9UNP0447SD_Elizabeth_French_

- Nadia Dominguez: Thank you for joining me for Wednesday Nite @ the Lab at UW-Madison Biotechnology Center. Tonight we welcome Elizabeth French from the Department of Dairy Science here at UW-Madison. Elizabeth French is currently a third-year doctoral candidate in the dairy science department. She completed her Bachelor's degree at the University of Illinois-Urbana/Champagne in Animal Sciences. She decided to continue her graduate studies in dairy nutrition at the University of Wisconsin-Madison after working with a UIUC dairy farm and extensive research within a dairy nutrition laboratory. Upon finishing her Master's degree at UW-Madison working with distillers' grains as a component of dairy cattle diet, she decided then to pursue her doctoral degree here at Madison. Her research has shifted from the feeding side of dairy nutrition to relating milk components with products from rumen fermentation. In addition to her research, she is long known here at Madison as a science outreacher. She has been the assistant coach for the North American Intercollegiate Dairy Challenge team for UW-Madison. 

She has coached the Midwest Dairy Challenge team for the past two years, has organized the Dairy Science major. For those of you who have participated or know about UW's Grandparent University, and has held an interactive laboratory at the PDPW Youth Leadership Derby. So please join me in welcoming Elizabeth French to Wednesday Nite @ the Lab. 
(applause)

- Elizabeth French: Thank you, Nadia, for that introduction. Am I okay? So, as she said, the title of my talk is "A Link Between the Rumen and Milk," influencing rumen fatty acids and their appearance in milk. Before I can get to this side of the research, I feel that I should step, walk with you through the digestion of a ruminant, and what is a ruminant in the first place. So, looking through an overview of tonight's talk, first I'll go through an overview of ruminant digestion, and from there I'll focus solely on the rumen and discussing the microbiology that occurs within the rumen. And from there, I will hopefully answer a little bit more, there is a link between the rumen and milk. 

And using fatty acids as a way of detecting if that difference does exist. And from there I will go into some future research based on some experiments that I've performed both on campus and off campus concerning these fatty acids. So first, as I said, I will move into, what are ruminants in the first place? So, displayed on this slide are a wide range of ruminants. So we have cows, sheep, goats, camels, alpacas, llamas. They even include wild ruminants such as deer, buffalo. So it's a huge suborder. And what's special about ruminants is that they're considered foregut fermenters. So unlike humans, where we have a gastric stomach, the cows instead, while they do have one of the compartments of their stomach is considered like our gastric stomach, there's also a huge fermentation vat and microbial fermentation which occurs prior to that digestion. So that's why they're given the name as a foregut fermenter. 

This is opposed to animals that are considered to be hindgut fermenters, such as horses and rabbits, even, which have an enlarged cecum, lower, closer to their large intestine. And even us as humans have some kind of fermentation, and other monogastrics or single-compartment stomached animals. And that's because in our colon we actually do have some microbial fermentation occurring. And some of the products that cows have sustained themselves on are actually produced within our colon, but not at the same levels as what is seen in the dairy cow. So as you can see here there are a wide range of ruminants. And my research focuses specifically on dairy cattle. So moving into an overview of the digestion, here is a picture of the digestive tract of a dairy cow. So first we start with the feed entering in through the esophagus and flowing into the rumen. And that is the largest compartment of the animal's four-chambered stomach, which is also an important point to make, is that sometimes you'll hear people say that cows have four stomachs. That's actually not the case. 

They have one stomach, but four compartments. Just a little bit of clarification there. So the feed will enter through the esophagus and go into the rumen, the first chamber, and from there it will flow into the reticulum, or the second chamber of the digestive tract, and from there move into the omasum and the abomasum. And that's actually considered to be the closest to our stomach. I'll go into detail a little bit more about what occurs there. And so then from there, the nutrients flow into the small intestine, or the nutrients are absorbed within the small intestine, and then any undigested feed will pass through the large intestine and be excreted as fecal material. So looking a little bit more closely at the individual compartments of the digestive tract. First I wanted to show here, here are epithelial samples of each of the different compartments within the ruminant tract. And you can see, based on what the epithelial structures look like, that they have different functions within the animal. And I brought with me here actually a model of a sheep digestive tract. 

