program, which is Sustainable Agriculture Research and Education is a national program that's funded by USDA. We're a grant and education program, so we give a grant to our five programs, farmer ranchers, youth educators, graduate students, research and education and professional development. If you want to find out more about San Diego to our national website, www.sar.org, you can click on Project Report and see reports from all of the presentations that have been here and unless they're so new that they haven't put a report up yet. So a great resource for you and also information about all of our grant programs in case you're interested in applying. So right now I'm going to introduce Sharon Lashour. She's going to grant a student grant recipient and she's going to talk about her project and using grant promises and the actual report machine. So good afternoon everybody. I'm just going to share basically a brief snippet of my dissertation research that I've been doing over the last year, looking at great comments. I'll explain what that is a little more in the presentation as a natural dewormer for sheep. So just an overview of what my talk will be about. I'll give you an introduction and background briefly why I did this, what's the problem, things that can. Some preliminary research that helped strengthen why we wanted to move a little bit further in doing this research. My SIR project overview, the results and discussion and then we'll go over some general conclusions. So the introduction and background, I'll give you background on gastrointestinal parasites, natural products in medicine and agriculture and what great promise it is. So gastrointestinal paroxysm or GIP has become a concern in the U.S. and globally because we have parasites that are becoming resistant to current commercial conventional deworming methods. This is mostly because of an increase of reassortment of certain genetic material within those parasites and also misuse of emblematic, some farmers don't really know how much to use or when to use it or aren't giving exact amounts. So that also increased the resistance within those animals. As well as with sheep and goats, there's a limited number of drugs that are actually approved by the FDA for those animals to get rid of intestinal parasites. So the issue that comes up is small ruminants are actually the ones that are having the most issue with resistance in gastrointestinal paroxysm. So there are common alternatives to chemical emblematics that are out there to control gastrointestinal parasites such as breeding to genetically resistant animals, animals that are naturally resistant to getting infected and dealing with the point to the point of disease. Also proper pasture management, making sure your pastures don't get overgrazed, making sure that you have proper pasture rotation between your animals as well as not overcrowding your fields, not having too many animals on those fields that can increase the spread of and transmission of those parasites to more animals when you do that. And there are also natural learning methods which I will talk about in this presentation. So just to give you an outlook, this right here is actually a dew drop. In this dew drop are parasitic larvae. They can be up to a thousand larvae within just a dew drop on a blade of grass. So just to give you some background on how the transmission and how it occurs, you have the larvae that are in the dew drop on a blade of grass. You have your sheep or goat come and because their browsers are going to nibble off the top more so they're just pulling straight up and grazing and they ingest those larvae. Once they ingest it, the larvae will go into several other stages before they become adults. They will reproduce and produce eggs that come out in the fecal matter which is put back on the field. Those eggs in the fecal matter will then develop into other larvae stages and eating organic material until they get to a pre-living stage which is the third stage larvae that become infective that are on that blade of grass. You have to cycle, start all over again. So this is just a list of common parasites and ruminants with the strontolith parasites being the most important which includes ostentatia or a brown stomach worm. You have humongous contortus which is also called the barbara full worm and also trichestandillus which is the bank web worm. Other parasites that are not worms would say and other parasites that are worms that are harmful and that farmers are worried about are coxidia which causes coxidiosis in your sheep and goats and cattle as well as tapeworm. So getting back to natural products and medical uses, natural products are basically those chemicals, compounds derived from living organisms, plants, animals, or insects that have some type of biological activity. They help in some way but they just come from natural sources. So natural products and agriculture has become a growing interest because it's a new way to control those GIP or parasites in small ruminants. There's also a public perception that residues from chemical dewormers are left in the meat so more people are wanting to go to natural products or go to looking at different alternatives. It's not necessarily true but it's part of propaganda. There are also limited to no commercial antimetric use with organic farming practices so going natural also helps with controlling those parasites that are on organic farms and there's also an increasing economic benefit in both plant and animal industries when you're able to reuse waste products or reuse certain agricultural products in a way that will help benefit not only your animals but help you get more money in your pocket. So some compounds of interest that we've seen in natural products that are out of the literature are condensed tannins, flavonoids, and antioxidants. So condensed tannins are just a chemical that's in plants, that's a plant secondary metabolite that just basically has been shown in certain research to increase absorption of essential amino acids in the small intestine and also that would lead to increased wool growth, body mass, milk production, and the amount of