it attracts proteins such as histone deacetylase or methyl binding proteins as well as other coverage receptor repressor proteins that blocks transcription factors to bind to the promoter region of the genes and therefore mRNA synthesis is suppressed so at the end of the day methylation reactions can control to some extent protein synthesis by controlling at the level of transcription and this leads to what we call gene silencing so that's one aspect of one carbon metabolism another aspect of one carb metabolism is that methyl donors play a role in phospholipid synthesis and we know that phospholipids blaming by multitude of roles in cell biology and phospholipid can be synthesized de novo through this pathway that's called CDP choline pathway in which choline of dietary origin can be activated to produce phosphocholine by the addition of a phosphate group from ATP and this can undergo reactions to form CDP choline that then condenses with a set of fatty acids usually a group of long-chain fatty acids in diacylglycerol to form phosphatidylcholine which is the major phospholipid in most a million species there is a second pathway that's typical of deliver that's called salvage pathway and as the name says it is used to recover substrate or molecules that are part of the phospholipid group so in this particular case phosphatidyl ethanolamine can undergo a series of methylation reactions coming from s adenosylmethionine to produce phosphatidylcholine in phosphatidylcholine is one of the phospholipids that make up what we call letting in less sitting or phospholipids in general they are building blocks for cell membrane but they have this feature of being none fulfilling with amphipathic properties so they have a polar head that is usually hydrophilic and they have a tail that's usually lipophilic and that becomes very important when you're transporting lipid like or FET like compounds in a food solution so for example here I ate a molecule or a compound called very low density lipoprotein that is secreted by parasites to carry triacylglycerol cholesterol cholesterol esters from delivering to the general circulation to redistribute lipids to other tissues obviously those lipids would not easily mix in blood unless they are in mollified and one way of combining these lipids in a compound that can be transferred transporting blood is by incorporating proteins or phospholipids into this big compound and here Alice trait the location where phosphatidylcholine or this lipotropic compounds such as phosphatidylcholine phosphatidyl ethanolamine can be present that combines this polar head with the lipophilic tail so this becomes really important for fatty acid transport not only in the blood as the liver secretes lipoproteins but also absorption from the gastrointestinal tract as well as transport across the Attilio cell in the mammary gland so it has multiple roles there and there are multiple pathways that are interrelated and I'm gonna give you one example here of those pathways using a central molecule that's called choline but I could pick something else so in this particular case here choline which is a water-soluble molecule can undergo a set elation to form acetylcholine in neurons or in the nerve tissue which is an important neurotransmitter in the central and peripheral nervous system for communication between neurons or you can undergo oxidation to form bating which is one of those methyl donors as you can see it contains three methyl groups and when bating donates a methyl group to homocysteine it can form the methyl glycine which now has only two methyl group in one of those methyl groups is transferred to home thing homocysteine can be used to reset the size methionine through an enzyme called methionine synthase in which homocysteine receives a methyl group from 5-methyl tetrahydrofolate and just to be clear this morning there was some discussion I just want to point out that this enzyme here methionine synthase requires a cofactor which is cyanocobalamin which is vitamin b12 so this is an enzyme that for proper function requires B vitamins and many if not all of the B vitamins work one way or another is a cofactor in those biochemical reactions so to no surprise in this particular example cyano cobalamin is a cofactor in this reaction that facilitates the transfer of a methyl group from five methyl methyl tetrahydrofolate to methionine resynthesizing tetrahydrofolate so here we see an interplay between compounds that originate eventually phospholipids B vitamins and amino acids methionine can undergo a further conversion in accepting a dimaggio group in form s adenosylmethionine which is another methyl donor that can donate multiple it can donate a methyl group per step to synthesize phosphatidylcholine in what's called the salvage pathway as you will see so I showed you before that choline can be synthesized through the the novel pathway that's called CDP choline pathway which is this here or choline can be synthesized in the paddock tissue through the salvage pathway that requires phosphatidyl ethanolamine that receives methyl groups from s adenosylmethionine to be converted eventually in phosphatidylcholine so here we see the connection between or among B vitamins amino acids in phospholipids becoming very important so that's another all of one carbon metabolism becomes really important there's another one that's called the transpiration pathway that becomes really important in terms of providing antioxidant molecules that's not illustrated in this diagram here just for the sake of simplicity otherwise we would spend