AWA CONFERENCE 2015 OCALA, FL

Transcription

1 AWA CONFERENCE 2015 OCALA, FL DAY 1 SPEECH TRANSCRIPTION The Latest in Reproduction Technologies Dr. David Greiger KSU Dr. Gabriel Gomes University of Florida [00:02:54.08] Thanks Jerry It is good to be here. I've spent the last 20+ years teaching college students about the back end of the cow - that is pretty much my career. So if you want to make me feel at home at 2 minutes in slap your head down on the desk and get some sleep. This wasn't that easy to put this talk together. I'm trying to give you some things on new reproductive technology. Some of you will be doing these things at your ranch and others not as much but I'd like to stimulate conversation and if there are questions after this I would like to take them. [00:03:56.05] - In the latest technologies you can't get away from some things - the only thing I have to add to the sleeves, lube and the hand is that now we have colored sleeves. Extrosyncronisations, there are all kind of systems, it gets confusing. We use prostaglandin, GRH, cedars, MGH, it gets pretty confusing. A lot of you it depends if you have a smaller herd you use different techniques. I'm just going to give you one thing on timed AI. If you were going to set up cows next week I'm going to give you the best timed system where you don't have to check heat - and it still comes down to getting females bred, you want them to have a calf, and have emenestrous sp? is key. [00:04:56.27] - How many people here have used CIDRs before? So, you know what they do, they hold cattle off before coming into heat until you want them to, alright. So if we were going to set cattle up we would put a CIDR in next Monday, we would wait one week we would

2 pull that, then to those animals we would give them Prostaglandin and then an Estres patch. This isn't higher tech but these patches have helped and I'll talk briefly about that. [00:05:31.16] - So for timed AI we would set them up like this - 2 days later if you had heifers theres a bit of a timing difference - you would AI those cattle - give them a shot of GRH whether they were in heat or not and you hope that they conceive. So heifers it will be a little earlier than cows. This really works well. There are advantages and disadvantages. So if you think about it, you are not checking heat and you are breeding 20 head, all of those 20 will be inseminated, you give them the drugs and you hope they conceive [00:06:15.01] - A lot of people I work with have larger herds they've got a lot to do. They got sorgum they're getting out of the fields, they have all sorts of diversified operations. They want to go in and if they can get a 50% calf crop for something like this it's worth that right. But you've got to decide if it's worth it or not. [00:06:39.13] - These estress patches, they go on the tail end of the cow, even when you're not checking heat, these things are useful. If you have a program that you set up and you want cattle to come into heat after you pulled the CIDR and give Prostaglandin, you put these patches on there, say you got 50 head, and you go out and there are three patches that are Red - that is a bad sign. You're not breeding according to these but they indicates are your animals cycling? are they responding to your synchronization system? they have a better chance of breeding. I [00:07:26.29] - One thing that I have found out during this talk is that your breed is fertile - that is a great thing for any reproductive technology. these patches will help and if you have any questions I'll answer them after. [00:07:58.16] - This next part is prenatal gender selection. Prenatal really means before birth, before implantation, and even before fertilization in this case. Fetal stage would be ultra-sound and many of you.. who in hear does Ultra sound for their cattle? You can do that at 30 days it's a highly used practice - if you wait til about 2 months of age you can get fetal gender and that gives you an opportunity if you

3 are going to sell those cattle or market them you could say that this dam is going to give a bull calf and that helps with marketing for buyers and sellers. [00:08:47.21] - I can explain to you how these things work but it will be up to you on whether you can make money on it or not. [00:08:59.08] - For the embryo stage there is a biopsy that is done. Where a few cells are taken out and we'll talk about the PCR genetic analysis. In both of these stages here fertilization has taken place, the gender is set, we don't have any Bruce Jenner thing going on here. So the advantage is you know what the pregnancy is going to be, the bad news is that you can't control anything from here. [00:09:32.17] - This last thing where we get to the gamete part, we can sort sperm, now this becomes a little more powerful because even before we have fertilization we can skew things - I would like a heifer calf over a bull calf. Some of these things have been around for a while but I want to give you a background on how these things work. [00:10:22.12] - For cell sorting - we got an X carrying sperm and a Y carrying sperm and we're going to sort those according to what sex chromosome they are carrying. I appreciated Holly's talk a lot. I'm a reproductive physiologist by trade, you know the sleeves and lube thing, but there is overlap between things that Holly talked about and what I'm mentioning today. [00:11:15.18] - So gender determination of embryos, we do a biopsy of that embryo, pre-implementation; so if you've had them flushed for embryo transfer you would do a biopsy of those and the holder on the left sucks the embryo there and we can extract a few cells from the embryo right? The goal is to determine what it is, extract cells and still have that embryo still viable. I'm not a graphic artist but you pull the cells out, put them in a tube, and extract the DNA that they are carrying [00:12:03.13] If there are a couple cells - just a couple copies of certain genes - if there are a million cells you have multiple copies. But in an embryo we don't have the source there to take out

