BRTF Monthly Webinar - Dr. Pedro Fontes - 05/19/21

all right goodnight everyone and welcome uh to our last webinar of the beef repetition task force uh this spring uh we are very pleased with the results there's been a lot of um you know a lot of good talks and a lot of good um attendance so we'll be very pleased with the results of this webinar series and we're planning to to continue um the series uh next spring so i want to thank everyone that has spent the time and join us every third wednesday of the month and we plan to keep that going for for next year um our last speaker of the series uh last but not least dr pedro fontes um he's a good friend and we would know each other uh from from way back in brazil and then from grad school but dr pedro fontes he's a got his dvm from sao paulo state university in brazil then got a master's from the university of florida and a phd in beef cattle reproduction with the physiology from the texas a m university and is currently an assistant professor at university of georgia in the department of animal animal science so dr fonte is going to speak to us today about pregnancy diagnosis and new technologies on pregnancy diagnosis so we're really looking forward to your talk in pedro the floor is yours um i forgot before uh before i turn to you uh we are live on facebook and also uh if you have ever missed one of our webinars they're all recorded and they're available on our youtube channel uh the be task force youtube channel and this webinar will also be available um tomorrow morning on youtube and at the end of the webinar we're going to have some questions and uh question and answer time so please use the q a tab of zoom to send your questions okay pedro now the floor is yours all right vitor um thank you for the introduction as we go through here let me know if you have any issues listening to me or anything like that but let's go ahead and get started first of all good evening everyone thanks for thanks for joining us today i think that's a that's an interesting topic that i really enjoy talking to producers about different pregnancy diagnosis tech technologies are available and and just as sort of a brief introduction as victor mentioned um i went to vet school in brazil so i graduated from sao paulo state university then i went to grad school at the university of florida as well as texas a m university so i got my phd from texas a m university and i have recently started here at uga at the university of georgia in 2020 and i'm i'm really happy with the with the invite from vitor because we're going to be able to share some of the work that we started doing here um looking at some new technologies to diagnose pregnancy uh more specifically we've been working with um color doppler ultrasonography so trying to diagnose pregnancy a little bit earlier than we currently uh do with our conventional methods but for a conversation today i want to go over um not only the current the the new technologies that are arising but also and some of the work we've been doing but also the more traditional methods of pregnancy diagnosis right so before we we we go ahead and get started um i want to have a brief introduction just to kind of get our conversation started with regards to beef reproductive efficiency so here i put together some data that i got from file and i uh organized that database on different decades here that you can see here in the y and the x-axis of this figure so here on the left we have the number of cattle slaughters here here in the u.s and here on the right we have the amount of beef that we produce and then you can see that as we move from from the 60s all the way until the last decade so 2010 here represents um all the data collected from 2010 all the way to 2020 and what we see when we look at those overall industry trends um is the re that we have experienced a decrease in the number of animals that we've been uh slaughtering um over the years and that decreases about 16 and what's interesting is that in the same interval of time we're able to increase beef production by about 30 percent uh meaning that we're basically producing more beef with fewer animals and and really highlighting that we're getting more efficient uh with our process of producing beef right um there's a lot of factors that have been driving those differences in efficiency but definitely one of them has been the changes in carcass weight right so when we evaluate the changes in carcass weight over the last 50 years or so we can see that we increase carcass weight by an average of about 50 percent basically meaning that for to produce the same amount of beef that we get from a carcass between the years of 2010 and 2020 in the 1960s we had to slaughter one and a half animal right so meaning that we really really changed the characteristics of our animals and so when we consider all that and we think about some of the future directions of the beef industry and some of the things that i think are relevant for this group here um we know that it's very unlikely that our herd numbers are going to increase so it's likely that we're going to have our herd numbers stabilize or potentially decrease our animal numbers and as we look at the carcass data that i showed you in the previous slide it's very likely that that really a rapid increase is going to probably plateau right we know that that increase it's not uh sustainable uh on the long haul looking forward uh for several reasons right we we know that we'll have to have some serious adaptations in our in our packing plants to process uh carcasses if they start getting um too heavy and we also know that there are several implications to the cow herd as we start thinking about cow maintenance and and things like that but besides of that scenario the specific scenario here uh we know that uh the demand for beef and not only for quantity but also for quality of beef is likely gonna continue to grow as we move forward right and you know as a reproductive physiologist uh the way that i like to approach this is that as you think that we're gonna as cow calf producers we're gonna be playing an important role on that overall increase in efficiency they're going to be observing in the future right so if you want to keep if you want to be able to meet the demand for quantity and quality of beef we're going to be able we're going to have to improve our efficiency in a cow calf segment of the industry right and by improving efficiency in the cow calf segment of the industry i'm talking about producing more pounds of calf uh per cow exposed breakout exposed in the breeding season and that's pretty relevant as we start talking about the use of reproductive technologies in the united states and this is a survey that was put together by the usda where we have a different herd size here on top so we have small medium and large operations and then we have different reproductive technologies here on the left okay so you have astro synchronization efficient insemination and road transfer but what i want to draw your attention to here is the three reproductive technologies here at the bottom because they're relevant to our discussion today right so when you look at the rage of relatively simple reproductive technologies right a pregnancy diagnosis through rectal palpation or ultrasound and more recently blood tests the rate of the percentage of our operations are adopting these technologies are considerably low right and what we see as well that it's interesting about the stable is that as we increase the size of the herd we also see an increase in the rate of adoption of these technologies as the herd increases right vito i'm