protein in the milk. Now this is actually due to the fact that condensed tannins will make a complex and bind with proteins during mastication or eating and then when they are able to pass through the gut so they don't get broken down and they're able to disassociate so you're getting more protein for the small intestines that would benefit the animal. It's also been shown to counteract protein losses caused by gut parasitism and stimulate the immune system with that extra protein that's available. Other research has shown that it's inactivated parasite larvae during the passage through the gut as well. Flavonoids have been shown to paralyze the parasite so it's able to be passed through the gut naturally and does not have a chance to attach or find a preferential niche. It also inhibits eggs from hatching which would decrease transmission and you also have a decrease of parasites expressing that multi-drug resistance and basically changing their genes so that they can become drug resistant. Antioxidants work in animals as they work with us. They are a way to scavenge for radicals within the body and they help increase immune function so the animal is able to fight off the infection naturally so it helps with that resilience to parasite infection. So grape pumice basically Ohio grape industry consists of about greater than 1500 acres of vineyards producing 1.5 to 4 times per acre since 2004 within the state. Grape pumice itself is what's left over after juice and wine making so it's the seeds, the stems, the skins and the pulp that's left over. And wine grape pumice has shown to have high levels of flavonoids and up to 4% of condensed tan which I just stated in the previous slide helped with gastrointestinal characteristics and has shown some type of efficacy so with these compounds being in grape pumice is why we wanted to look into it. So several animal studies have actually looked at grape pumice as utilizing as an animal feed but not necessarily at the parasite level that I'm looking at and what they saw was there's only a marginal amount of digestibility lowered when you put paired with other things so if you were to put grape pumice in your feed that has alfalfa hay or sorghum hay in it what you'll get is you don't have a lower digestibility it still pretty much stays the same within 1.6% and when given in moderation up to 10% of the diet being grape pumice it can actually help in fattening your lambs. So getting on to the next part of my talk which is the preliminary study that I did with USDA and cultural research services in Beltsville, Maryland and what we did was we took Shiraz and Cabernet Sauvignon wine grape pumice from a local vineyard here in Ohio in Madison. We dried those samples and ground them and then extracted them with alcohol so that we could get those active compounds of interest that I discussed earlier and then removed off the ethanol and actually put it in a saline solution which is a salt water solution. We looked at those third stage larvae of the brown stomach worm in an excheved state which means we took their outer cuticle off to simulate more what would be in the body when before they transitioned to the next stage larvae and we used about 100 larvae per treatment well. We looked at them at zero to for 24 hours after introduction of the extract on those parasites and they larvae were assessed for how they moved, how well were they alive, were they sick, were they inactive and were they dead. So this is just a tabular version of my result looking at the activity at two different concentrations of the Shiraz or the Cabernet Sauvignon grapes. There was actually no difference within concentration but we saw there was a decrease of almost 49 to 50% within four hours of those worms being in the extract and there was no difference in the results between four and 24 hours and at the actual Cabernet Sauvignon grapes had a higher efficacy of about 51% versus 48% reduction in activity of the Shiraz grapes and that was mostly due to their higher level of condensed tannin within the Cabernet Sauvignon grapes than the Shiraz but that was all relative to a control that only decreased about 20%. So when you look at that overall you're able to see that the extracts of the wine grape pumice actually showed some efficacy in decreasing the overall health and viability of those parasite larvae and there was no difference like I said between concentrations but there was a difference from the control. So this led us to our next studies which was we saw there's some efficacy with wine grapes on the parasite themselves so what happens when we look at them in the animal. We also did fecal culture which was to look how those extracts would be on the eggs that are in the feces so to help break that cycle of transmission that I talked about earlier. So with the animal trials, the first trial we used 54 growing lambs that were naturally infected they were tested for the level of infection that they had before they were starting to treatment groups. There were three treatment groups in the first trial. We wanted to test it against a commercial dewormer such as Cydecton. We wanted to test it against also the grape pumice that we formulated in a diet that we gave in five day dose that would give about 28 grams of condensed tannin per kilogram of dry matter. So we also wanted to test both of those next to a negative control which means they were given nothing these animals are just living as is eating regular normal feed.  