This Real Science Exchange podcast episode was recorded during a webinar from Balchem’s Real Science Lecture Series. You can find it at balchem.com/realscience.
This Real Science Exchange podcast episode was recorded during a webinar from Balchem’s Real Science Lecture Series. You can find it at balchem.com/realscience.
Feeding rumen-protected choline in early lactation has consistently increased milk yield and energy-corrected milk yield, which is more pronounced when cows are fed diets low in metabolizable methionine. Choline feeding also increases milk fat and protein yield, minimizes body condition loss in early lactation, and reduces postpartum disease incidence. Dr. McFadden presents three topics about choline biology in the dairy cow. (01:45)
Fatty acid nutrition to optimize methyl donor efficiency. (4:02)
Fatty liver is a concern for fresh cows because of its relationship with ketosis, poor fertility and compromised milk production. Cows with fatty liver exhibit low circulating concentrations of phosphatidylcholine, which is a component of very low-density lipoproteins (VLDL) that transport triglycerides out of the liver. Feeding rumen-protected choline lowers liver triglyceride deposition by supporting the synthesis of phosphatidylcholine and thus, VLDL.
Dr. McFadden goes on to explain the two different pathways for phosphatidylcholine in the liver and how those interact with fatty acid metabolism. He describes several experiments that have investigated how rumen-protected choline and supplemental fatty acids interact in lactating cows.
Low phosphatidylcholine supply is a key feature of fatty liver in dairy cows, likely due to low polyunsaturated fatty acid (PUFA) and low choline supplies. Delivery of post-ruminal PUFA may support phosphatidylcholine synthesis with accompanying improvements in insulin sensitivity, body condition maintenance, and inflammation, but interactions with dietary fatty acid digestibility should be considered. Dr. McFadden gives a list of considerations for fresh cow diets incorporating fat and choline supplementation.
Gastrointestinal choline degradation and trimethylamine N-oxide (TMAO) (16:58)
Unprotected choline is almost totally degraded in the rumen. Microbes convert choline into trimethylamine (TMA) which is then converted to TMAO in the liver. Rumen-protected choline allows for a large proportion of choline to reach the small intestine intact. However, research shows that choline can also be degraded by microbes in the small intestine in the same pathway, limiting choline bioavailability. Plasma TMAO accumulation is associated with non-alcoholic fatty liver disease, inflammation, insulin resistance, obesity, oxidative stress, and cardiovascular disease in rodent and human models. Little research was available regarding if the relationship between TMAO and poor health was causative or just associative. Dr. McFadden’s lab infused cows intravenously with TMAO and found that TMAO did not modify milk production or glucose tolerance in early lactation cows.
TMAO does not appear to influence energy metabolism or health in early lactation cows. Choline is subject to both ruminal and lower-gut degradation to TMA, and that influence on choline bioavailability needs to be defined. Data in non-ruminants suggests that unsaturated fatty acid feeding can shift the gut microbes to slow TMA formation.
Lysophosphatidylcholine and immunomodulation (28:45)
Dr. McFadden gives an overview of neutrophil activation and the oxidative burst that contributes to pathogen killing. The ability to elicit the oxidative burst is diminished in pre-weaned calves and transition cows. When cows were given endotoxin to cause an immune response, circulating lysophosphatidylcholine was decreased. In rodent models, lysophosphatidylcholine promotes the oxidative burst and suppresses long-term inflammation in response to bacterial infection. Dr. McFadden cultured neutrophils from pre-weaned calves with lysophosphatidylcholine and observed an enhanced oxidative burst.
Immunosuppression is characterized by low circulating lysophosphatidylcholine concentrations in dairy cows. In vitro data suggests lysophosphatidylcholine can activate neutrophils, and rumen-protected choline increases circulating lysophosphatidylcholine. Future research is likely to define an immunomodulatory role for choline.
Dr. McFadden takes questions from the webinar audience. (38:07)
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Moderator (00:01):
The following podcast was taken from a webinar presented by Dr. Joe McFadden from Cornell University, titled Methyl Donor Nutrition from the Transition Dairy Cow. To view the full webinar and access slides referenced during this podcast, visit balchem.com/realscience and use the search bar to jump down to this webinar presented on May 4th, 2020.
Dr. Joe McFadden (00:24):
Welcome everyone. Please take note of my email in case you wanna reach out to me after I can direct your attention to any one of the three meta-analyses at the bottom of this slide. I think what we can we have a general appreciation for RU protected choline feeding in early lactation and the benefits that can result the practice has consistently demonstrated increases in milk yield and energy corrected milk yield. The effect is certainly more pronounced in when cows are fed diets slow and metabolizable with IE is estimated that approximately 50% of this response is due to increases in dry matter intake. However, endos mechanisms, which we really don't fully understand quite yet explain the other half. Choline feeding also supports increases in milk fat and protein yield to explain gains in energy corrected milk. It also supports the ability of rumen protected choline to minimize body condition loss in neuro lactation and reduce the incidences of postpartum diseases.