getting just want to double check everybody's seeing my slides uh i'm getting some messages here in the chat can you see my slides okay peter yeah pedro um it seems that um some people are getting your video instead of those lights i think if um your video is spotlighted so i'll try to fix that but please keep going we'll fix it all right sounds good so yeah so we know that as we look at this data we see that as the size of the operations increase the proportion of producers that are utilizing reproductive technologies also increase right and as we start thinking about the reasons of that there's likely several reasons for that but one of that is likely that the percentage of the annual income of those families that run those larger operations likely a greater percentage of their income is coming from their cattle operation right and and those folks are uh are usually aware of the benefits of the utilization of these technologies so so they're taking advantage of these technologies so as you think about improving efficiency moving forward hopefully um in a few years from now if we conduct a survey again hopefully we'll be able to see an increase in the cooperation of these technologies so why should we perform pregnancy diagnosis why should we worry about running cows through and collecting uh that information um so there are several reasons right why we want to collect pregnancy diagnosis information right and you know we don't only want to collect that information but we also want to use that information to actively make management decisions right so as we know the pregnancy status of our cow herd we can make changes for nutritional plan uh but not only that right we use that pregnancy status information to make um marketing decisions right so if you think about uh traditional cow calf operation usually about 15 to 30 percent of their income are going to be coming from coke house so having the pregnancy diagnosis information and establishing our culling criteria based on on lack of reproductive efficiency uh it's it's substantially important right not only that we know that having that information uh together with knowing you know that there are seasonal variations in cold cow prices and combining that with different prices in commodity can really help producers uh develop strategies with regards to their um with the cows with the grass of the cows that they're calling right there's several options and several things we can do uh ranging from adding weight to our cow herd or immediately marketing them depending on market institute on the market situation so having that pregnancy diagnosis information is really important and as we start talking about you know evaluating reproductive efficiency so a couple days ago we were crack checking some of our cows that we transferred some embryos so i really want to evaluate how we're doing with regards to the use of these reproductive technologies either if we're talking about embryo transfer or fissure insemination or whether we're trying to evaluate the performance of a specific sire from a fertility standpoint so there's a lot of value to have that pregnancy diagnosis information and and usually when we think about um the consequences of neglecting uh the importance of pregnancy diagnosis you know if we fail to detect those open cows we're going to be holding cows that theoretically shouldn't be kept in our herds right so we ultimately increase the number of animals in our operation and as we increase the number of animals in our operation we also increase the expenses associated with cal maintenance right which ultimately increases our production costs not only that as i mentioned earlier those cocals usually represent about 15 to 30 percent of our income right so if we fail to market those cows to properly recognize those less fertile cows and color them and market them we're going to ultimately decrease the income that is coming from those whole cows if we're not utilizing pregnancy diagnosis not only that we by doing so we're going to fail to call some some of our last fertile animals and we know that as we keep those less fertile animals on the long haul we're going to decrease reproductive efficiency and reproductive performance over time so before we go ahead and start talking about specific methods uh for early pregnancy diagnosis i just want to talk a little bit about uh what's going on right during early pregnancy so here i have a figure of a representation of some of the physiological events that are happening during early pregnancy so here you have the uterus uh actually the uterine horn of a cow okay then we have the oviduct here and this is then these yellow structures are are the embryo here and here more towards the left side of the diagram we have the representation of uh orbee and oxide right so we know that when a cow ovulates that oxide is going to arrive here in the oviduct and hopefully when it's kind of halfway through that obvious duct it also is going to get fertilized right and after fertilization we have the formation of an embryo that is going to arrive in the uterus usually around five to six days after ovulation right and for those of you that have done uh some embryo transfer work and have seen folks flushing cows um and you you you get a chance to look at the uh at the scope while the embryologist was you know sorting those embryos you've probably seen a figure similar to this one so that embryo arrives in the uterus uh in a spherical shape so this is day seven embryo and then as you can see um around that angle there's a protective layer which is what we call a zona pellucida okay and as we progress in gestation uh that embryo is going to hatch from that zona pellucida so this is a electron microscopy figure which is basically a fancy way to visualize those embryos in a research setting where you have that spherical embryo hatching from that protein protective layer from that zona pelican a day aid of development okay so as we progress in pregnancy that embryo that was once spherical starts elongating gradually elongating it requires this tubular form so this is a electron microscopy as well of an embryo on day 13 in a tubular shape and then as we progress in pregnancy through day 14 that embryo keeps elongating all the way until day 17 here where we have a flesh conceptus in what we call a filamentous form okay so um you can see that these conceptus and that embryo undergo some dramatic morphological changes and not only morphological changes we know that they undergo a series of functional changes and every time we're talking about some pretty substantial changes in biology in early pregnancy development we're talking about uh the very high probability of things going wrong right we know that uh there's a lot of pregnancy loss that occurred not only uh from day seven to day 17 but even before that now uh and also after day 17 as that embryo uh starts attaching and and and forming the placenta within uh against that urine lumen here that we have represented here on top so there's a lot of things that can go wrong there's a lot of research efforts that it has been the folks have been putting um on trying to understand what are the factors that regulate uh the developing of those embryos and what can we do from a management standpoint to optimize all these events you to maximize fertility but what i want to share with you now it's it's a meta-analysis that was done by uh dr poland's group where he basically characterized what's going on in the beef in our beef cattle population with regard to those pregnancy losses that occurred not only during early gestation but also towards more advanced stages of