a lot of time just talking about those things so why is this important for the transition cow well we know and you know very well that transition cows have reduced intake as they approach for iteration particularly in the last week of gestation and they have inadequate intake of dry matter in the first weeks after cabin which lead to negative nutrient balance because they because the diets that we feed prepared that typically contain a large amount of NDF and in many cases very undigestible in the earth and because intake is low it's no surprise that there may be a possibility for inadequate intake of essential amino acids because microbial synthesis is reduced because there's just less substrate in the rumen for microbial growth in methionine can be decrease in supply and we know the meta means typically one of the limiting amino acids for milk protein synthesis in typical diets based on corn and soybean but also because the synthesis of phospholipids require transmetallation reaction in a feature in the ruminant is that when cows or calves or heifers consume dietary ingredients that contain choline it turns out that the rumen microbes degrade most of that choline in the rumen therefore very little reaches the small intestine to be absorbed so the pathway that I showed you initially very likely the de novo synthesis of phosphatidylcholine from dietary choline most likely is in short supply during the spirit because degradation of choline in the rumen and the low dry matter intake on top of that we know that amino acids are limiting because right at this time is synthesis of protein in callosum that's sucking up a lot of the amino acids that have been absorbed so very likely the supply of methionine for transmetallation reaction through s-adenosylmethionine may also be in short supply so we would expect that availability of phospholipids in this period would be lowest compared to other stages in the lactation cycle and I'll show you some data on that well this is important because in the many aspects of intermediary metabolism in transition dairy cows lipid become lipids or fatty acids become a central point and we know that during this period in which the mammary gland sets a priority for not nutrient utilization cows rely on adaptation to increase the rate of lipolysis of fat tablets that are needed to supply calories for the mammary gland so there will be mobilization of triacylglycerol that results in circulating free fatty acids that are transport in blood associated with albumin primarily that can be transferred well I don't know why my computer connected with this let me close this that can be transferred to the mammary gland and provide mobilize fatty acids mostly long-chain fatty acids c16 or longer for milk fat synthesis but some of those fatty acids can also be taken up by a parasites through a transport system and they will be a source of energy to the paddock cells and during this process not only will generate ATP but some of them can undergo ketogenesis once the energy needs of that cell is met so now the cow generates ketone bodies that can carry carbon sources in a water-soluble form that can be used by other tissues such as the muscle the brain or even as a substrate for synthesis of looks at through the de novo pathway of milk-fed synthesis but if the amount of trials of fatty acid is taking up overcome the ability to synthesize ATP synthesize ketones in export those fatty acids they eventually accumulate estrella steel glycerol because those various will be raised terrified within the cytoplasm of the parasite this triacylglycerols can eventually be exported from the liver as very low density lipoprotein but it turns out that ruminants have limited ability compared to monogastric stew secrete VLDL and as those triacylglycerols accumulate in the cytoplasm of the cell it can induce inflammatory response we can induce acute phase response and it can damage the cell and outer it's metabolic function so we don't want excessive amounts of triacylglycerol glycerols accumulating in the parasites of dairy cows although most of them will have some degree of accumulation so are there nutritional interventions that can play a role in this this processes that would benefit cows and we're going to spend some time talking about them so first I want to touch on how common is fat accumulation in the paddock tissue in dairy cows and there is a really extensive review and literature published about 15 years ago in which they summarized multiple XOR studies in the literature that had collected a paddock tissue and what they showed was that if we use the cutoff of 5% triacylglycerol in the wet tissue basis of the liver many many cows will have that amount so typically in a well-fed cow they have one perhaps 2% triacylglycerol in the paddock tissue but as you can see here anywhere from more than 15% of the curls to as much as 65% of the cows had between 5 to 10% triacylglycerol in another fraction of the cows and more intent intransitive literal so if we put it all together what we see is that almost half of the cows will develop some degree of moderate fatty liver during the first weeks after calving so usually that accumulation is maximum between the first and second week and then slowly those triacylglycerols it'll get eliminated or they get oxidized from the liver so are there things that we can do to reduce this okay so obviously phospholipids have lipotropic effects and by having this lipotropic effects they play a role in transfer of triacylglycerols from the appellate issue among other tissues so here you can see in