They were assessed for body weight, average daily gain, feed intake, pack cell volume, and for matcha which would help assess the anemia score and the number of eggs per gram within their fecal also known as fecal egg counts. Trial two we came up with after trial one it was a little different in the beginning when we wrote it out but due to the results that we got from trial one our next step was to look at a dose titration of grape pumice in the diet. So the treatment so we still have 54 animals a different set of lambs and three treatment groups there's that negative control again that's given nothing then we had a middle ground which is given about two pounds per head of the grape pumice diet that gave that same 28 grams of condensed tannin per kilogram of dry matter but it was supplemented with a pound and a quarter of alfalfa pellet. So what their normal feed would be we're just giving it in a lower dosage than the next treatment group which was a three and a quarter pound per head of grape pumice that gave a dose of 35 grams of condensed tannin per kilogram of dry matter. We also assessed them for the same parameters as the first trial. So with trial one there was actually no difference between treatments with the body weight average daily gain or feed intake but we did see an increase in body which is expected for growing lambs as the weeks progressed the average weight gain over the 21 day period was about 6.8 pounds give or take 0.3 pounds per lamb the average daily gain for the different treatment groups was about 0.17 pounds for the ones given sidectin 0.33 pounds for those that were given nothing or a negative control and 0.32 pounds for those given the grape pumice the pack cell volumes or the red blood cells that helped us determine anemia decreased over time but overall the highest for the positive control there was no difference between the negative control and the grape pumice treatment. So with that being said the pack cell volume was at around 28 and usually you want to do warm your animals when you see that pack cell volume drop to or below 20. With the Fremontia which is an observational examination of anemia within those animals there was no difference between treatment that stayed relatively high around 2 the highest was 1, 5 being the lowest you usually try to do warm around 3 or lower and that corresponds to a pack cell volume of below 20. So you have what you can assess on farm, the Fremontia, what you can assess in the lab the pack cell volume. Beakle egg count was actually lowest for the sidectin treatment and there was no difference between the negative control or the grape pumice treatment. They were actually relatively high, they didn't really change much during the trial but of course you expect sidectin to bring the people egg count down. The surprising result that came up, sidectin should bring the amount of eggs per gram of feces down below 100 when it's working properly and it did not in our positive group and that was a bit of a problem on our farm because that was increasing resistance occurring in those lambs to a commercial dewormer. So that's what led into the next trial which was see if we can give more condensed tannins and if we can bring the egg count down and see some type of dose response. So in trial 2 there was once again no difference between treatments for body weight, average daily gain or feed intake. The body weight still increased over the weeks that progressed as they grew. The average weight gain over 21 days was a little higher at 7.7 pounds and the average daily gain was highest for the great pumice that was supplemented with alfalfa and the next after that was the negative control, the lowest being the group given a great pumice diet at the highest concentration. The paxil volume for these animals increased over time but there was still no difference between treatments and the Fremontia scores actually stayed slightly higher. There was no difference between treatments at all and it stayed around 1 to 1.5 so generally healthy animals. The fecal egg count showed no difference between treatments but the middle ground diet, the alfalfa, the great pumice that was supplemented with the alfalfa actually had a tendency to reduce the fecal egg counts which was a bit of a surprise to us because your hypothesis would be more condensed tannins you would expect a lower fecal egg count instead of the middle ground. But when I did further analysis I realized that there was actually more protein given with the second diet of the great pumice and alfalfa supplement so when you're increasing the protein and you have the condensed tannin you have a positive interaction happening that by increasing that nutrient value you're able to decrease the egg count and give the animals a little more to help fight the infection and decrease the amount of fecal egg. So moving on to the next part of the study which was the fecal culture and this was looking at adding the extract to the infected poop itself that had the eggs in it we wanted to include farmers so I went to five farms three conventional and two organic farms to collect the poop from their herd of sheep. We did two treatments looking at giving them nothing just mixing poop and water and doing a normal larval development type culture and also we used great biomass at 38 milligrams of condensed tannin per milliliter and that dose was decided on how much condensed tannin would be undigested left in the poop after that highest dose diet of the 45 grams of condensed tannin per kilogram of dry matter. And we looked at basically was there a presence in the presence of acids of those extracts did the egg hatch into larvae. So what we actually saw was that the fecal cultures that were done in the extracts actually had kept eggs from hatching or developing at 100% inhibition. No eggs hatched whatsoever when compared to the controls. There was no difference in the effective extracts between farms so it didn't matter where they used commercial and they were seeing resistance on a conventional farm where they weren't using any dewormers at all on an organic farm. We saw no difference in treatment between the farms, meaning that the extract work regardless of farming practice. One of just an observational but not quantifiable result that I saw was there was no fungus or mold that grew with the culture and the ones given the extract which was kind of interesting to know that it had some type of antifungal or antimicrobial effect and it also decreased the smell of poop. We now love that in working with poop.  