gestation as well the take-home message that i want you to have in this slide is that um as gestation progresses uh a lot of pregnancy loss occurs right so if you look during early gestation here and this is a series of studies that they compile in this matte analysis where folks um we're going there and actually um collecting the reproductive tract of a cow you know bringing to a laboratory and flushing those tracks and and visualizing the percentage of cows that actually had an embryo an embryolike structure during different stages of gestation as well as data from the field where they go there and perform pregnancy diagnosis with ultrasound uh and later stages of pregnancy it should really characterize when those pregnancy losses are occurring so basically if you go here and you synchronize a group of cows and fix time area settings and officially inseminate them we are here at a hundred percent of the cows being serviced right and as we move forward in gestation pregnancy loss start gradually occurring right so for example based on that meta-analysis if you go here day 10 let's say we we develop the best pregnancy diagnosis method possible that can detect 100 percent of the cows pregnant here at day 10 of gestation if you think about the real value of that pregnancy diagnosis for a cattle producer it's not really valuable right because naturally some of these cows are going to experience embryonic mortality here as gestation progresses so as we talk about trying to diagnose pregnancy early this is something to keep in mind okay and you know for uh for better interpretation of this idea of pregnancy loss uh what folks have been doing in the literature is dividing the pregnancy loss in different periods and usually when you look at most of the beef research looking at pregnancy laws usually they classified the pregnancy loss that is occurring from day zero all the way to day 30 so we're doing our first pregnancy diagnosis as early embryonic loss and then we were talking about the pregnancy laws that are occurring between day 30 and day 60 or day 30 and day 100 depending on the experiment usually we're talking about what we call uh late embryonic or early fetal loss okay and basically you can see that the rates of early embryonic mortality are substantially high compared to the rates of late rates of late embryonic mortality and this is going to change a little bit here on the late embryonic mortality side of things if we're talking about dairy herds that's a little bit different those rates tend to increase not only that if we're talking about folks that are utilizing in vitro and reproduction we know that the rates of embryonic mortality is a little bit higher here as well but something to keep in mind is that you know those cows that experience early embryonic mortality they're very likely going to rebreed within the same breeding season right however those cows that experience late embryonic mortality are those cows that you know kind of break our hearts you know you go there and synchronize your cows and use a really high quality sire produce a really high quality uh you think you're producing a really high quality offspring you go there and they 30 back check that cow is pregnant but then you run that cow again 30 days later or at the end of the breeding season and she lost that pregnancy right so that's uh really frustrating you know from from from a producer's standpoint but not only that the economic consequences of those pregnancy losses are pretty uh substantial as well so when you start trying to um when you start looking into some of the research that is doing comparing different methods of pregnancy diagnosis you see a lot of different terms that are quite complicated so for a conversation today i like to use two main terms as we start describing uh the accuracy of different pregnancy diagnosis methods right and these terms are false positive and false negative okay i'm not going to talk about sensitivity specificity and positive predictive values i'm going to be focusing mostly on false positive and false negative terms and basically what i mean by a false positive is uh when we go there we have a cow that is open and we use a specific pregnancy diagnosis technique and we go there and you call that cow pregnant but she's actually not pregnant right so that would be what we call a false positive whereas the false negative would be a cow uh that we know that she's pregnant right um but we go there and we use a specific method to diagnose pregnancy and that method tells us that cow is not pregnant so that will be what we call a false negative right so as we think about an optimal pregnancy diagnosis scenario we want to avoid both false positives as well as false negative results okay with regards to false negative results these are likely uh more important as we think about uh the management practices of a brief operation right so if you're resynchronizing cows we don't want to have false positive results false negative results and also if you're thinking about marketing cows we also cannot afford having false negative results so these are things to keep in mind as we move forward so let's jump in and start talking about some of the current methods that are available so likely the most commonly utilized uh method for pregnancy diagnosis is rectal palpation right so uh when you're performing uh pregnancy diagnosis with direct palpation we're gonna bring the veterinarian in and one of the things that i want to brought up before we start diving into these different methods is that regardless of the method that we're utilizing you know we really it's really important that producers work with their veterinarians right to come in and provide that accurate result that you can really trust and and you know develop that relationship with their veterinarians which is really important right as we talk about these uh different methods make sure you're working with your local veterinarian to bring them in and perform those pregnancy diagnosis results so as you think about rectal palpation and compared to some other methods we're going to talk about rectal palpation um usually it's performed a little bit later right you've got to wait around until around day 40 of the station to start being substantially accurate with the rectal palpation again it requires a trained personnel to perform the test so we usually recommend that you work with your veterinarian that's really important and one of the good things about performing uh rectal palpation compared to other tests so for example the blood based pregnancy diagnosis is the fact that we get shoot side results right we go there and we palpate our cows and and we can make a decision right there whether we're going to sort that cow we're going to send it to market or whatever we want to do another positive uh thing about the recto palpation method is the fact that you know depending on how trained the veterinarian that's performing those um um diagnosis is they they can detect quite a few pathologies such as you know mummified fetus and things like that uh and and that's definitely a positive of rectal palpation and so basically when the veterinarian come in and he's uh regularly palpating her cows he's looking for some specific landmark so here these are some figures from the draws project so here i have a pregnant track a reproductive track on the left and an open reproductive tract on the right so here you have the different uterine horns and i want to draw attention to the fact that these urine horns are pretty symmetrical right and here when you go to this pregnancy here and this pregnant tract this is uh probably around the 15 