a case control type of study in which this group they looked at the amount of microsomal transfer protein the abundance of a polical protein be 100 in a polyp of protein E those are our April proteins that are important for VLDL assembly and secretion and this is a transfer protein that transfer triacylglycerols across cell membranes and what they showed was that cows that developed fatty liver they had smaller abundance of those important proteins for fatty acid or triacylglycerol metabolism with in the etheric tissue in monogastric cin mice supplementing choline to animals that were fed they calling the fish and diet as you can see enhance the ability of the paddock of the intestinal cell the entire site to secrete a poly pool protein b48 and this is the main April protein that's incorporating into chylomicrons during the absorption of fatty acids in the lymphatic system so this would make the case that sentences of April proteins or VLDL assembly can be compromised in causal developed fatty liver and maybe there is some relationship in terms of cause and effect there and this also shows that fat absorption in the gastrointestinal tract can be compromised when the availability of substrate for phospholipid synthesis is it's less than ideal okay so as I mentioned to you it's no surprise that because of what happens during the transition period it's right at a time when the availability of phosphatidylcholine is leased in dairy cows right between the week of calving in the first one or two weeks after canning so we would think that if I can enhance the availability of phosphatidylcholine one way or the other perhaps this would benefit transition cows and we'll touch into that during this presentation now we know that phosphatidylcholine has lipotropic effects and we know that if we deliver substrate for its synthesis in this particular case by supplying choline and a rumen protected form might be beneficial so this is what these two experiments showed in the first one they use a dry cow feed restriction model and they looked at cows are received or did not receive choline in a room in protected form and then they measure triacylglycerol in the liver during the feed available time and then after cows underwent feed restriction and what they show was that by causing cows to not consume feed they mobilize body fat that accumulates in the liver but the accumulation is last when they receive supplemental remain protected choline the same was observing this experiment by Marcos and OB here in Florida but now he titrated the dose of choline ion in a woman protected form he fed from zero grams of choline ion all the way up to almost 26 grams of choline so this would be approximately 30 or 32 grams of choline chloride being fed in a room protected form and what Marcus showed was that during feed restriction this cows had a linear decrease in a paddock triacylglycerol accumulation in the liver as the dose of choline increase we just replicated this experiment recently with about a hundred with exactly one hundred and ten pairs free part and cows this cows were at two hundred and thirty two days of gestation and they were assigned randomly to one of five treatments so we had 22 cows per treatment and I'll go through the basics of the experimental design so we had a control group in which cows received no choline ion and then we had treatment win which cows received twelve point nine grams of choline ion in a form that is has low concentration of calling chloride in the entire product so there's a lot of coding lipids or they receive almost 26 grams of choline ion in the same form the next two treatments we use exactly the same dose either twelve point nine or twenty five point eight but now they were supplied in a room protected form with a high concentration of calling chloride the experimental design was quite simple cows underwent five days about Lipton intake and then we impulse feed restriction for nine days in which they were allowed to consume fifty percent of their net energy for lactation required for maintenance and pregnancy assuming a 42 kilogram newborn calf so I'm show you some of the results so as expected we control intake during feed restriction this cows were consuming about seven mega calories of net energy for lactation and this is exactly 50 percent of the estimated amounts of net energy required for those cows based on NRC so to no surprise concentrations of nifA and BHP they were elevated to for a dry cow but they were exactly the same across the different treatments so we were interested whether the amount of choline or the form in which the room protect choline was supplied would affect the paddock triacylglycerol accumulation and as we had seen before in previous experiment we had exactly the same response so when we look at a paddock triacylglycerol after nine days of feed restriction on an ass's on a tissue basis in the control group we had more than in the choline treated cows and as we increase the dose of choline we can see a further reduction so we had an infection an effect of choline supplementation and amount of choline supplementation the Zacks same response was observed when we correct the triacylglycerol to tissue dry matter basis when we look at glycogen content it increased when cows receive rumen protected choline compared to the control and the same was seen on a dry matter basis so when we look at the ratio of triacylglycerol to glycogen it was more favorable in the cows the receive room protected choline in particular those that receive a larger amount of room and protect : so we can see that