Dr. Joe McFadden (01:26):
I think this is great news. I'm sure you think the same as well, and somlecithing that we have heard before about rumen protected choline feeding, but this is not your average choline talk. So I'm going to present somlecithing new information about choline biology in the dairy cow. I'll cover three distinct topics that will each end with a conclusion. For topic one, my study has recently focused on fatty acid nutrition. I'm going to make a case for why we should consider fatty acid feeding when we feed methyl donors like choline, but also methionine to cows. For topic number two, we'll discuss choline degradation in the rumen, but also the intestines, and focus our attention on the role of trimethyl mean oxide and its potential effects on health. And lastly, for topic number three, we have a series of trials in development studying a complex lipid called lysophosphatidylcholine. You may recognize this as lysin. I'll make a case for why we should consider this lysophosphatidylcholine as an immunomodulator in fresh cows, but also pre-wean calves. Overall, my goal is to talk to you about somlecithing new, somlecithing that you haven't heard about before. But please realize I think we are certainly at the forefront of the science, and there's much more research to do.
Commercial (02:48):
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Dr. Joe McFadden (03:51):
Topic number one, why does fatty acid composition likely matter when we feed choline and other methyl donors? First evidence that choline was an essential nutrient in humans was derived by work, performed by Charles Best and Frederick Banty. The work of these researchers led to the discovery of insulin. They utilized diabetic dogs with their pancreas removed, but they observed in these dogs is that they developed severe fatty liver disease. Although I don't understand sort of the reason why, for why they gave lecithin as a dietary treatment or, or as part of a component of the diet. Otin contains phosphatidylcholine, and when they gave lecithin to these dogs, they observed mark reductions in hepatic lipid composition. When they removed the lecithin, the liver lipid content was around 22% in the dog. But when they gave that lecithin to the dog, kept them, kept the lecithin in the diet liver and fat content was just 4%. I think this body of work initiated decades worth of research focused on the relationship between dietary choline, phosphatidylcholine in the cow, and as well as humans and fatty liver progression.
Dr. Joe McFadden (05:02):
Now, flash forward to present day. Fatty liver is a concern for fresh cows because of its relationship with ketosis, poor fertility and compromised milk production. Our lab has routinely characterized the bovine lipid to better develop nutritional approaches to prevent this disease. So in this figure here, we work with a commercial dairy farm in Southern Pennsylvania. Livers and cows were biopsied during the fresh period. We then analyzed over 300 lipids, not just phosphatidylcholine, but other lipids as well. Liver and plasma tissue cows were classified as having liver lipid content of less than 5%. So healthy animals or high liver lipid content greater than 12%. The top features in plasma that reflected the fatty liver condition were low circulating concentrations of a complex lipid called phosphatidylcholine. Now, what we notice is, is that we detected over a hundred phosphatidylcholine, but the majority of those that were low had a very low degree of saturation.
Dr. Joe McFadden (06:02):
They were, they were rich in unsaturated fatty acids. Acetylcholine is a membrane lipid composed of two fatty acids, and again, most of them can be unsaturated. Lipid has a metabolite within it called phosphocholine. This is a common storage form of choline, and it's a metabolite that's often responsive to rumen protective choline feeding. So the take home message here was that the development of fatty liver in cows develops with low circulating concentrations of phosphocholine. We also observed somlecithing very similar in the liver, and I promise not to dwell too much on the past, but many studies have demonstrated the ability of rumen protected choline feeding to lower liver triglyceride deposition. For instance, Harwell and coworkers demonstrated this in over conditioned cals L like and colleagues observed this effect with lower circulating BHB concentrations. Others have shown evidence for improved VLDL assembly with choline feeding.
Dr. Joe McFadden (07:03):
Now, this is particularly important when we talk about choline and fatty acid nutrition, right? Because rumen protected choline feeding is believed to prevent fatty liver disease by enhancing the secretion of triglyceride within very low density lipoproteins, VLDL from liver. It does this because choline supports the hepatic synthesis of phosphocholine. In fact, there are two key pathways for this, right? The first pathway is referred to as the cytidine diphosphate choline pathway. CDP choline pathway initiation of this metabolic route relies on choline and non ruminants. Majority of phosphatidylcholine made is believed to be generated by this pathway. The second alternative route, often referred to as the compensatory pathway, is the phosphoethanolamine, the methyl transferase pathway, abbreviated PEMT, which is an enzyme. It's an enzyme involved in trans methylation. It relies heavily on methyl groups, three of them exactly to methylate phosphoethanolamine informed phosphatidylcholine. These methyl groups can come from methyl donors such as methionine choline or beane, but the process relies on folates and B vitamins as well.