4 thousands of cells because at this stage you have a couple thousand at the most or a few hundred. [00:12:28.10] - So what I'm going to explain is the gender selection but this application goes back to everything related to DNA markers or mutations that cause disease, anything that's related to DNA this PCR assay is the basis for that. It's a lot more complicated than that but this is the basis for all genetic analysis. [00:12:58.12] - So we're going to take the DNA, we've got it in the tube here and I just want you to thinkg of this - each of these strands represents a certain copy. This is very simple again. My graphics art is not great, but that blue spot is the target we want. Alright, and in this case if we want to know gender that target comes only from the Y chromosome. So it's not in the X chromosome, it's Y specific. So we know if that genes there it's got to be a male embryo, theoretically we only need one cell to do that but it's very difficult. [00:13:35.27] - What PCR does is it amplifies that little piece, from a few copies to a million and billion of copies so that we can look at things more closely in the lab. Like Holly was talking about genetic markers if that gene is there PCR will amplify that in ways that we can determine that. See those strands of DNA I drew there, if they are intact than you have a good chance of the analysis working. If that DNA, hair or a blood sample, if that stuff is broken up this assay doesn't work. [00:14: I love going to cattle meetings and putting up mice. This particular gene that they would do for determining gender of these few cells that came from an embryo is this Y specific gene, it's called SRY and it means Sex Determining Region - Y - it comes from the Y chromosome - I put this slide up because when they discover these things- a lot of the times they find them in mice and it applies to humans, cattle, pigs, whatever, and you talk about scrotal circumference on that mouse on the right... both of these mice are XX - if you took their cells out they would be female genetic mice, they were born XX - except in a genetic mutation the mouse on the right got a little piece of that SRY gene from the Y chromosome, some crossover that Holly was talking about, and that little piece of DNA produces protein that allows that female mouse to grow extremely

5 large testes. [00:16:28.16] - So a lot of times we do research and it takes years for things and things are discovered in rodents and we apply those to large animals and generally our genes are close enough across species that they work. [00:16:40.14] - Jerry, did you tell me that you sent some cells to GeneSEek or you did some DNA work... [00:16:56.03] - GeneSeek is NeoGen now - it's a very cool place and I asked them about certain things. So if you think about single cell genotyping, and that just means can you take a single cell and get a genotype and it would be more than just gender or parentage, it would be all of these other markers- it's a very difficult thing to do. Now they work on that, but the fewer amount of starting material that you have the better chance you can keep the embryo alive. So analysis from a single cell is doable and the reason to do that is that you can genotype an embryo prior to transfer-to-recipient - thing about that DNA thing is that if you have just a couple cells in there and that DNA is bad, you just don't have any backup, if you have a million cells in there - it doesn't matter if a few are bad you will still have enough information to work with. [00:18:08.13] - Talking to the GeneSeek folks, how cells are handled prior to DNA extraction - this could be anything from hair extraction to blood, they get thousands of samples a day. And part of these things they'll run the assay and if things don't work in the end it could be that the DNA is broken up. It's not that you have to learn this technique but for single cell or a few cells genotyping we almost need a program to train that to that so that the DNA remains in intact. handling is critical. [00:18:58.04] - This is kind of coming I think into flowing into genetics where the more you learn about your breed the genes that are doing certain things the more information you have the better impact you can have on production. [00:19:24.09] - Alright Semen sexing. Who has had bulls in here where you've sorted for gender selection? This technique works. I've