Some general conclusions and what's next. The effects of wine grape pumice have an efficacy on parasites. We saw that looking at the extracts in the larvae directly. The next step would be in looking does this work against the barbical worm? Does it work against the bankrobe worm? Does it work against copcidia or tapeworm? So looking at other parasites that affect ruminants, will it help with those parasites as well and only look at the brown stomach worm as well as looking to see if we can pull out specific things in those extracts instead of just using a crude extract to see exactly what compound is causing this efficacy. We also, with the Envivo studies, they were inconclusive. This was mostly because the number of animals we had was too small. So we need to increase our replication in order to actually see a significant result from our treatment group. But also, when looking at the grape pumice diet when compared to the normal alfalfa diet, it was a lot lower in nutrient value. It had the same energy but it didn't necessarily give the same amount of nutrients and vitamins and minerals. So formulating a great pumice diet that had the optimum nutrition range and the optimum amount of condensed tannin would be beneficial in the next step in doing another animal trial. Also, the fecal cultures provided insight into decreasing the transmission on farms. If it's not allowing the eggs to hatch, it's not allowing transmission to occur. So you could then even use the extracts themselves or use the grape pumice into the diet for a few days to try to inactivate the eggs, deworm your animals, and then see if you can decrease the amount of transitions. The problem that occurs is you have constant reinfection happening. So you may only have one or two sheep that are highly infected and a few of them that are fighting off resistance but because they're over shedding eggs, in what they call super shedder, what are happening, you have one or two that are affecting your entire herd over and over and over again. So just helping to see if there can be other studies that can be done to help add grape pumice to the diet to decrease the amount of transmission. So here are the references for the information that I've given you. Are there any questions or comments? Yes, ma'am. Okay, so to make sure I understand your question, you want, your question is that in, there are certain things or compounds in grapes themselves that are toxic to dogs, and you want to know if anybody looked at it into these agricultural animals to see if there's toxicity within the kidneys. No, I have not seen anything out there about that. The other difference is the way things are metabolized in the dog or a non-ruminant animal are completely different as how they're metabolized or digested into a ruminant animal. So that may be another way that it's not really affecting kidney function that anybody has seen so far. If that helps me to answer your question. Anyone else? Yes, ma'am. So your, well, the question is that, could I just explain further what was the process that I used to get this extract? Is that correct? Okay, so what I actually did is collected the wine grape pumice brush from the vineyard, brought it back. The vineyard is Dubon, vineyards in Madison, Ohio. It's not from the grapevine. It's a wine, no, it's a wine grape pumice. So what's, it's basically the wine grapes have already been taken, harvested, crushed, fermented for about a week, and we're getting after you spin off that liquid on the first fermentation. So we're getting the waste that's left over. Right.  So it's the waste that's left over. It's not actual agricultural products if that makes sense. Does that help better? All right. So after that, we dried it to about 95% dry matter. And then we took sub-samples of our large group. We actually got about a half a ton of this grape pumice for our subsequent studies. So we dried it down once it was dry to above 95% dry matter. We ground it in a wily meal to pass through a two millimeter sieve and then take, it took a 70% aqueous ethanol solution, which is basically 70% ethanol, 30% water with vitamin C added to help keep the compounds in it from oxidizing. We wanted to make sure we kept our compounds active and extracted over 24 hours. So basically set the great dry grape pumice in that mixture, let it mix for 24 hours in the solution. And then after that, we took off all the liquid portion, basically took off the liquid portion, threw away the solids and evaporated off all the alcohol and then re-suspended it in a salt solution so that it would not harm the larvae themselves. Now as far as the animal studies, it was put in a diet. So the grape pumice was not extracted at all. Right. So you have to understand with the extracts that's bench work. That's the research in the lab that I need to look to see is it working against the parasites out of the body. When it comes to the practicality of using grape pumice, you would basically get the waste material from a vineyard and when you sent it off to get your diet formulated, it would be incorporated into your diet. So eventually, hopefully it'll be commercially available but until that happens, basically what's happening is they're taking the raw material and mixing it in as a feed additive.  You're just adding it as a part of the ration itself. Would you, would it be a daily part of the ration or would it be a treatment? So the way I look at it, I think it would be something that is a little bit of both. So it's a daily treatment for a given amount of days until you start to see the decrease. And then the reason we looked at that was, was there an extended effect than it was? So just that five day dose would be enough or seven days would be enough and then you continue to use your natural or whatever your normal feed would be after that. That's where the trend is to think of what it could be daily. Right. Right. So you're now a part of the ration that will offer a treatment. So what would be something that would need to be in consideration for anyone who's certified, you know, what, how does that change that? Right. And that's why we looked at it as a five day dose is what we looked at and that, you know, it did have an extended effect. So you could do it once a month for about a week or business week. And then you should be able to still see some type of advocacy within your heart. Is there any other question? One more? Thank you for your attention. I appreciate it.