So this is the rumen and if I was a sheep or cow, this would be located on my left side here. And so then on the other side is that omasum and abomasum that I was discussing. So this is how it lies in the animal. Put this down. So moving on first to the rumen, the largest compartment of the digestive tract, and I'll show here, it's a very muscular structure, all these lighter colored area represents muscular folds that help the rumen contract. And it can hold in dairy cows up to 50 gallons. With a sheep rumen, the model that I'm holding, that normally holds approximately five to ten gallons, but a cow's rumen can hold over 50 gallons. And its nickname is the fermentation vat. And this is because this is where the majority of microbial fermentation occurs. And then what is present on the screen here are papillae, or finger-like projections which project from the rumen and are used to absorb products from microbial fermentation. 

And the pH of the rumen is typically 6 to 6.7, so it's relatively closer to a more neutral environment. And then in the corner what is displayed is the actual phases that occur within the rumen. So at the very top of the rumen, there are more gasses that are located, so methane and carbon dioxide. In the middle there's the longer particles or the freshly ingested feed that the cow has consumed probably that day. And on the bottom that's where more, or the ventral part of the rumen, that's where the grain and smaller particles are located. And depending on where papillae are located in the rumen, will influence their shape and size and that's just based on the different phases within the rumen that exist. And so from there, feed moves from the rumen into the reticulum, and this is also known as the "honeycomb." On the slide there, it's apparent, the honeycomb structure. And even in this model, it's a little far away, but it also has that honeycomb structure. And this is the area where feed is compressed and formed into boluses and eventually regurgitated, which is what gives the ruminant its name, is that it regurgitates its feed and re-chews it to reduce the particle size, re-swallows it, and it's available back in the rumen for microbes to utilize and hopefully be digested and move down the tract. 

So the reticulum is a lot smaller. So the rumen was 50 gallons, the reticulum's closer to five gallons in the animal or has that capacity. So it's smaller, but it plays a vital role. And so from the reticulum moving into the omasum, which is also called "many plies," and that's because it has leaves, the tissue has leaves, which is shown in that picture, like a book. And this is where most predominantly water absorption takes place in this compartment. Some mineral absorption as well as some organic acid absorption produced from the microbes. And I'll go into that a little bit later on. And so then the abomasum, or the "true stomach," that's where the acid production and enzymatic digestion takes place, so enzymes used to break down proteins, for instance, are produced within that abomasum. And that is what is called more of the, more similar to humans and their stomach. And so from here, the feed will move down to the small intestine, nutrients will be absorbed, as I mentioned earlier, and to the large intestine. 

So since I've gone through more of the digestion side of things in an overview of that, I'd like to move back into the rumen and some of the microbiology which occurs within the rumen. And making that link between the rumen and the milk. So the microbial habitat in the rumen is home to thousands and thousands of species of microorganisms, predominantly bacteria, which range in 10 to the 10th cells of bacteria per gram of rumen contents, and protozoa, a little bit lower in number, 10 to the 7th number of cells per milliliter of rumen contents. And while protozoa are fewer in number than bacteria, they can represent upwards of half the amount of the mass, so they're equal in mass, the bacteria and the protozoa. And then of the total microbial mass present in the rumen, 6% of that is actually composed of fungi. And so in this top picture, this is bacteria attached to a piece of straw. And this is a picture of a protozoa and a fungi here. And so these bacteria, an overview of some of the degradation that takes place in the rumen. First you have the feed entering in and from there the microbial population will degrade that feed or utilize that feed, the protein fiber, starch and fat, and degrade that into its individual building blocks, such as amino acids, glucose, and fatty acids. And from there, focusing first on the amino acids in a protein, those amino acids can be used by the bacteria themselves to synthesize their own protein, as well as being degraded to what are known as volatile fatty acids, which are a major energy substrate for the dairy cow. 

And then fiber and starch are also common or essential in feeds for dairy cows, and are degraded down into glucose and then volatile fatty acids and energy. So just a brief overview of ruminant digestion as a whole. And so going back once again to the phases of the rumen, at the top we have the gas phase. In the middle is where the longer particles are, and then the bottom is the grain and smaller particles. Approximately 3/4 of the total bacterial populations in the rumen or bacterial species are considered "solid associated," and that means that they reside within the long particles side. So in that picture I was showing you where the bacteria were attached to the straw, most of the time the solid associated bacteria must adhere to the feed to be able to digest them. And then also, a quarter of the bacterial population is used to digest grain and smaller particles. So there's many different species that are used to degrade a variety of substrates and are essential to ruminant fermentation. And so, as I mentioned, volatile fatty acids are a major end product of fermentation and so for the remainder of the talk, I'll be abbreviating volatile fatty acids as VFA. And the three major VFAs are acetate, propionate, and butyrate. 