Dr. Joe McFadden (08:18):
Phosphatidylcholine is important because it forms a monolayer of the VLDL particle that contains triglyceride within inadequate phosphatidylcholine. Synthesis may limit VLDL assembly and thus triglycerides secretion, right? So choline feeding has the potential to prevent this women protected choline feeding has been shown to increase phosphatidylcholine in VLDL fraction in plasma we, we collaborated with the University of Florida and these, the study involve feeding RU protected choline ion as reassure for reference, 60 grams per day is equivalent to 13 grams per day of choline ion. In this particular study, we saw an increase in phosphatidylcholine within VLDL. We also observed increases in hepatic phosphatidylcholine concentrations. An increase in triglyceride within VLDL and plasma and XBee and colleagues at the University of Florida demonstrated the ability of this practice to decrease liver triglyceride deposition. And this was in prepartum cows experiencing negative energy balance. So collectively here, data suggests that room protected choline feeding can increase phosphatidylcholine synthesis to support BLDL triglycerides secretion from liver to prevent fatty liver disease.
Dr. Joe McFadden (09:42):
So this brings me to why we should consider fatty acid nutrition when feeding methyl donors. First, the figure illustrates the two previous pathways I described. The CDP choline pathway in blue relies on choline. As you can see, you'll see a lot of any methionine there and non ruminants. This pathway utilizes diacetyl, glycerol rich and saturated and omega six fatty acids like little lake. If the same is true in the transition, cal, these fatty acids are coming from the diet, but more importantly from adipose tissue mobilization. So there's an ample supply of fatty acids, I think to support the CDP choline pathway in fresh cows. Right? The PMT pathway is shown in red. This pathway relies on methyl donors including choline, methane, and methionine. It also utilizes phosphoethanolamine formed from DI of glycerol that has a very low degree of saturation and non ruminants. This pathway prefers very long chain polyunsaturated fatty acids like DHA.
Dr. Joe McFadden (10:41):
However, in the transition cow, the proportion of these fatty acids relative to the total is at its lowest. I hypothesized that inadequate post ruminal pufa supply likely limits PEMT activation. And thus you can have these methyl donors in the diet like choline and methionine. But PEMT would remain inactive because of inadequate availability of unsaturated fatty acids. Okay, so we started to explore this possibility in dairy cows. In this study we have macally infused all cows with a high dose of a tic acid emulsion, right? So there was a lot of tic acid going. Post-partum cows would then add macally infused with or without choline chloride and panel A choline infusion was able to increase or decrease plasma phosphatidylcholine concentrations. But this response varied based on the saturation of the phospholipid and panel B. We show that choline infusion increase saturated phosphatidylcholine like the one shown, which we proposed to be originating from the CDP choline pathway. But we actually observed decreases in rich phosphatidylcholine choline like this one that contains DHA in panel C. Perhaps choline was unable to activate the P-E-M-M-T pathway when saturated fatty acid supply was more than adequate. But the proportion of DHA available to the cow was low.
Dr. Joe McFadden (12:03):
In the same study, we also infused cows with fish oil rich in DHA. We started to do this as well with different types of rooted protected fish oil supplements. We observed increases in fascial cholines containing DHA other FoST choline rich in pufa that were not found in the fish oil. Were also responsive, suggesting that it's possible that that fish oil treatment was able to promote PEMT activation and just take up those other polyunsaturated fatty acids. And for phos to support phosphatidylcholine production or the same was not true for saturated phosphatidylcholine. We gave this fish oil where we did not see a change in those that were saturated.
Dr. Joe McFadden (12:48):
Now in, in humans, DHA and choline, dietary cos supplementation together works better than feeding only DHA or choline alone at activating PEMT, right? So this is actually a pretty hot topic right now. Looking at the synergy of these two nutrients. We're considering the possibility that increasing post al unsaturated fatty acid delivery, not just DHA, but other omega threes like linoleic acid, but also like oleic acid, how might they help boost the activation of that PEMT pathway and support the ability of choline, methionine, or beane to promote phosphatidylcholine production and alleviate that liver disease, you know, to show the ability of dietary fatty acid feeding to modulate choline supply. We recently fed lactating cows, one of two pried palm fat supplements. An MPA contained 72% tic acid and 12% oleic acid. HPA was pure palmitic acid at 99% feeding cows the palm fat containing oleic acid.