6 taught this for a long time. I was talking to Darrell about this too and they have started this in California in a laser lab - sometimes there are engineers and biologists who have nothing to do with cattle that come up with things. There are animal scientists and USDA that used this to develop the sorting technique. [00:20:15.01] - If you look at the circle up here that would represent one cell and that's a carrier type with all the chromosomes in that. If that was from cattle there would be 60 pairs in there but this is just an example. As Holly talked about where one half of chromosomes go each way... The same amount of DNA goes one way the same amount goes the other way. And it's all the same amount of DNA until you get to the X & Y - the red circle is around the x chromosome and the blue circle is around the y chromosome - look at the difference between those two chromosomes. and because of that difference the total amount of DNA in an X chromosome carrying sperm is different than a Y carrying sperm. that little difference is about 2 or 3% depending on species and that means that the total DNA in the cell that they can detect. To me this is one of the more fascinating things in taking laser technology, biologists, reproductive physiologists and applying this through something that is commercially viable and it does work. Does that make sense? [00:21:56.27] - All of that DNA is packed into the head of the sperm. This is something that I use in class - microscopic view of sperm - this is the same dye that the laser can detect. So if you put this under UV light then this is what it looks like. Only the heads show up and fluorescents is what the laser causes - the computer detects that and says this is 3% more or 3% less DNA and it sorts it accordingly. It's a very cool technique. In class it's very easy to thaw a straw out, incubate the sperm with the dye and show the students the fluorescent. [00:23:03.16] - Here's the basis - the sperm are dyed and it goes into the top of the column and it's very much a single file process. The bottle neck of this application is the sorting rate. Now they are still screaming by at a few thousand per second but how many viable sperm do you want in an AI straw before you breed a cow? You want about 10 to 20 million after thaw. It takes hours of time to get single straws of this and that is why sexing technologies have sorters set up

7 in a big building that do this and they are running 24/7. [00:24:05.27] - to finish this then.. they come down there, the sperm have dye, the laser hits the sperm, the computer detects how much DNA is in there and a little droplet of either + or - oil goes into each of those cells and at the bottom they go through these charged plates and it sorts them on charge either right or left and that's how you get X & Y chromosomes sorted on a single ejaculate. [00:24:41.17] - I have one picture to show it more complicated. The machine is shown right here and there is a regular tube that catches that, magnetic plates, lasers, and there are spermatozoa that are cruising right through there, that they can be dyed, that can be passed through this and be collected in this tube down here and that they are still viable is quite an amazing thing and it just took years to figure this out. [00:25:27.29] - Therese computer monitors, there will be peaks and you can almost see individual sperm going through this thing and they have technicians that monitor this to make sure that everything looks alright. At this point things get very complicated, I get confused and I really want to go have a sandwich. Very good stuff. This works, it will be 88-92% accurate. It's solid. We had a lot of things before where people were trying different things...does X carrying sperm swim faster than Y and all these different things, different size? different shape? the % DNA thing works. [00:26:39.10] - Another thing, this is not that big a deal, I'm a nerd so I love biology. You'll notice that the sperm are sorted down through here and there s a middle tube and a lot of waste goes in that. And waste sometimes there is 52% of the ejaculate they cannot sort. They've improved on that but that waste is because spermatozoa they are not really balloons they are more like a tennis racket. I wanted to bring my model on my carry on luggage, my prop, but it didn't fit and I didn't want to check a bag. These sperm are flat instead of perfectly round. You see on this slide here you see some that glow intensely like that one right there...and others. IT depends on how they go through that - if they're not flat against the laser the computer cannot detect the difference. As long as they get enough that are sorted and are viable it's a good process