day 60 of gestation and you can see that there's a clear asymmetry between the horns so whenever your veterinarian is in and he's performing direct on palpation he's trying to palpate those both horns and trying to feel for fluid um inside the hearing horn but also that asymmetry uh between the two different uterine horns some other things that we're going to be looking for as well is of course the fetus itself and not only the fetus actually the placenta right so this is a pregnancy that was dissected out of the uterus and you can see that the fetus is free floating here inside uh it's floating here inside that placental structure here and you can see this button-like structure this is what we call the cotyledons because here we have only the fetal component uh present here and the cotyledon together with the with the coronicles form what we call the plasma tone so whenever your vet goes in he can feel some of those classroom terms depending on the stage of pregnancy and use that to diagnose pregnancy another method that it's commonly utilized uh in beef cooperation is ocean sonography and i'm calling conventional oceanography here because we're going to talk a little bit about color doppler or sonography as we move forward in the presentation and you know as we think about the advantages of utilizing conventional ultrasound we can detect pregnancy a little bit earlier in heifers we can go as early as day 26 and cows as early as day 28 depending on how trained the person is potentially even earlier than that not only that but with the ultrasound we can have a pretty good idea on the viability of the pregnancy depending on the stage of pregnancy as well but not only that we can pretty accurately age that pregnancy a little bit more accurately with uh ultrasound than we than with the erector palpation itself maybe when you're talking about early pregnancy diagnosis not only that we can also determine uh the sex of the fetus depending on the stage of the pregnancy that we're evaluating those cows and those fetuses causal variables very similar to what happens in the rectal palpation and from uh uh how those when you think about how those ultrasound examinations work so basically we have an ultrasound machine here on the right and then we have a trans-rectal probe so we're going to introduce that probe in the rectum and the probe is going to send ultrasound waves those waves are going to hit an object in our case here we're talking about the reproductive tract that object is going to bounce waves back they're going to be received by that same probe right so depending on the length uh and the characteristics of that wave that bounces back that receiver will interpret those waves and translate that into an image that has different shades of gray and basically those waves are going to be changed based on the density of our object here so if you're talking about a fetus that has bone has bones and it's it's pretty dense let's call it like that uh the image that we're going to be visualizing the oxygen is going to be bright right whereas if we're talking about a tissue that is less dense or something that's not dense at all like water uh the type of image that that ultrasound is going to generate is going to be a little bit dark right so this is an example of some of our uh of a video that we record recently here in one of our track checks this is a day 85 or so pregnancy and here you can see the skull um of the fetus the head of that fetus surrounded by this black image here which is um the fluid contained within the uterus right so that the fetus is floating within the uterus then we're visualizing fluid as well as the fetus itself so that was a day 85 ish pregnancy and now i'm going to show you an early pregnancy so if you're going around day 28 day 30 this is something this is where you're going to be observing so this is a cross section of the uterine horn we have a pocket of fluid right here and then you can see a button-like structure popping up which is that developing embryo here with about 29 days that's when we perform the specific pregnancy diagnosis here okay so once you know the the person get trained uh on how to visualize these images it's quite easy to interpret what's going on and recognize those pregnancies as long as you have substantial experience palpating cows right so that's why it's important uh to bring someone in that has those skills and that's why we encourage everybody to be working with their veterinarians one of the things that i want to show you that i've showed you all that i thought it was cool we were recently doing some final prank checks from one of our fall breeding herds and this is a a pregnancy with about 110 days and this is a cross-section of the thorax of a calf and just to highlight the quality of the ultrasound machines that are available now for us to utilize and how detail the images uh are getting um so here i'm going to play that video and i want you to draw your attention right in the middle of the screen you're going to be able to see the heart of the fetus beating but not only that you're going to be able to also see the different chambers of the heart so if we pause our image here you can see the different ventricles and and you can not only see the ventricles of the heart but you're also going to be able to see the bulbs of the heart as the the heart of the fetus be so pretty interesting to see how how accurate our images are getting and the kind of quality of ultrasound equipment that we can utilize to generate some pretty trustworthy results with regards to pregnancy diagnosis methods so not only that through reco through transrector ultrasonography we can pretty accurately age pregnancy so um this is a couple of images from fetuses at different stages of gestation so here we have a day 53 pregnancy here we have a day 84 pregnancy and as you can see uh they're pretty substantial differences right here is just the head of the calf and here you can see the whole fetus fitting within the screen so whenever you bring your veterinarian in they can go in and pretty easily distinguish those pregnancies and be able to age those pregnancies pretty accurately not only that if they come depending on how on the timing in which those pregnancy diagnoses are being performed you're also going to see we are also going to be able to uh determine uh distinguished male and female fetuses okay so now let's switch gears and talk a little bit about uh the blood-based pregnancy diagnosis and usually when you talk about the blood-based pregnancy diagnosis folks who come and ask what are we detecting right in those blood-based pregnancy tests and and and usually people wonder if it's the same thing as we detect in humans uh tests that you can buy at cvs or walgreens and the reality is not so we're detecting something different here so we're talking about pregnancy detection in cal encountered with blood through the blood tests we're talking about the quantification of what we call pregnancy associated glycoproteins so this is an image of a of a pregnancy here okay so have the fetus here with the different uh layers of the placenta here you have the amnion the coriolen toys here but what i want to draw your attention here is should these button-like structures this is what we call the plasma tones so if you were to grab a and tone from a pregnancy like this and you know cut it in half and bring into the lab and and look at it into the microscope you would see something like this diagram here on the right where you have a layer of fetal cells on top here represented in marine and a layer of maternal cells here represented in yellow and then in specific