Dr. Joe McFadden (13:52):
The increase total phosphatidylcholine, total single myelin and total choline supply relative to cows that were fed the palm fat containing only palmitic acid. So I know this doesn't test the PEMT hypothesis, but it does show that depending on the dietary fatty acid composition, we could potentially influence endogenous choline status. So I think this deserves further consideration moving forward, especially for fresh cows that are fed commercial fats that are high in saturated fat fatty acids. So to end for conclusion, one, low phosphatidylcholine supply is a key feature of fatty liver and dairy cows, right? It's likely due to low pufa and choline supply that definitely in situations where post ruminal availability of pufa and choline or limiting delivery of po a protected pufa that are protected from ru degradation could potentially support choline synthesis in the cap. We should consider the interactions though with fatty acid digestibility, which I'm not gonna get into at the moment.
Dr. Joe McFadden (14:54):
The other added benefit of pufa is that there's an improvement of insulin sensitivity and improvement of insulin sensitivity has the potential to reduce body fat mobilization as well as inflammation. And the reduction in body fat mobilization can further decrease the hepatic fatty acid uptake and thus also provide an additional mechanism to prevent fatty liver. So here are some fresh cow sort considerations. I gave a talk last week on nutrient partitioning and the role of fatty acids in the diet. You know, we should be keeping dietary fat four to six, six point half percent. You know, when we think about commercial fats, we're starting to see a lot of these different types of commercial fats that are available. They're, they can be mixed with unsaturated fatty acids. It's important to ensure though that any addition of commercial fats the diet typically at one third of total dietary fat, that those unsaturated fatty acids are protected for that talk.
Dr. Joe McFadden (15:48):
I went through the literature and started studying the, the ratios of saturated fatty acids, unsaturated fatty acids in the diet. It's pretty clear that as you get closer to one-to-one proportion of saturated fat, to unsaturated fat in the diet, you're gonna increase nutrient partitioning to the mammary gland, but potentially accelerate body condition loss. And that could exacerbate the fatty liver condition. Looking at the omega six, Omega-3 fatty acid ratio, less than five in fresh cows is best, you know, trying to increase the corporation of, of Omega-3 fatty acids like oleic acid. And certainly linolenic acid has likely has similar effects on PEMT pathway and choline and methionine efficacy. But, you know, dish oil is fish oil is certainly gonna be more effective at a lower doses feeding 12 and a half to 20 grams per day of room protected choline ion. And keeping the metabolizable methionine high in fresh cow diets will also ensure that we're we're providing all the necessary components for the pathways that generate syl choline and liver. Topic number two, only degradation is something that we want to avoid. Okay? This is why RU protective supplements were developed to prevent choline degradation in the rumen.
Dr. Joe McFadden (17:06):
The next few slides will describe new data obtained by our lab that suggests that choline degradation may also occur posturally, and thus limit bioavailability.
Dr. Joe McFadden (17:17):
So in the gastrointestinal tract, choline also, but also carnitine, choline and carnitine together are metabolized by gut bacteria. And this figure actually comes from biomedical research paper focused on human nutrition. But in non ruminants, it's fairly well established that in the small intestine choline and carnitine can be converted to a metabolite called trimethylamine by an enzyme called trimethylamine lyase. Now, not all bacteria, as far as I know, have this trimethylamine lyase. Different bacterial populations will express it, but that trimethylamine lecithin can convert choline to trimethylamine TMA. That TMA and non ruminants is converted to a metabolite called trimethylamine oxide in the liver, abbreviated frequently as TMAO. And that's done by an enzyme called flattened mono oxygenase. A-T-M-A-O can be absorbed by tissues once it's released from liver or excreted in urine, or in the cow, or even the postnatal woman. It could be found in milk. At its core, TMAO is a marker of gut choline degradation because it can only come from the formation of trimethyl mean by gut bacteria.
Dr. Joe McFadden (18:29):
So what about the cow? Well, I'm sure at least a few of you're aware of this table did unfortunately I forgot to add this additional site. This is from Sharma and Erman In 1988, I believe. They were looking at feeding choline unprotected choline and looking at rumen choline degradation. They fed rumen unprotected choline at low and high levels from 24 to 326 grams per day. That's crazy high. The flow of choline through the EDA was only slightly responsive, increasing from one to 2.5 grams per day. The authors evaluated ruminal choline degradation, and it exceeded 97% when choline was unprotected. Nearly all of it was degraded. So this is why we have protection. Well, the authors did not measure trimethyl mean or TM ao, but they did reference previous papers. Research as early as 1959 has been demonstrated that the addition of choline to in vitro room fluid cultures resulted in the marked degradation of choline, but increases trimethylamine.