And as you can see, acetate represents on average 60% of the total VFAs produced. Propionate is approximately 30%, and butyrate is about 8%. And I do have samples of these, so I'm not going to pass them around because everyone's spread out pretty far, but after the talk if you'd like to come up and smell some of these, you're more than welcome to. These are major end products of rumen fermentation. And so within the rumen, the acetate and butyrate are primarily used for fat synthesis and energy by the animal. Propionate, on the other hand, is used to synthesize glucose. We as humans will break down carbohydrates into glucose and absorb that directly, since the ruminant has that foregut fermentation, it's unable to absorb glucose directly, because the microbial population degrades it. So the cow instead must use propionate to synthesize its own glucose. And so the other VFAs which are present only represent approximately 2% of the total VFAs produced. And this includes valerate, anti-isovalerate, and isovalerate. 

And some of these VFAs are produced actually from degrading amino acids, in particular anti-isovalerate and isovalerate, and that will come up a little bit later on when I start tying in the fatty acids produced in the rumen with milk. And these are also used by the animal for energy and some glucose production. So this moves into the next area that's so fascinating about ruminants, is the symbiotic relationship that occurs between microbial populations and the ruminant, or host animal. So those VFAs being a major end product of fermentation represent about 50% of the total energy used by the animal daily, whether for maintaining its tissues, for milk production, just for daily maintenance of that animal. So without those VFAs it would be unable to survive, without its microbial population. Also, 40% of the total protein the cow takes in each day comes from the microbial population. So both the VFAs and the microbial protein being produced are very important for the animal and difficult to predict because of the fact that the only way to measure them is actually within the rumen. So this is where it gets a little trickier for being able to estimate production of protein and the VFA proportions. So some of the techniques that are currently being used for estimating these two products. First is the analysis by gas-liquid chromatography. 

This is where you would take a sample of rumen fluid and you would analyze for the profile of VFAs present. So here you need a sample of rumen fluid and this will vary between diets and everything. I'll get into that just a little bit later, how diet can influence production. So also, there are internal markers that can be used. So within the case of protein, there are markers that are found in the urine that are called purines that make up some of the nucleic acids of DNA within the microbial population. Those are detected in the urine and are used to, if you know the concentration of the urine, you can estimate the amount of microbial protein. However, the assumption is being made that all the purines from the feeds are actually degraded, but that's not necessarily the case. So there's definitely room for improvement. And then some of the other markers available, an external marker, such as N-15, that's giving a known amount into the cow and it's measured later on in the omasum, samples are taken from the omasum to see the amount of N-15 present, which indicates the amount of incorporation into microbial protein.

- How do you get the samples?

- Good question you asked, I'm going to show that on the next slide so I'll answer that in just a few moments. I'll get into that. But then lastly, based on all this work measuring within the cow, computer models have been made to, based on the diet composition, how much fiber is being fed, the amount of protein, fat. These models are used to estimate the amount of protein being produced as well as the VFA profiles present. So going back to your question, all this work requires the use of cannulated cows, or most of it, aside from the computer models. That's the external marker I was mentioning, N-15, as well as measuring rumen fluid, requires the use of a cannulated cow. And so the rumen is actually, a hole is made right around here, so this would be in the cow on the left side. It always has to be on the left side, 'cause that's where the rumen is present. So a hole will be made into the rumen, and then from there you can sample directly into the rumen and my research, I'll be discussing in a few moments, actually I was infusing the solutions directly into the rumen. And then also, sometimes in research if you want to completely bypass the rumen, there also have been contraptions made such as this. 