stages of gestation we're going to see that some of these fetal cells will start differentiating okay and some of these differentiated cells they will migrate from the fetal side of the placenta into the maternal side of the placenta and once they get into the maternal side they'll tap that basement brain membrane here and they will secrete some some products into the maternal stroma here in the uterus and some of these products will make their way into the maternal circulation and pregnancy associated like proteins is one of them okay so we can go there and collect a blood sample from a cow from the jugular vein or from the coccygeal vein and the tail and we can take that symbol to a laboratory and quantify the circulating concentrations of those pregnancy-associated glycoproteins which again it's coming from those cells that are derived from the fetal placenta so serving as a bio marker for pregnancy so here's some of some of the research from uh kai polar's master's work when he was still in missouri with dr smith and here we have the concentrations of pegs at different months of gestation okay and you can see that as gestation progresses the concentrations of pegs will gradually increase right not only that if we were to zoom in during uh early gestation here this is what i got here in this figure at the bottom so we have the circulating concentrations of pegs at different days of gestation here starting at day 24 going all the way to the 60 of the station and usually at day 24 is when we see the first increase and the first significant difference between pregnant and non-pregnant cows with regards to the circulating concentrations of facts okay so we can actually go in here day 30 28 and day 30 and collect a blood sample and send it to a laboratory and quantify those bags and determine which cows are pregnant and which cows are open so um there's three main companies three different companies working with boiler-based pregnancy diagnosis so we have the product line from idex bioprine has their own assay as well as gen x have their assay and basically the way that these assays work is these are antibody based assays this use technology called elisa or basically utilize antibody to recognize those pegs in the circulation of the cows and then based on the recognition of those circulating pegs we determine whether cows are pregnant or not and where we get asked a lot is is there better is there a better kid or or is there a kid that works better and the reality is there's a lot of research backing up each one of these technologies and you know even though they use different antibodies and that's pretty much the main difference between them all these tests can pretty accurately detect pregnancy and yield some pretty reliable results one of the things that would was one of the with withdrawals of these blood-based pregnancy diagnosis is the fact that we had to ship those samples to a laboratory more recently idac's probably a couple of years ago made it available in a market a shoot side blood test which is basically a budget a blood test that you can perform shoot side it takes about 30 minutes uh basically we have a series of steps from dispensing the sample into specific wells to adding specific types of reagents and those samples will go through a series of washes and then at the end you're going to see a change in color and there's different wells for each well represent a cow and depending on the change of color that you observe in the well you determine whether cow is pregnant or not and this is uh you know it's a little bit it's not as it's not super easy so you need to be really organized you get those going and you get those uh done make sure you're not getting uh samples mixed here as you start pipetting these into the plates so these are things you keep in mind as you think about potentially utilizing these technologies and again you really benefit if you decide to utilize the technology like that work with your veterinarian let them guide you through the process of utilizing technology like that so um how accurate are these commercially available blood-based pregnancy diagnosis and we when we look across the literature the accuracy of those tests are really good right so these are two in the independent peer review publications with more than 90 accuracy uh for uh for these blood-based pregnancy diagnosis and usually you know when we talk about 97 or 93 accuracy folks who go back and these are craig checks performed blood samples collected around the 28 day 30 of gestation folks will come back and say well why it's not 100 right and and there's a variety of reasons why it's not 100 and one of them being embryonic mortality i'm going to show you some data for that and the other uh one that it's important it's cows with early days falls apart so if you get a group of cows that we breed them when they are early in their postpartum period what will happen is as you remember from that figure that i showed you before the concentration of these circula of these pregnancy associated glycoproteins and the end of gestation is substantially high right so once these cows calve it takes about 60 to 70 days for those cows to clear that pregnant uh those pregnancy associated glycoproteins from their circulation so if you breed cows really early in the postpartum period and we collect those blood samples a little bit earlier than we normally do what might have what might happen is what we did we'll be detecting some some of the pregnancy associated glycoproteins that are coming from the previous pregnancy so that ends up uh leading us to have some false positive results which are not the end of the world but you know if we follow the recommendations from from those companies with regards to the timing for postpartum period we can really mitigate those false positive results so you know i mentioned the the idea of embryonic mortality and pregnancy associated glycoproteins so this is some other research from from dr pollard's group where basically we have two groups of cows here cows that maintain pregnancy and cows that experience what we talked about what we define as late embryonic mortality so basically here we're talking about cows that were diagnosed as pregnant via ultrasonography here at day 30 and then when we ran those cows through again the day 60 day 100 of gestation those cows lost their pregnancy at 8 30. even though they had a healthy uh fetus embryo here um with a heartbeat and fluid and you know were considered pregnant through the ultrasonography so and what's interesting here is that you know when we collected the when we collected samples and we did the same thing here in georgia but when you look at kai's data um here are samples collected at day 30 okay and those cows are pregnant when we're collecting those samples but if they're going to experience embryonic mortality they already have lower circulating concentrations of fats we observe something really similar here in one of our studies that we're running right now and this is some preliminary data but also those cows are experienced late embryonic mortality at day 29 when they're still pregnant on average they had lower circulating concentrations there's some work being done with regards to trying to predict pregnancy loss through the utilization of those pregnancy associated glycoproteins but this is uh at least at this point um not at a stage where we can utilize that information commercially what we do know is that those cows are coming in and they have lower circulating concentrations of effects and you know i mentioned earlier that we can see an increase in circulating concentrations of pegs at 824.