Dr. Joe McFadden (19:32):
That's what you see in panel B. This would be caused by the actions of trimethylamine lyase. Others that investigated the fate of trimethyl mean what happens to it while during fasting. And ruminants that trimethylamine is rapidly converted to methane data suggests that during feeding the conversion of methane decreases substantially, meaning that trimethylamine may pass the ruminate. Collectively, these data confirmed that unprotected choline can be converted to trimethyl mean in the ruminate, which may be available to the cow. Instead, the implications also extend to choline in more complex forms. We recently completed a lecithin feeding trial, and lecithin contains in part phosphatidylcholine right cows are fed D oil, soy lecithin, and zero to 0.36% of rash in dry matter. Our fatty acid data suggested that lecithin was extensively degraded in the rumen. I'm not showing it here that phosphocholine was released in support. We observed mark increases in circulating TMAO concentrations.
Dr. Joe McFadden (20:32):
This TMAO had to originated from choline degradation in trimethyl formation in the gastro tract. Can't say conclusively whether or not that was from thein or the intestine. Up until this point, I've discussed ruminal choline degradation, right? But that same infusion abomasal infusion trial that I described earlier will be abomasal infused choline chloride in the unprotected form to lactating cows. We observed marked increases in plasma choline concentrations like we would expect. Choline metabolites like beene and dimethyl glycine were also responsive to choline infusion. That also makes sense because when you feed Rubin protected choline choline, you may not see changes in free choline concentrations. But those choline metabolites, beane and di dimethyl glycine are often responsive. But we were surprised to see an increase in plasma TMAO concentrations. These data suggest that choline was degraded post ruly to trimethyl mean by bacteria. We can say this because we have may infused the choline chloride and that that trimethyl mean was subsequently converted to TMAO in the cow's liver. This is certainly not a desired response because our hope is that postal choline is reserved for lecithin absorption only and is not degraded. Recent work, however, by Dr. Veth not presented here, suggests that the bioavailability of choline and enters the AUM is not 100%. It's possible that some of that choline is degraded to trimethyl mean. We just don't know the exact amount quite yet.
Dr. Joe McFadden (22:06):
So the one concern is that choline is degraded in the intestine and thus limits its absorption. Okay? And that's certainly an issue. At the same time, however, my lab knew very little about TMAO, so I had my graduate students do a PubMed search, started reading the literature, and it was enlightening soon after the reports of trimethyl mean in cows research focused on TMAO. Different studies have increased in the literature substantially, and it's, I think it's considered a, a relatively hot topic at the moment. The reason is is that TM AO is associated with poor health and rodents in humans. For instance, non-alcoholic fatty liver disease, inflammation, insulin resistance, obesity, oxidative stress, and cardiovascular disease are all associated with the accumulation of TMAO and plasma. Where recent studies have aimed to determine whether TMAO causes these poor health outcomes. 'cause For the longest time, these were clearly just associations.
Dr. Joe McFadden (22:59):
In some cases, this has been modestly proven that TMAO can cause health impairment. The strongest case is for the ability of TMAO to promote oxidative stress and cardiovascular disease. And this is predominantly in rodent models. There are also some studies demonstrating the ability of TMAO to promote fatty liver in diabetes, right? Insulin resistance, again, in rodent models, this is certainly alarming to us because when you think about it, cow has fatty liver disease, you're feeding RU protected choline to prevent this, right? And severe insulin resistance could accelerate body fat mobilization in cows. But we certainly recognize that women protected choline feeding has consistently proven to have a positive effect on health. Can't ignore that. But we were certainly cognizant that not all transition calci studies see a triglyceride lowering effect with room protected choline feeding. I think that might relate to the fatty acid feeding in the diet, but we've considered TMAL as well.
Dr. Joe McFadden (23:57):
So we searched the literature again and we did find a single metabolomics paper demonstrating a positive correlation between circulating TMO and fatty liver and transition cows. Alright, so this had us a little bit nervous. So we recently completed a study to explore the effects of TMAO on milk production in health and early lactation cows that are replicated four by four Latin square design eight cows are continuously intravenously infused TMAO at one of four doses, zero to 60 grams per day for six days. We collected plasma liver, milk and urine during each experimental period. In panel A. We clearly demonstrate the ability of this approach to increase plasma TMAO concentrations in a stepwise manner. As expected, we observed marked increases in urinary and milk TMAO concentrations. The good news is that urine is the origin of disposal and not milk. Just by comparing the concentrations to date, we've actually quantified TMAO yield in milk. And even at these high doses the yield is not going to exceed a hundred milligrams per earlier, per total day per day in milk at room protected choline feeding levels in the field. I suspect that it's less than 10 10 megs per day. That's total milk value. That's, so it's not a lot because, so we wanted consider that though, considering the negative implications of TMO and human health. Now, we're currently quantifying though the yield of TMAL in urine, but I think most of it's going to the urine.