8 [00:27:48.18] - So candidate bulls, we've gotta small bull stud that is part of K-State University, they'll send semen to have it sorted...candidate bulls they want that to be progressively motile, like when you do a semen check and if you get the bulls check those boys should be swimming on a slide, right forward, they want more than 65% of those to be motile, K? For a breeding bull, there is different standards for different breeds, but if 40% of those are motile that bull can usually breed cows just fine. But to do this technique they need to be really good. Morphology they can't have misshaped heads or bad tails - you need 80% to be normal morphology - and if they don't meet those criteria semen technology won't sort those for you. [00:28:59.00] - I think this comes back to in the other breeds, angus, simmental, I wish we had more bulls that we could select from that are semen sorted, sometimes it's due to that if his boys aren't very good to begin with then you can't improve them with technology. In sexing technology I think STG genetics now, there are a couple doses, you might see something that says 5.0 dos or 2.1 that really means that there are 5 million sperm that have been sorted in that dose and usually people will use that for embryo donors, IBF, and 2.1 is the lower number of sperm. These are the costs that I got from the group up there I don't know if they are that close but if you sort a bull and you choose that you want his Y chromosome sorted you can get the X chromosome for half price. Yay- Wal-Mart, half price! [00:30:12.07] - If you use sexed semen and you've done a very few numbers of cows some producers are not happy, now if you used sexed semen, remember that 90% - this should be 9-1 kinda ratio. If you've used this on 10 cows versus a 100 should that ratio be the same? You would think so right? Here's a beautiful graph here - I have students - If i took a quarter and flipped it 10 times - how many heads and how many tails? 50/50 right? Now out of 10 though if you just had 10 calves in a year and it's supposed to be 9/1 - it should be 50/50 - and if they do it 10 this year and 10 next year - you that one of those times just by chance they got 9 tails and 1 head. Now that generally not going to happen right? But I've heard people say that if you used sexed smen and you were hoping to get all bulls from this technology and 6 calves were bulls and 4 were heifers, you're not

9 going to be happy. But if you do 100 and it really is sorted the same, that number should be close. So does that make sense? [00:32:15.28] - What does not work... this stuff I saw - 2 or 3 years ago, heifer plus and some that was philly plus or bull plus. And this was supposed to... how does it work, right on the label it says, the sexing process stimulates the motility of X chromosome baring sperm while slowing the motility of the Y chromosome baring sperm, when inseminated the female arrive at the egg before the male sperm, result is more fertilized by the X baring sperm resulting in more heifer calves. [00:32:59.26] - In semen sorting, you'd think you could thaw a straw out, mix it in this bottle for 30 seconds, put it back in the gun and you've changed the way and how fast these guys can swim? You cannot do that, this does not work. When you look for quality in research, some may swim faster than the others but if it's based on DNA and just 3% it doesn't affect with the speed of how these guys are swimming. I don't know how these companies start but there is some research to check these guys out and it doesn't work. You see this for a few months and then it tanks because it doesn't work. [00:33:46.13] - I want to finish a little bit on cloning. I won't go through the whole process - it's been around for a while and it's actually called Sematic Cell Nuclear Transfer, we're cloning animals. We did one project years ago, we had a Herford cow that we cloned at Kansas State. For me it was good to see how efficient and detailed the process was. You almost think like in regular embryo transfer you've got embryos from a donor female that are going into a recipient, in this case we have donor cells from a female cow here and the recipient is really eggs from an ovary that we have taken the DNA out of. There is no male in this process - men you have been replaced. So these cells here - they are skin cells, they are diploid, they have two sets of their chromosomes pairs that s all they need. These unfertilized eggs, they just come from slaughter house ovaries, you pull the eggs out you pull the DNA out of them, they are really serving as the house or the capsule for this embryo to develop. I'm not big on math but generally you would have 1 set of chromosomes and sperm and those are haploid right, 1+1 is two and things are happy, in this case these skin cells are 2, so if you have you have 3, and

10 that is lethal, it's a very technical process - there are companies that are still doing this and they can clone adult animals with this process. [00:35:44.25] - Just so you know, they are improving on this, it's hard to get data on this. When we did this process if you had conventional embryos and transferred those you would hope for 50,60,70% conception, and that's what the line shows there, and this is going from just one month to term - if we put in the cloned embryos you could see that it just drops down, and we had maybe one calf born from this group. It's a little better than that, and you need to use this technology in a very valuable sire or cow that you are only going to increase the genetics from that animal. [00:36:39.23]- I think that the Wagyu breed is a fertile breed and maybe there s folks in this room that have animals cloned. The smaller breeds the lower birth weight breeds do better and that efficiency may be at 30% for live calves born which is actually excellent for this process. Now that doesn't come near normal pregnancy rate but it's coming closer all the time. The other breeds are not as good. [00:37:09.13] - When this cloning thing came down... Will this cloning process replace natural breeding selection from people like you. It does not, right. If you have, just think about the dairy folks, would you clone the same cow today that you would have cloned 25 years ago? No, because we are always getting better. Using information. If you clone this animal you can make more of him but here is the level you're at. The line doesn't go up. It can propagate individual animals but it's not going to replace what we do with EPD or visual observations with genetic markers down the road. [00:38:14.02] - How we doing on time? Okay, clones are safe to eat. Go ahead and eat em. There s nothing different than conventionally made animals. Are clones GMOs? Here s the 50/50 answer - there s nothing... you took a cell from the ear, you grew it in a dish, you used that DNA and put it an unfertilized egg, there was no intentional tweeking of the DNA, we didn't add anything or take anything out, in GMO crops you are adding something in. So for as far as we know they are not GMO animals. And that is actually why they are safe to eat. That next step in creating GMO animals. they are trying to clear