So this is where the plug would go in the outside of the cow, and if you want to bypass the microbial fermentation all together, this can actually be inserted all the way into the abomasum. So you can bypass that fermentation all together if you wanted to see the effect of the feed, if it wasn't digested in the rumen. So with using N-15 as a marker, you actually have to go in and sample from the omasum, the third compartment, and that requires a special tool, which I didn't bring with me to suck those samples out. So it's a very, slightly invasive technique, and so this wouldn't be applicable on the farm by most producers or nutritionists to estimate the amount of protein being produced. And therefore, it's important to try to find other techniques, other markers that are easy to collect and noninvasive so more people can use them, as well as certain products that directly relate to microbial populations. And so while the cannulated cow is an excellent research tool, it's not the most applicable on the farm, which leads into the next area of research that I'm in, the link between the rumen and the milk and using fatty acids as a marker of this. And the fatty acids in particular we're talking about are -- fatty acids and before I go into how they are synthesized in the animal, I'd like to discuss why we're interested in them in the first place. Well, they were first detected in milk fat in about the 1950s and after this time they were detected in multiple other ruminants as well as other herbivores, specifically in the adipose tissue as well as milk fat. And it normally wasn't present in rats or monogastrics, but they noticed it was present in rat adipose tissue after it consumed milk from a dairy cow, so this led people to wonder, what's going on here? So later on in the '60s, a lot of in vitro experiments were performed using pure cultures of rumen bacterial species. 

And it was found that these -- fatty acids were a major component of the bacterial lipids, specifically this cell membrane within the bacteria. And then these fatty acids turn out to be important, particularly the -- fatty acids, for maintaining the membrane fluidity, so making sure the bacteria can transport nutrients across, back and forth adequately and maintain that fluidity. So these fatty acids seemed like a prime candidate. And so how are they formed in the rumen? So, those VFAs I was mentioning earlier, propionate, while primarily used for glucose synthesis in dairy cows can be used in the microbial population to synthesize odd-chain fatty acids, or OCFA, as they'll be abbreviated on the slides to come. And this means that there's an odd number of chains within, or odd number of carbons within that fatty acid chain. And then isovalerate and anteisovalerate, it's the branch chains that are sticking off here, lead to the production of branch chain fatty acids, so a class of fatty acids called iso and anteiso fatty acids. So collectively the branch chain fatty acids are BCFA. And so all together these represent the odd and branched-chain fatty acids. So already research has been conducted that has demonstrated that these odd and branch chain fatty acids can be related to microbial protein flow. 

And so in this slide, on the Y axis we have the flow of bacterial nitrogen to the small intestine, this is in grams per day on the Y axis. And then on the X axis we have the amount of milk odd and branched-chain fatty acids, measured in grams per day. And it was found, and this was a meta-analysis of three studies, so different diets, that the bacterial nitrogen was approximately nine times the amount of milk odd and branched-chain fatty acids. So they were able to create this relationship between the bacterial nitrogen and both odd and branched-chain fatty acids. Some of the work that I've done here actually estimates that closer to 10 to 12 times the amount of odd and branch chain fatty acids, so there are still some kinks that need to be worked out here and figuring out how these are really related to these fatty acids. And also, it's important to note, the study shown here was performed in Europe and the feeding systems there and the feeds that are given to dairy cattle are different than what are given here in the United States. We have different forage crops and grain crops. So also, while it does seem promising that we will be able to relate microbial protein production to odd and branch chain fatty acids, we were also able to find, and based on some of the literature, that rumen volatile fatty acid proportions in fact can be predicted from the amount of milk odd and branch chain fatty acids. So the Y axis on each of these is the observed volatile fatty acid proportion or percent, minus from the predicted VFA proportion, predicted by the model. These darker lines represent a one-to-one ratio, so what was observed was the same as what was predicted in the model. 

And so we have acetate, propionate, and butyrate. Along the X axis is the -- proportion of these VFAs. So on the side here are the fatty acids that were used within the model to predict proportions of these volatile fatty acids. And so it's apparent that, looking down the line, there are differences into which fatty acids can be used to predict other VFAs. So this may indicate that there are certain species, the species that produce more propionate also have more of those particular fatty acids, or it could mean something else. And once again, in some of the research I've done here, it isn't as clear cut. The different fatty acids relate to the VFAs differently, so once again, more data needs to be accumulated to really detect if these fatty acids can be used to estimate this. Since we've already demonstrated that production, literature has already shown that VFAs and microbial protein can be predicted from the odd and branch chain fatty acids, some of the influences that have been studied on milk odd and branch chain fatty acids. And so, this is where I like to go over just a little bit of what has been done, also in the literature, also looking at, how can you manipulate these fatty acids? So nutrient composition is one area that is shown to change fatty acids. 