Dr. Joe McFadden (25:29):
We then study the effects of acute intravenous TMAO infusion on milk production. There was no changes in dry matter intake and these early lactation cows no changes in energy intake, no change in milk yield yields of fat protein and lactose did not change. Milk content. Fat protein or lactose did not change. Fat corrected milk yield energy corrected milk yield or the efficiency to produce milk did not change with fruit. And so granted, this is an acute intravenous TMAO infusion. These animals were not fresh cows, but it appears as though that TMAO does not influence milk production.
Dr. Joe McFadden (26:09):
You know, we perform glucose challenges to evaluate glucose utilization and insulin response in these early lactation cows treated with or without TMAL. Recall that TMAL accumulation causes insulin resistance and rodents. You know, and we don't want that to happen in in postpartum cows. Although we did not measure insulin, the good news is that we did not see a change in the glucose response 'cause insulin suppresses adipose tissue mobilization. We studied circulating fatty acids in response to that glucose challenge. We did not detect a difference in treatment. We also measured plasma glucose triglyceride and total fatty acids every day of the experimental period and samples collected before feeding. We did not see a treatment response. We performed a serum liver panel to look at different liver enzymes that we could better understand hepatic health in these animals that were given TMAO, we did not see any change with treatment.
Dr. Joe McFadden (27:02):
And we lastly, just, just to be safe, we analyze over 50 polar metabolites using metabolomics and over 300 lipids using untargeted lipidomics, a fully comprehensive approach using mass spectrometry. And for the most part, we observed very little change. So, second conclusion. Trimethyl oxide does not appear to influence energy metabolism or challenge health in early lactation cows. We really need to address that question because certainly when you feed room protected choline, the goal is to improve health and milk production. So we're moving on from this. We do not think TMAL is causing any kind of health impairment or impairment on milk production. We still have a challenge ahead of us. That's bullet number two. Choline is subjected to rumen ruminal, but also lower gut degradation to trimethylamine. We've, we've shown this with the infusion of choline chloride. The influence of this lower gut choline degradation. A choline bioavailability needs to be defined.
Dr. Joe McFadden (27:58):
I'll say though, we're starting to think about how might diet influence that microbiome, whether it be ruminal or even lower gut microbiome. And thus influence choline degradation to trimethyl mean data in non ruminant suggests that unsaturated fatty acid feeding as a means to shift the gut microbiota and slow trimethyl formation and improve choline bioavailability. So this ties in nicely with the fatty acid feeding recommended earlier postal availability of pufa has the potential we need to test this to reduce choline degradation and increase choline by availability. Alright, final topic, topic number three. A few of you may know that I have this sort of interest studying lecithin phospholipids and fatty acid digestibility. Well, lysophosphatidylcholine is a component of lysate. Alright, looking at the structure of lysophosphatidylcholine shown there you use as a, you use as a single fatty acid right in its production, right?
Dr. Joe McFadden (29:00):
And ruminants, the most abundant lysophosphatidylcholine include those that utilize tic steric and oleic acids, right? Lysophosphatidylcholine also contains phosphocholine. Alright? And plenty of data has shown that ruin protected choline feeding is able to increase lysophosphatidylcholine concentrations. It's one of those choline metabolites that's responsive to choline feeding. While this topic focuses on how lysophosphatidylcholine may modulate immune function in dairy cattle, but first I'm going to provide some background about immunity. Recall that cows and as humans as well, have innate adaptive immune systems. Adaptive immunity is specific for pathogens, but slower to act. The innate immune system is fast acting, but non-specific composed of different cells like neutrophils. The activation of neutrophils involve somlecithing called the oxidative burst, which is the rapid release of reactive oxygen species, including superoxide ion and hydrogen peroxide. This involves the enzyme NA DPH oxidase. This oxidative burst contributes to pathogen killing, but the process also involves low pH and enzymes found in phagolysosome.