so can we accurately go there and collect a blood sample on day 24 in in in evaluative cows are pregnant in inaccurate determined pregnancy status and so this is some research uh out of texas and american university as well uh here we have day 24 circulating concentrations of pegs for cows and heifers divided in pregnant and non-pregnant animals and you can see that the pregnant ones do in fact have higher circulating concentrations of pegs at day 24. however when you try to utilize that information to actually predict their pregnancy status at day 30 to figure out whether they're going to be pregnant at day 30 comparing to the north sound of day 30 which is our gold standard method right we have some false positive results which are likely driven by some embryonic mortality that occurs between the 24 and day 30. but not only that we have some false negative results so you know the reason why i want to bring that up is that it is really important as we think about utilizing those blood-based pregnancy diagnosis to follow the recommendations right if you detect if we try to collect those samples a little bit earlier it's more likely that we might run into issues so make sure you follow the recommendation of whatever um blood-based pregnancy diagnosis you utilize okay so from a practical standpoint you know if we consider all that and we're thinking about uh scheduling pregnancy diagnosis if you're thinking about a scenario with 75-day breeding season we can you know after we remove the bowls 30 days later run cows through perform a transrectal ultrasound or collect a blood sample or potentially wait 40 days and perform rectal palpation and by doing so we're going to be able to distinguish pregnant from open cows and figure out potentially calling decisions based on that based on that information but not only that if you're thinking about a an operation that has a longer breeding season let's say 75 days or longer and you want to not only distinguish pregnant and open cows at the end of the breeding season but also recognize females that are pregnant to ai and differentiate them from the ones that are pregnant by natural service ideally you want to go in and perform a pregnancy diagnosis earlier during your breeding season around 8 30 day 40 of gestation and recognize those ones that are pregnant and then perform a final pregnancy diagnosis to figure out which ones got pregnant by the cleanup pool one way to do it uh for the operations that have a short breeding season and want to distinguish pregnant and open cows uh but not only that also distinguish those ai pregnancies uh when we're considering um operations with a short breeding season we can come here in and perform a single transaction ultrasound around 90 days after breeding in this example the 60 day breeding season that will give us 30 days between the removal of the cleaner bowls in our pregnancy diagnosis which allowed us to differentiate um ai and cleanup bowls through the ultrasound here so that's that's another option as well as you're thinking about you know if that information is important for you so now let's move forward and talk a little bit about how we can utilize strategically some of these pregnancy diagnosis technologies and the context of resynchronization right so for folks that are utilizing astro synchronization and fixed timed ai in in traditional i guess systems uh most of the folks are utilizing ai are exposing cows to one round of fixed time artificial insemination and and the beef uh cattle population right usually those cows are exposed to natural synchronization program and then we go there officially inseminate those cows and about 10 to 14 days later we're going to turn it in a cleaner pool right and you know there are several uh benefits for utilizing artificial insemination but one of the main ones is the fact that you know we can capture some value from those pregnancies that are generated through ai right so how can we potentially maximize uh the number of ai pregnancies in our operation so there are several different ways that we can do that and one of one of the ways that we can do that is by performing a heat detection after our first round of fixed armored fission insemination and this is something that we do uh in some of our herds here at the university of georgia mainly with our replacement heifers because it's easy easier to perform that heat detection we usually perform the heat detection between 15 and 25 days after breeding and then we will ai those heifers following an ampn role and we usually get a pretty good pregnancy rates and that and utilizing this approach and we really increase the number of of pregnancies that are generating through ai but not only that we know that that process of heat detection can be a little bit challenging depending on the size of operation and the logistics of each operation so we see some operations and we tend to do that as well in some of our operations is to utilize resynchronization right so we go there exposed calcium one round of uh astrosynchronization followed by fixed time the ai we go there and perform an oceanography examination on the 28th day 30 of the station then we get our pregnant cows and uh we don't do anything with the pregnant cows and and our open cows get exposed to a resynchronization program right so basically those cows will get a cedar in a general age and follow a regular synchronization protocol in this example here i have the five uh the seven day cosine plus cedar protocol but something that has been uh done recently probably over the last 10 years from a research standpoint and there's some operations now utilizing that commercially is uh starting to resynchronize cows while they still have an unknown pregnancy status right so basically go their exposure calls to a regular synchronization program fixed time artificial insemination then at day 21 here in this example you go there and start resynchronizing all your cows give a generation injection put a scenery in and when it comes time to give a prostaglandin injection which would theoretically abort a pregnancy we go there and remove that cedar perform a a conventional ultrasound and we detect the pregnant cattle the petting cows are like all um the open cows are going to receive a prostaglandin injection and then we fix time artificial insemination artificially inseminated so by doing so we can decrease that interval between our artificial insemination events so one thing to notice here is that what's driving how early we are re-breeding those animals in a fixed-time ai setting right in a resynchronization setting is our ability to detect pregnancy earlier right because we're limited to day 28 of gestation here we cannot decrease that interval between the stream breathing events and this is something we've been trying to tackle based on some research that it's done in brazil and we're trying to apply those results here in the u.s so and that's through the use of color doppler ultrasonography so what is the what is the rationale for utilizing color doppler ultrasonography so as i mentioned earlier uh when we're working with conventional ultrasound we're trying to visualize fluid right uh inside the uterus as well as the presence of an embryo but in that context we're limited to when uh there's there's a legit accumulation of fluid that we can go there and detect with the transvector ultrasound right however we do know that you know the open cows uh they will start undergoing what we call ludiolysis earlier than that right and by ludiolysis we mean the regression of the corpus luteum and the corpus luteum is this ovarian structure here on the right so this is the structure that produces progesterone so this structure is required for pregnancy maintenance right so uh when a cow is open uh we have uh the endogenous secretion of that prostaglandin that will cause varioly so the prostaglandin is coming from the