So increasing the amount of forage in the diet has been shown to increase branch chain fatty acids. And also increase odd chain fatty acids. So the nutrient composition is varying the amount of fiber, starch, excuse me, protein, fat, and then also, ingredients have been shown to play a role in changing the odd and branch chain fatty acids. This also ties back into the nutrient composition, so it's all interlinked, and so the idea behind this is, the driving force changing these odd and branch chain fatty acids are in fact the microbial populations in the rumen, because certain species utilize different feeds, or use various feeds more effectively. So a species that degrades more starch might not degrade protein as well. So depending on what you're feeding that animal will support different species. So it seems that the changes in milk odd and branch chain fatty acids are actually reflective of changes in microbial species. And so also, I brought with me some different feeds that are commonly fed to dairy cows here in the Midwest. We have some fuzzy cotton seed. And at the end if you'd like to come up as well, you can feel free to take a look. 

But if you look at, say, a grain versus a forage, it's apparent why there might be different species that would degrade them because the form of the feed is so different. And then I have here the total mixed rations, so there are many different feeds being fed that support different species. So a lot can manipulate these fatty acids. But my question was, what about the precursors? What about the volatile fatty acids, that pathway I showed you. How about those? Will those manipulate odd and branch chain fatty acids? So to answer that question, fell into one of my first experiments. So just to review once again, propionate is thought to contribute to more odd chain fatty acids, and then isovalerate and anteisovalerate are thought to contribute to more of the branch chain fatty acids. So altogether, the odd and branch chain fatty acids. 

So in my experiment, the hypothesis was that infusing solutions into the rumen of different VFAs would increase the appearance or proportion of the respective odd and branch chain fatty acid. So in three of my treatments, for instance, propionate was thought to increase the odd chain fatty acids. And then isovalerate and anteisovalerate, products from amino acid degradation, but they also can be produced directly from the microbial population, but predominantly from degrading amino acids. Those are used to synthesize the branch chain fatty acids. And just to show you how this sampling took place. Solutions were approximately, I made 15-liter solutions of each of these volatile fatty acids, and they were infused over an 18-hour period. So zero hours when they began being infused, milk, the animals were moved from their pumps because they were pumped directly into the rumen at 11.5 hours after infusion, being milked. And then at 18 hours the infusions were ended and a rumen sample was collected. And then 23.5 hours after infusions, or about 6 hours after the infusions had ended, milk was collected again. And then another 12 hours after this and 12 hours after that. 

So there was 30 hours in between the end of the infusions before the animal was infused with the next round of solutions. So my treatments included propionate, the isovalerate, anteisovalerate, as well as acetate. So as shown on the other slide, I predicted that propionate would increase odd chain fatty acids and iso and anteisovalerate would increase the branch chain fatty acids. But with my rumen samples, when I sampled them, just kind of explaining the reasoning behind that, the whole rumen contents were collected. So I took grab samples using a cannulated cow from five different areas within the rumen to have a representative sample. These contents were brought back to the lab and kept warm, and then strained through cheese cloth where they were separated into the liquid and solid phase. And so once again, with the idea that the bacteria within the solid phase, those would be different than what was seen in the liquid phase. And a fatty acid analysis was performed on both of these samples. And in addition, milk fat was extracted at the different time points and composited by day, because cows are milked twice a day on some farms and on our farm. So we would take a sample at the PM milking and the AM milking and composite those, so we'd have one daily composite and extract the fat from those samples and measure it. 

So first I'd like to move into the rumen results that we have. And so in each of my slides, the total branch chain fatty acids will be represented in blue, and the total odd chain fatty acids are represented in gray. And this is the fatty acid concentration measured in the rumen liquid. So the percent of total fatty acids is on the Y axis and then the treatment infused is on the X axis. And also, the asterisk represents any treatment effects that were detected. And at the top on each slide showing these results, I have what we predicted would occur. So propionate, the odd chain fatty acids, and the other two for the branch chain fatty acids. But looking at the slide, only one treatment really seemed to alter the concentration within the rumen liquid, and that was the anteisovalerate. And while we expected it to increase the branch chain fatty acids, we sure didn't expect it to increase the odd chain fatty acids to that extent. So not quite as clear cut as we were looking for. 

And the other infusion treatments didn't seem to alter the concentration very much. And even propionate, which was thought to increase odd chain fatty acids, was lower than the other treatments, so unexpected. But then moving into the solid, well, we didn't see much of a difference there either. So once again, the Y axis is the percent of total rumen solid fatty acids and infusion treatment on the X axis. And once again, the anteisovalerate led to the only difference detected among treatments, and this was in the branch chain fatty acids, which was what was predicted. However, it just didn't seem that isovalerate really increased the proportion of those fatty acids, so this was a little bit interesting to see. And numerically, propionate did have a greater amount of odd chain fatty acids, but it wasn't statistically significant. So lastly, looking at the milk odd and branch chain fatty acids, there are a few more treatment differences detected. Once again they were unexpected than what we had anticipated. So the percent total milk fatty acids on the Y axis, it's apparent that anteisovalerate and propionate has significant increases in the odd chain fatty acids. 