Dr. Joe McFadden (30:09):
Why is this important? While the neonatal calf relies on passive immunity from colostrum, but this immunity fades and because innate and a death of immunity are underdeveloped, pre pre-weaning the calf experience is what is referred to as a gap in immunity. In panel A, these data demonstrate that the ability of neutrophils to elicit the oxidative burst and promote phagocytosis is diminished pre-weaning. We also like to talk about immunosuppression and transition cals panel B. The oxidative burst is diminished in multiparous cows and declines with time in prima Paris cattle. So researchers, researchers have sort of been on the hunt here for dietary approach approaches to bolster immune function and pre wean cals and transition cows. Dr. Bradford at MSU is certainly the immunity guru. He recently had a study where he evaluated the effects of choline supplementation on bovine neutrophils and monocytes. In brief, he observed that the phagocytic and killing ability, alright, of isolated neutrophils were linear diminished with increasing doses of colon. He detect dec decreases in oxidative burst and pro-inflammatory 10 of alpha. He also had provided evidence that there was an increase in choline metabolism with this colon supplementation. Now the conclusions were a bit broad because it's a bit early to make more definitive conclusions, but it was clear that choline was able to modulate neutrophil immune function.
Dr. Joe McFadden (31:30):
My interest in lysophosphatidylcholine actually stemmed from a collaboration I had with Dr. Baard. He gave endotoxin lipopolysaccharide to some lactating cows that were experiencing hyperlipidemia, high circulating fatty acids. He saw the typical response, an increase in rectal temperature, an increase in circulating fatty acids. And response to the, the hyperlipidemia induction protocol he had, he infused them with triglyceride to try to mimic the fatty liver conditions that you'll see in a fresh. Well, what was important here is in panel C, that when he gave the endotoxin there was the activation of the acute phase response, an increase in serum amyloid A. He also saw changes in LPS binding protein. His animals were experiencing an an, an infection pathogen infection and were responding accordingly. While we took these samples, we took all the plasma samples from all the time points and liver tissue that are omic work. And long story short, we were noticed marked reductions in lyo phospho coatings. Sure, this what happened there. And phosphocholine. Now this decrease was about 80% decrease by hour 16. But the decrease in lysophosphos choline occurred as early as four. And in transition cows, they're experiencing that immunosuppression. They also have low levels of lysophosphatidylcholine, right at Partion. So for now, we've established this relationship between endotoxemia immunosuppression and lysophosphatidylcholine status that LPCs are low at this time.
Dr. Joe McFadden (32:57):
So we got back into the literature because we really, we didn't have an hypothesis up until this point, but in the literature, people that die from a bacterial infection sepsis also have low lysophosphatidylcholine concentrations. And there've been plenty of studies looking at lysophosphatidylcholine therapy as a way to protect against a, a bacterial infection. This is just one study they gave. Basically they used this approach where they use, it's called a SQL ligation procedure. They open up the abdomen, they puncture the gut, they let the bacteria enter the gut, and they close the gut. And then eventually the mice are gonna die. They'll basically all die within 10 days. But if you gave those same mice lysophosphatidylcholine at the start, mortality was prevented. And at the high dose here, it was nearly, almost completely prevented by, by day 10. And in panel B, they also subjected mice to puree coli injections, live e coli.
Dr. Joe McFadden (33:50):
Certainly that is going to proliferate in the animal. But the addition of lysophosphatidylcholine was able to increase e coli killing data. Looking at the mechanisms, has identified the ability of lysophosphatidylcholine to activate neutrophils, right? It promotes the oxidative of burst it. It can suppress long-term inflammation in response to bacterial infection. It can clear out e coli faster. It also has been shown to help bolster adaptive immunity. It can increase immunoglobulin production and interferon gamma availability. So we took the opportunity, we have, we took some, we just started a series of experiments looking at lysophosphatidylcholine therapy and diet as a way to help boost immunity, alright? In animals that are experiencing immunosuppression, right? And so here we had pre wean calf, we isolated some neutrophils from pre wean calves, right? I think this is around week two, week three of life.
Dr. Joe McFadden (34:47):
And we cultured those neutrophils with lysophosphatidylcholine. And that was, and we had two types here, TIC and sta. We've also done this with the oleic acid version. And what we observed was, is that saturated lysophosphatidylcholine was able to enhance the oxidative burst of these bovine neutrophils. So we gave P-M-A-P-M-A is four blown ATE acetate. And what it does is it, it activates the neutrophils promotes the oxidative burst, but the presence of the lysophosphatidylcholine amplified this response at increasing doses. Well, just to remind you, we protected fo choline feeding is able to increase lysophosphatidylcholine supply re currently considering how not just choline feeding might influence immunity, right, especially in transition cows. But looking at are those emulsifiers less thin and ly. So to conclude here for topic number three, immunosuppression and endotoxemia are characterized by low circulating lysophosphatidylcholine concentrations. In dairy, cows vitro data suggests that lash saturated lysophosphatidylcholine activate neutrophils and that room protected choline feeding is able to increase lysophosphatidylcholine.