in the cow as well as in the monogastric in which the literature is much more abundant choline in fact has this lipotropic effects we just don't know the exact mechanism yet and we are working on that as well as other people are now let's just look at what happens when we feed this to the transition period here I have three experiments that show different responses in the first one the concentration of triacylglycerol hepatic tissue was less for the choline supplemented cows in the second one there was no difference in the third one choline supplemented cows actually had greater concentration tries to glycerol so this heterogeneity in the data in general is associated whether choline stimulated or did not stimulate dry matter intake because in almost every experiment choline has been able to stimulate milk yield in particular fat cracked milk yield so when milk you'd increase without a concurrent change in dry matter intake we see less of a positive effect or no positive effect on a territory so blister all but when intake is stimulate we see a marked reduction interest of glycerol in the liver we recently reviewed literature to look at responses to choline supplementation starting pre Partin and carry on through the early postpartum period in during this process you go through searching the literature in which we found lots of papers that cite the word choline but many of them were either duplicated in different engines that we use to search eventually we screen records many of them had data that we were not interested so we were interested in choline for pre parting cows fed in a room in protected form so we were able to find 20 papers with 21 randomized experiments in which choline was randomly assigned to cows yes or no and we were interested in looking at animal responses such as production responses as well as health responses just for the sake of time I won't be able to show you everything but I'll touch in some key aspects we were interested in answering a question what is the dose of choline that optimizes the animal response and whether there was an interaction between choline and supply of methionine in the pre or post partum diets of those cows and I'll show you data on milk yield energy correct meal killed and milk components so here's just to show there were 30 treatment means in which cows received no choline in every single experiment there was an untreated control group in which they receive zero grams of choline ion and then there were 36 treatment means in which cows received some amount of choline that ranged from five point six to twenty five point two grams so this is the range of choline intake in the cows given that this cows were supplemented with choline but there was always a control group in within the same experiment and you can see here what was the intake of methionine in those cows pre part an impulse part now show you a histogram here for the cows that were in the those experiments during the prepartum period so you can see intakes of metabolized methionine that averaged approximately 25 grams and that represent approximately 2 to 2.1 percent of the metabolizable protein and there were some experiments that supplemented protected amino acids to to a very large amount so they got to values that were quite high if we look at the postpartum diets we see larger quantities because these cows were eating more dry matter but if you look at the ratio of metabolized methionine to the metabolizable protein of the diet it follows somewhat of a similar pattern with a few experiments in which Paul Spartan diets were supplemented with a substantial amount of room protective material so let's just look at what happened to intake you will see figures like this the dots that fall on the zero grams of choline ion are the control treatment means or the zero grams in this y scale you're gonna see the response in the x-axis you're gonna see the amount of choline ion in those mix models you the size of the symbol represents the weight of that treatment mean in the statistical analysis we use meta analytical approach to this the lines connect treatment means within the same experiment and the continuous dark line represents that there wasn't a statistical effect and here is the direction of the response which you can see it's a linear response with an increase of this level for each gram of choline that was added to the diet during the transition period if we use the median amount of choline ion fed in those experiments of twelve point nine grams we observe a two hundred and thirty gram increase in dry matter intake prepartum a half a kilogram increase in dry matter intake postpartum so that's dry matter intake there's a slight increase it's not a huge increase so here's yield some milk and energy correct milk we see a linear response to choline supplementation in terms of milk yield and in terms of energy corrected milk EO okay but pay attention to this there is an interaction between choline and the proportion of methionine as the metabolize were protein in the diet so this interaction here means that the response to choline was to some extent depend upon the supply of methionine so here we see that pathway that we just discussed in biochemistry playing a role in cow biology and animal responds so here is a forest plot that shows the energy corrected milk across the multiple comparisons so you can see what direction that response went so if the point estimate which are the black dots fall to the right of the central line it means that supplementing choline increase milk yield or energy correct milk you if the