uterus of the cow and it's gonna break down that co and decrease the progesterone and what's that and what that's going to do is it's good that cows started to recycle again or express estrus again i guess that's a better term so as this concentration of progesterone go down the concentration of estradiol will go up those cows are going to show asteroids and potentially be re-bred um in a heat detection setting or a natural service right but you know the idea of utilizing the color doppler ultrasonography it comes from the fact that the blood flow to that luteal structure here to that corpus medium decreases earlier than the actual structural ludiolysis occur right so the question we're trying to ask you is can we go in and using colored doppler ultrasound and can we determine which cows are undergoing ludiolysis by visualizing differences in blood flow to the ovary more specifically to the corpus luteum and this is a video with an example of a of a color doppler ultrasound examination basically what the color doctor examination will do is not only visualize from from from a brightness mode standpoint uh the ovary and the structures within the ovary we're also going to be measuring and visualizing blood flowing through different structures and we know that the corpus luteum here is highly vascularized so the idea is that we can go in with that new technology and visualize those differences in circulating in blood circulation so as i played this video here we have two different images one here on the right and one on the left uh the one on the right um is an image on a traditional ultrasound b mode and the one on the left is an image with the color doppler and the color doppler signal for vascularization is gonna show up in your screen uh red or blue okay so every time you see a red or blue that means that you have signs of blood vascularization so initially that you can see this is noise here because of the motion of the probe but as we play that video here we can see that if we look at our conventional ultrasound here we can see that corpus luteum kind of resembling what i showed you here so this is an ovary with a corpus luteum here in yellow and this is the exact same thing we're seeing this b mode image here so this uh gray structure here is the corpus luteum and then when we look at the color doppler here on the left side we're trying to visualize signals of blood flow within that structure here right so as i play that video you can see that this is a corpus luteum of a cow that is highly vascularized which based on this method suggests that she will be pregnant at that stage even though we cannot visualize the embryo or fluid inside the uterus by visualizing blood flowing into that specific structure we we would recognize cows that are not undergoing luniolus and more specifically we'll be able to recognize how they are in fact undergoing radiolysis and determine those cow should be open okay so you can see here i have uh differences in co area but not only that differences uh in blood flow measured through color doppler ultrasonography and different stages of gestation and you can see that there's a significant um there's a significant decrease in the signals for color doppler blood flow around 820 of gestation right and not only that there's some research there's no research performed in brazil out of kellerman publishers group showing that there is actually a greater correlation between the percentage of cl that is vasculized through color doppler also ultrasonography examinations and the concentrations of progesterone then when we compare the concentrations of progesterone and the actual area of that corpus luteum indicating that color doppler ultrasonography might be a better estimate than conventional ultrasonography to visualize those scales they're going with dialysis there's research that has been done in south america trying to visualize and those differences in blood vascularization to the corpus luteum but not only that trying to determine pregnancy status both in beef and dairy herds and one thing that i want to draw your attention here is there are differences in accuracy when you look at the dairy data and the reason for that is because there's a higher rate of false positive results in the dairy data sets and the reason for that is very likely uh the fact that we observe a little bit more embryonic mortality on those dairy cows so basically when we're skating those cows at day 20 of gestation they could potentially be pregnant but as we wait from day 20 to day 30 to compare the color doppler ultrasound with the conventional b mode ultrasound there's some pregnancy loss occurring that is decreasing the accuracy of those tests then we look at the beef data the beef data is way more accurate uh with with really relatively low false positive rate results uh one of the things to keep in mind is that with the caller doctor we we observed zero uh here we had half percent but here we have zero percent of false negative results so if you think about utilizing that technology in the context of resynchronization it seems like it's a promising technology right but unfortunately there's field data on our beef population and this is something that we've been trying to tackle we know that there's some physiological differences uh in our posterior beef population here when we compared to the vascenicas population in which a lot of these technologies were developed down in south america or not necessarily develop or validate it in in larger experiments so and not only that there are differences in subspecies with regards to embryonic mortality as well as characteristics of the cl when it comes time uh when those uh corpus luteum are are undergoing with aeolysis right so we wanted to validate those results within our beef boss towers population that's something we've been tackling here at uga and this is the experimental design uh for that study so basically we're exposing those scouts a round of synchronization followed by fixed time to ai and then we go in at day 20 and 22 of gestation and we perform a colored doctor evaluation of the corpus luteum we record videos of those cls using the color doppler and then on day 25 we collect blood samples for pegs and then at day 30 we go there and compare all these three methods so the color doppler at day 20 the color doppler day 22 and pegs at day 25 with the gold standard um trans-rectal ultrasound utilizing the b mode at day 30 of gestation right not only there we're also evaluating pregnancy at day 100 to see if there are any differences in these parameters associated with lead embryonic mortality i'm not going to show a whole lot of that today but i am going to present some of our um data with regards to the color doppler so we we have videos for more than 200 cows now and and all those videos are recorded and with regards to our criteria just to differentiate pregnant from open cows in both day 20 and 22 with the color doppler uh we're defining cows being non-pregnant a cow that either doesn't have a cl right or a cow that do have a cl a corpus luteum but that corpus luteum had uh 25 or less of its area being vascularized okay now our another layer uh and that criteria is the fact that if we had a corpus luteum smaller than two centimeters the cow is also considered non-pregnant based on these criteria here and again we utilize this criteria and we compared with our pregnancy diagnosis perform at 8 30 with conventional voltage sound so this is again the example of our color doppler ultrasonography examination here so here is just noise at this point then when we grab the ovary you can see the cl and you can see quite a lot of vascularization in that signal right so this will be a cow that we will consider pregnant based on that criteria and here we have a cow that we consider uh open based on their previously mentioned criteria okay and what i want to draw your attention here