Dr. Joe McFadden (35:58):
Dr. Bradford has shown that unprotected, not unprotected, but free choline is able to modulate immune function of neutrophils. So there's certainly a, a connection here that's between immunity and choline status and the involvement of phosphatidylcholine. As we start to look more at this work with lecithin and lecithin, I fully suspect more of these kinds of studies in the future, please be aware that that lecithin does have phosphatidylcholine, which can be degraded to lysophosphatidylcholine and absorbed, but it's not the only phospholipid found in lecithin. And the same thing with lecithin that it not, does not just have phosphatidylcholine. In fact, crude forms of these will also have triglycerides, which is, is it's gonna have different response on immunity, obviously. One last thing. Some of our speakers there, Dr. White point her out here, but we had a bunch of collaborative authors put out this review paper one carbon metabolism and methyl donor nutrition in the dairy cow. I think that's a decent summary of the current state of the literature. You can catch this in JDS thanks to our collaborators, the late Dr. Staples under BA guard and Dr. Howie at Johns Hopkins.
Commercial (37:18):
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Scott Sorrell (38:07):
So we have quite a lot of questions that have come in from our audience, so let's get right into those. First one, can milk TMAO be considered a marker for choline degradation?
Dr. Joe McFadden (38:19):
Yeah, so I mean, I'm, I this is, this is something I'm looking into. I think TMAO is a marker, whether it's plasma or, or milk could certainly be a marker of choline degradation. But I wanna, I wanna be careful here because we don't necessarily know where that TMAO is coming from, right? We don't know if it's coming from the intestines or the rumen quite yet. So I think if we're, if you're thinking about ru protected colon supplements that have a high rumen degree of rumen protection and that most of it is being provided to the intestine, then we could certainly make assumptions that what you see in milk could be majority derived from intestinal degradation. I also think it's just a possible means to, to compare supplements. But I think you have to compare it within the context of bioavailability. And so that requires a little bit of intricate trial design to do. Yes, it's certainly possible.
Scott Sorrell (39:12):
This one's a little complex. So lecithins are reasonably effective surfactant and there's literature out there relating surfactant feeding to changes in nutrient absorption. So to what extent have you been able to separate shifts in nutrients supply at the, the Lumin wall from a, a more direct mechanistic effect of the eth than molecule?
Dr. Joe McFadden (39:35):
Yeah, I mean that, it's a very complicated question.
Scott Sorrell (41:09):
Alright, so let's you mentioned undigested fatty acids a moment ago and, and this question was about those getting lost somewhere and the relation between ufas and choline. So requests, if you could elaborate on that relationship a little.
Dr. Joe McFadden (41:25):
Yep, yep. So I'm thinking about it in, in the liver. That's the, the key take home message here. Then if you can provide more polyunsaturated fatty acids, particularly Omega-3 fatty acids to the liver, to the cow, then you could support phosphatidylcholine production. One of my arguments here is, is that you could be feeding methionine choline methyl donors in general, and one of the intended outcomes is to activate methylation to produce phosphatidylcholine. But you might not be able to do that, particularly in a fresh cow that it has limited endogenous supply of pufa to begin with. And so if we're able to increase the post ruminal availability of pofa and are protected from rum degradation, then we could potentially support the ability of methyl donors to generate phosphatidylcholine and prevent fatty liver disease.
Scott Sorrell (42:17):
Alright, so along those lines would it be possible to isotopically label choline to look at TMAO in milk or plasma to resu measure A rated degradation?
Dr. Joe McFadden (42:30):
Yeah, absolutely. I think you, maybe you're my graduate student. We we're, we're exploring that, yes.
Scott Sorrell (42:37):
All right. One of your colleagues has a a another question here about the connection between choline incubation and LPC incubation. So why did LPC increase the oxidative burst of neutrophils, but choline decreased it?
Dr. Joe McFadden (42:52):
Yep. All right. So, you know, the thing with the choline is, is that, you know, first of all, there are two different metabolites for one and choline most likely is getting taken up by those cells being metabolized with side inside the neutrophils. The way the lysophosphatidylcholine acts is that it binds two receptors on the plasma membrane, right? And so there's two different modes of action there. And so choline iss not gonna be able to interact with the lysophosphatidylcholine receptor. Now, I also will say too, that the effects that they're seeing with the choline in those in vitro cultures, you might not see that in vivo in response to room protected choline feeding, because often you do not see a free choline response with feeding room protected choline. You will see the lyo fosil two of choline response. So I think we just need, I think we just don't have enough data there to sort of distinguish that, but there's just sort of a something to be aware of.
Scott Sorrell (43:45):
All right, Dr. Mcfadden, that's where we're gonna have to end it. Great answers and thank you to all of our attendees for those questions.
Moderator (43:53):
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