point estimate falls to the left it means that it numerically decreased milk you okay you can see the confidence limits in the overall response which is the dashed red line with the random effects here showing that there was an increment in energy correct milk across all comparisons of almost point four standard deviations which translates into approximately two point two kilograms of milk but I mentioned to you that this was depend upon supply of metabolized vomit alanine because we observed some heterogeneity in the response because Magne to the response was depending on supply of methionine and this is how the data looked like when diets had moderate to low proportion of the metabolizable protein as metabolized Matheny the response to choline was substantial okay but when diets had a quite large amount of the metabolized protein as metabolizable methionine now the response to calling was to present but became smaller okay and the same was true for energy corrected milk so if we look at when metabolized well methionine was 1.8 percent of the diet of the metabolize of protein of the diet I'm sorry the response to twelve point nine grams of choline ion was 2.1 kilograms of energy corrected mule when cows received the same amount of choline ion in a room protected form but the diet now contains two point three percent metabolized protein they still respond positively but the response was less now 1.2 kilograms of energy corrected milk so here we can see that there is a relationship between these molecules but there is also some independent effects because we see responses regardless of the level of metabolizing methionine but they attenuate at the very high amounts so here's concentration and yield the milk fat milk fat goes up there was a quadratic relationship with : with the optimal amount somewhere here in terms of concentration of fat but the yield respond exactly like energy correct milk or milk you owe the same was true for milk protein we saw a linear response to amount of choline supplemented to cow so this is telling me that choline is able to stimulate meal production the interesting thing and I'll touch on a couple of other aspects about this source of methyl is that the response girls above and beyond the period of supplementation this this is one of two experiments that we conducted here in University of Florida in which girls were supplemented during the transition period starting three weeks before the expected day of calving up to 21 days postpartum so an average disc out stay in prepar died about 18 to 19 days and until exactly 21 days postpartum and you can see that there was an increment in milk yield but this increment in milk yield lasts for a quite long period of time in fact for the first 40 weeks Falls part and there was a tendency for an increase in milk you dove about 2 kilograms in this particular experiment in the second experiment in which cows are supplementing again during the same period of time in their lactation cycle we saw an additional 2 kilograms milk response after 25 weeks Falls part so to no surprise things that we do during the transition period that benefits cows they tend to last longer than the period of supplementation because many of those intervention have impacts on animal health and they have effects that are other than just a nutritional component of the particular supplement that you're providing one of the pressing questions that many people ask is what cows respond to choline are only the ones that are more prone to fatty liver so we pull together data from two experiments in which we collect body condition score twice before enrollment in the experiment and we had cows they receive no choline supplementation or they receive 12 point 9 grams of choline ion in a women protected form so you can see here the distribution of body condition score of those cows and you can see that they are quite similar across the two treatments so we had cows though who had moderate body condition score and we had cows that were over condition so we would once perhaps would expect that this cows would be more likely to respond to calling because of the notion the choline has this lipotropic effect and over conditioned cars usually are more prone to in disease that are associated with intermediary metabolism or lipids but what we found was that the response to choline which is the difference between the blue line which is the control and the orange line which is the choline supplemented cows for milk yield and for energy correct milk yield as well as for every response that we measure was consistent across the multitude a body condition score so each dot here represents a cow in the data set there is about 215 cows here and the point I want to make is that you can see the response it's an increment in milk yield an increment in energy corrected milk yield of approximately 2.2 kilograms irrespective a body condition score we saw no interaction with body condition score so my point here is that the cows cows respond to rain protect : whether they have low or they have high body condition so because choline is interrelated with supply of metabolizer amino acids in particular Mitani i'm gonna finish up just showing you some data of protein in prepartum cows we did exact same exercise as we I had shown you before for collecting data in the literature from published randomized experiments in which the cows were supplementing or not with choline we did the same with protein pre Partin but now this experiments had to feed different diets pre part and manipulate only protein either the amount of protein