11 GMO salmon - that's the tricky thing. [00:39:23.02] - these are some cells on the slide - if you do Genetically Modify them they will look something like this. You take a jellyfish gene you stick it on those cells, the cells use those genes to make a protein they normally wouldn't have, and that would be a GMO. [00:39:54.02] - Are clones really identical? Look at these, the coat pattern is different. This group from Australia did a great job. If you look at the white and red they are a little bit different - why is that? If these are truly genetically identical - why isn't the coat pattern identical? I think we're getting there...in epigenetics that would be part of that - environment right. How those cells grow in utero because in utero they grow a little differently - they migrate different. We think they perform the same in meat quality, marbling, and carcass characteristics - that's a good thing in passing the genes on. [00:41:03.25] - And I want to end with this... should we clone Jerry Reeves? I would say maybe- Jerry's a good guy he's done a lot for the breed - I am where I am today because of Jerry - but could you imagine 100 Jerry s at the bar. But, Jerry is from Coosbay, Oregon, he's a logger, he hunted elk, he's a cowboy so that environment effected who he is - but if we cloned him and put him in NYC - he might not be the same. [00:42:28.22] Thanks for your attention I am finally done... (claps) Gabriel Gomes [00:43:07.20] - Good morning, my presentation will be talking about this project that us from the College of Veterinarian Medicine at the University of Florida have been conducting in partnership with Clear Creek Cattle Co. - the title of this project is "Superovulation in Embryo Transfer in Wagyu and Cross Bred Recipients" [00:43:24.01] - I believe this type of project is really important to improve efficiency of our reproductive problems. So let me try to explain why.

12 [00:43:31.25] - As you know reproductive technology like estrosynchronisation have been used for many decades in bovines and they are used for the objective to improve genetics and improve reproductive performance of the herds, however, even when this technology was successfully adopted from this match we have the formation of only one embryo and out of this embryo we will develop one calf so we are only able to obtain 1 animal per cow per year. So if we think about the Wagyu breed and we take into account the long period of time it takes to raise these animals having only one calf per cow per year represents pretty slow increase to animals to the market. [00:44:20.15] - More recent techniques have been developed with the objective to explore the genetic potentials of both males and females and one of these techniques is called superovulation of donor cows with superior genetics so then these cows are inseminated by superior bulls and because these cows are super ovulated they would have the formation of multiple embryos. These embryos will be collected by uterine flushing and these embryos will then be transferred to recipient cows which are cows that have lower genetic value but they are known for having good reproductive performance and good maternal abilities. [00:44:56.06] - So these cows will go through a normal gestation - they will deliver their calf and raise as their own although the biological mother of that calf is the donor cow, the cow with the superior genetics. So any breed involving superovulation and embryo transfer can promote the acceleration of genetic progress of the herd. Talking about Wagyu, this technology has an additional advantage, and that advantage is that we have the potential to scale the breed - so now we don't have one calf per cow per year - we could 3, 5, 15 calves. This really represents the opportunity to meet the demand requirements for these animals. [00:45:49.03] - In spite of all the potential that superovulation has to boost efficiency in our beef and dairy herds it is interesting to note how slowly these technologies have evolved. So if we look at the most recent reports available from the International Embryo Society and more specifically look at the number of embryos produced per