We expected that for the propionate, but we did not expect that as much for the anteisovalerate, and there's no difference in the branch chain fatty acids. So then, one thing that we noticed, the proportion of the odd chain fatty acids was greater in the milk than what was seen in the rumen contents. And this is consistent with other studies that have been done, measuring the transfer efficiency of the fatty acids from the small intestine to milk fat and there actually is a greater amount of odd chain fatty acids seen in milk fat, compared to the microbial fatty acids. So this would indicate that there is some synthesis occurring in the peripheral tissues of the dairy cow. And the branch chain fatty acids, while they remain relatively unchanged, they seem, they were a little bit lower than what was seen in the rumen, specifically the solid, but they were actually closer to the solid branch chain fatty acids than liquid, which seemed to vary a lot more based on what we infused.

- Did you try any combination in the infusion treatments?

- No, we didn't, but we have done some combinations in vitro, so in the lab, looking at some of these. And those have presented mixed results, and some of the other published research that's out there, when they did include a mixture of volatile fatty acids, they saw minimal changes in these, in the fatty acids produced. But once again, when you have an in vitro system with pure cultures, those bacteria might not respond the same way, as if they were in the total mixed culture. So that wasn't very promising, but there's been very limited research done. 

However, there has been some research looking at infusing propionate, and they've seen increases in the odd chain fatty acids in the milk. So it seemed that it would have the same result in this study. But they also noticed when infusing propionate in earlier studies that there was an increase in odd chain fatty acids in adipose tissue, so there is some synthesis occurring. So the conclusions of this study was overall that infusing volatile fatty acids tended to have minimal effects on odd and branch chain fatty acid proportions, and the changes that we did observe that were significant were not what we expected. So it seems that there still are some unanswered questions about what really drives the odd and branch chain fatty acid proportions. And maybe a more dietary influence compared to, say, the precursors available. So then some further conclusions, as I mentioned, the rumen odd and branch chain fatty acids didn't seem to match the milk odd and branch chain fatty acids. The odd chain fatty acids were greater in milk and then the branch chain fatty acids were greater in the rumen contents. So there's still, as I said, some unanswered questions, which leads me to the other areas of future research, so, are there other sources of odd and branch chain fatty acids? Well, yes, we do know that there are some. 

Even some of the odd chain fatty acids can be produced in the mammary gland. So research needs to be performed that really looks at how much do those peripheral sources of fatty acids contribute to milk. And for instance, in animals in early lactation, when they're mobilized, so after a cow has calved and she's mobilizing more body fat to support her milk production, you may see an increase in some of these fatty acids, so they're obviously mobilizing the fatty acids from adipose tissues. So more research needs to be done to determine how much that does influence. And so, then along those lines, would there be greater differences with different substrates or precursors, so in the case with amino acids. And I've performed a few in vitro experiments looking at that, but unfortunately, they varied across the board and were not consistent. It seemed that there might be greater production from amino acids, but a few more runs need to be performed to look at that. And so then, even if these milk fatty acids cannot necessarily be used to predict microbial flow, as well as the volatile fatty acid proportions, it may be indicative of other processes in the rumen. So for instance, vitamin status. So B-12 is one vitamin that is, all B-12 vitamins are actually produced by the microbial population in the rumen. 

And so the animal itself doesn't need to be supplemented with those vitamins. So perhaps the amount of odd and branch chain fatty acids could indicate how much B-12 is being produced in the animal. So it seems that there are some other areas of research, that even if these fatty acids can't be used for exactly what we were hoping for, we are getting better insight into rumen fermentation and metabolism in the dairy cow, because, yeah, there are just some other answers, other questions that need to be answered. So from there, I'm finishing up a little bit early. I'd like to acknowledge my professor for the opportunity to be doing my graduate research here at this university, Louis Armentano. As well as two people in my lab, Sandy Bertics and Jake Finer, which helped me with sample preparation and analysis of my fatty acids. And without their help I could not have had all these samples analyzed. So with that, I'd like to thank you very much for your attention and please open the floor up to any questions you may have. 
(applause)