in the diet the degradability of the protein source or supplement amino acids but only pre pardon the pol Spartan diets they were always the same ok so at the end of the day we were able to identify 27 experiment in which cows were randomly assigned to receive different concentrations of protein the diet or different protein sources or be supplemented with amino acids pretty pardon and I want to emphasize that because you were gonna see subsequent webinars in which people are gonna focus on supplement amino acids pre and post part in a lot more detail than tonight so we were interested in understanding whether protein pre Partin affects post part on intake yields of milk fat cracked milk and milk components body weight and body condition score so what we did is we pull the data from literature we identified the diets and the dietary ingredients we entered those diets into NRC we entered the observed dry matter intake of the particular treatment in the treatment meal and then we calculated the supply of net energy metabolizable protein and metabolizable amino acids and we analyzed the data for nulliparous separate from parents cows just because as you will see nulliparous eat less so the supply of metabolize protein is quite different so the dots here represent the diet's that were fed in the different treatments to nulliparous which are the triangles into the paris cows which are the open circles and you can see that for a given amount of crude protein in the diet there's a substantial difference in the supply of metabolized protein and the reason is quite simple cows eat more than half Earth's and by simply eating more there will be more microbial growth there will be more under gradable protein that gets to the small intestine so the supply of metabolizable amino acids goes up just because of the differences in intake so if you look at two particular amino acids metabolizable methionine and lysine we see exact same response Paris cows consume more of those in some diets cows were supplement with protected amino acids so then the amounts were substantially greater so that is true for metallian in lysine or any amino acid that we looked at in that data set so just quickly I'm going to show you the data on heifers or first lactation animals there were no Lipper's cows before calving now they became first lactation and in Paris cows the same system the size of the symbol represents the weight of that particular treatment meaning the analysis if you see a central dark line means that there was a statistical effect which I didn't place here but if that central line is not present there was no statistical effect okay and the dashed lines connect the treatment means within an experiment so to no surprise as the intake of metabolized protein pre part an increase in our upper limit observed was 1100 grams for the heifers there was a linear increase in milk yield in energy corrected milk EO but when we look to the data from the Paris cows we saw basically no response whether the cows ingested 900 grams of metabolizable protein or they consumed 1.4 kilograms of metabolizable protein pre part and we saw exactly the same milk response or energy correct milk response so no benefit to Paris cows a major benefit to newly Paris cows when we look at fat yield exactly the same as I showed you before protein yield exactly the same so major benefit to nulliparous cows and there are reasons for that not only they have a growing fetus they are creating a lean tissue so they need to add muscle mass during this period whereas if we look at the Paris cows we saw no response in fat yield in a small response and protein yield that was only observed in the experiments that had cows producing more than 36 kilograms of milk per day postpartum in experiments that had cows producing less than 28 kilograms of milk per day we saw no response to supply of metabolize protein prepartum so if a cow consumed 1100 grams of metabolizable protein prepartum the estimated supply of metabolizable lysine was approximately 92 grams or 94 grams and the supply of metabolizable protein was approximately 26 to 28 grams okay so if we want to translate that into individual amino acids okay just to summarize include supplementing choline ion as a room in protected choline to feed restricted cows it's unquestionably that it has major lipotropic effects and it reduces triacylglycerol in the risk of fatty liver supplementing : to transition cows benefit lactation performance and this benefit goes beyond the period of supplementation we saw responses observe in dives with low or high metabolizable methionine postpartum but there was an interaction suggesting that there is an interplay between them but there's also some independent MEK and is present there in the response were observed regardless of body condition score of cows we really don't know the optimum amount of choline ion we think that's probably closer to this 25 grams but we need more data on that just lastly the amounts of metabolized protein pre pardoned they differ between the nulliparous and Paris cows and I think there's good biology to justify that based on the published data we think that Paris cows need anywhere between 800 to 900 grams of MP prepartum which approximates to 20 grams of metabolized methionine whereas heifers nulliparous need at least 11 in grams we don't know whether they need more because there are no data on that okay in this result in approximately 26 to 28 grams of metabolized metal yoni pre pardoned for heifers so thank you for your attention and I'll be glad to take any questions [Music]