that i brought the video with the cow that we consider open that had the highest vascularization so just to serve as an example but you can see that there's some uh pretty clear differences with regards to the amount of color doppler signal within the luteal structure right we get a little bit of signal at the periphery of that cl and a lot of uh we got some in us in a specific signal here as we're moving our probe uh in the in that ovary but within that cl there's little to no signal so in that context we'll be considering that cow open right so when we compile the data and and we evaluate so this is uh what we call a receiver operating curve so don't worry too much about that focus here on the right so here we have the accuracy and the and the percentage of false positive and false negative results for the pegs that we collected at day 25 at day 29 as well but what i want to draw your attention here is should the results for the caller doppler so similar to what is observed in the data and the bosnica's animals our false negative rates using those conservative criteria are zero percent meaning we're not calling um any pregnant cow open right because ultimately through that color doppler ultrasonography we're trying to identify the open ones right rather than than diagnosing the pregnant ones right and we have a pretty decent accuracy with 86 percent a day 20 and uh 90 percent of day 22 and all those that decrease in accuracy was coming from false positive results meaning that there are a few cows in this case here at day 20 14 of our cows we're calling them bread but at day 30 when they're running them through again they're coming out as open with the conventional voter cell when you look at the 822 that number decreases to nine percent okay and one of the things you think about you know it's what's driving this why why it's not 100 accurate right when you go in with the color doppler ultrasonography and there are a few things that could be happening here right and we don't really know the answer but one of them is uh the lack of synchrony we could have some of these cows uh that failed to respond to the synchronization protocol and then whenever running them through a day 20 day 22 they are at random stages of their cycles so we're not scanning them at that specific time where lyriolysis is occurring not only that but what i think it's more likely happened is that the vast majority of those cows they are in fact pregnant at day 22 uh in a day 20 of gestation but when we run them through a day 30 we know that there's quite a bit of loss that occurs between day 20 22 and day 30. so that pregnancy loss is

likely driving some of those um false positive results so okay from a from a producer perspective right from a practical standpoint so is there a potential for the use of colored doppler ultrasonography in the near future and so this is the compiled data for uh our study here and here we have uh the results for our conventional b mode ultrasound so we had a 53 pregnancy rates in the study okay so we had 53 pregnant cows and 40 of them open out of the 40 open uh when you think about the total number of animals only 14 of the total number of animals uh represented cows that we we misdiagnosed that meaning that we call them bread and they were actually open on day 30. okay so that means that we were able at day 20 to detect 71 of the non-pregnant cows and on day 22 80 of the non-pregnant cows with this color golfer technology utilizing that specific criteria and as we think about potentially utilizing that in the industry right remembering that we have zero uh false negative results here uh we have the heat distribution uh of cows after uh timed ai but i want to draw your attention here to the cows that did not show heat so basically if we go there and and go in the literature and you know look at studies where folks were there for 10 days going there mornings and afternoons trying to detect those no non-pregnant cows to re-breed them their detect they were failing to detect about uh 31 about 30 of them okay in a heat detection setting meaning that you know we're getting some pretty some results are pretty similar to what you would get if you go there and detect those cows for 10 days every day mornings and afternoons so i consider that somewhat of an encouraging result with regards to detecting on pregnant cows but ultimately what drives or what's going to drive the potential utilization of that is the actual fertility that we're going to be seeing when we start resynchronizing those cows really really early right and that's something that's our next step from a research standpoint is to characterize that what kind of fertility can we get uh when we're utilizing color doppler ultrasonography in the context of re-synchronization okay and not only that how those those false positive results will play a role in the overall number of ai pregnancies we can generate we're going to be able to generate pregnancies early but how many more pregnancies are we going to generate and how how many pregnancy are we going to not generate because of those false positive results so these are things you keep in mind as we move forward you know when we keep working on this trying to establish resynchronization programs that combined up that utilize color doppler ultrasonography so in summary there are several methods available that work well right so we talk about rectal palpation ultrasound blood tests not only that but we know that you know all these tests are based on the literature that is out there and also our experience in the field they work really well right so you know if you go there and work with the vector in here and figure out what works best in your operation you can choose any of these methods and you know you have a successful result as long as you're doing things right right and you can actually rely on this in this techniques and in these methods as well so not only that we can utilize pregnancy diagnosis strategically in the context of resynchronization programs and the idea of that is to maximize the number of ai pregnancies and not only that as we think about the future of pregnancy diagnosis and potentially what kind of technologies are we going to be utilizing in the future are we going to be able to potentially predict embryonic mortality we don't know potentially maybe in the future we might develop techniques in which we can more accurately predict embryonic mortality not only that uh thus color doppler alternate is called doppler ultrasonography a feasible alternative to re-synchronize um estrous and increase the proportion of pregnancies they're regenerating uh through reproductive biotechnologies right so we're talking about both our fissure insemination and embryo transfer so in south america folks are utilizing this commercially but we need to validate those results here and see if it made sense there's a lot of physiological differences there's a lot of reasons why folks that have more from an influenced cattle in those parts of the world are re-synchronizing cows that might not make a whole lot of sense for us here but this is something that needs to be investigated so we need to try to tackle this from a research setting to see if we can come up to feasible and recommendations for producers in the near future so with that i'd like to thank the faculty members as well as the students and interns and forum staff that had helped us generate some of the doppler work as well as the folks that have been funding our program since i got here in georgia we really appreciate that and with that victor i'd like to thank you as well for the invite and turn it back to you to potentially answer some questions we might have i cannot hear your vitor not right now hello yes can you hear me pedro yes we can hear now apologize for that um just want to remi

2021-05-21 08:53

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