13 collection or superovulation procedures, you can see that after 10 years we have only increased the embryo production by 0.7. so that means that roughly compared to 10 years ago we have only been able to get 0.7 embryos of a cow that we were doing 10 years ago. This is a very slow increase and there are several reasons for that. [00:46:49.27] - One of these reasons is that these technologies are expensive so it is tough for universities to conduct experiments in this field and really evaluate different protocols different collection techniques..etc. However, again, specific market scenarios for the Wagyu breed makes this type of research not only possible but desirable, because each one of these embryos if they are worth thousands of dollars you better get as many as you can. [00:47:25.09] - Our main objectives of this projects is to investigate the main factors involved in embryo production in Wagyu embryo donors and gestation performance in cross-bred embryo recipients. Here at Clear Creek Cattle Co. they do a really great job collecting the information from their animals. Basically we have this huge data set that we can take and we can analyze and identify specific factors that are associated with greater embryo production by the donors and a greater pregnancy rate by the recipient cows after the embryo transfer. [00:48:05.11] - We collected information so far from 110 superovulation procedures that yielded about 2400 viable embryos so far. In this table you can find the factors we are evaluating in terms fo association of donor performance. So each one of these are factors we are evaluating. For example season of the year - for 110 superovulations that were done at Clear Creek - 33% of them were done in the Spring, 7% were done in the Summer, 32% were done in the fall, and 28% were done in the Winter. And the same is true for these other 5 categories. [00:48:52.19] - A few of the factors we are evaluating are Weather, was it hold or cold on the day we started our protocol, Tecnhician, there are three different technicians that conduct the experiments at clear creek so we wanted to evaluate which one of them performed better than the others; Flushing season, we wanted to evaluate if successive superovulation procedures would somehow affect the

14 performance of a cow, FSH type, there are two main types of drugs on the market that the technicians use so we decided to check if one of them is better than the other, and finally presence of a nursing calf, which means that she either had a calf or not and she is being subjected to this protocol or not. [00:49:46.19] - We did the same for recipients collecting information for 186 embryo transfers so far. The same idea here we evaluated the affects of Season, Technician, Recipient Location, Embryo Quality, and Embryo type - whether it was fresh or frozen. For our recipient cows what we do is to evaluate the pregnancy rate for embryo transfers. What we do here is put all the information in a spread sheet and we build a model - the idea of the model is to cancel out all the other factors so we can make the most fair comparison for one specific factor. For example if we want to evaluate the affects of season on recipient performance - we zero out all the other factors and in the long run it should be able to tell all the best factors for each category. [00:51:10.23] - Let me show you some of the results we have so far. As you would expect if you have any experience with cattle with Season and donor performance so far we found that the best season to do embryo transfer here at Clear Creek would be Spring and the worst season would be the Summer. And here just going back we are evaluating mean number of viable embryos per superovulation procedure for each collection this is the average number of embryos we are getting. We also found an affect of technician - so far one of the technicians has performed better than the others. We also found that in the FSH types one of the those drugs apparently works better than the other at least in this reproductive practice. What came as a surprise for us was that the presence of the nursing calf was associated with greater production of embryos by the donor cow. [00:52:08.21] - So we have 7.5 viable embryos as an average from cows that had a nursing calf compared to 5.2 embryos from cows that did not have a nursing calf. This is something that we definitely want to explore more for the future. [00:52:19.24] - For results for recipient cows here we are evaluating the performance - we did not find any main factors that affected the

15 performance of recipient cows except for weather. Which is not a surprise. The day that we transfer the embryos if it's hot we have lower pregnancy rate if it's cold we have higher pregnancy rate. For recipient cows we can't go too far from the basics that we already know. Keep those cows in body conditions, free of diseases, a good vaccination schedule, free from environmental stressors and so on. [00:53:04.23] - Some take home messages, we found some seasonal affects, technician, FSH, and presence of a nursing calf on performance of donor cows and we found affects of weather on the day of the transfer of recipient performance. Our idea with this project is to reanalyze the data after each reproductive season so we can gather more information and gather more statistical power so we can really tell what in the future the best season, technician...again so we can one more time make decisions on the farm based on numbers and facts on not personal stories and impressions. That's what I had to present - thank you University of Florida, Clear Creek Cattle, and I hope you enjoyed the presentation Jerry Reeves: We are open for Questions Ralph Valdez: On your results with FSH drugs which of the two did you find the best results from? And what were the two drugs? A: I didn't want to publish the real names because they are still partial results so I don't want to make any statements that will change in the future. I should point out that this drug is working better for their conditions, ideally we should do this kind of study at every farm, so that you can find out the factors that work out the best for your farm. Q: Goodell : I found in animals that when I do this kind of reproductive work, it's, there's more of a relationship with an individual animal than a particular group, I find some cows it actually works better with one particular drug than the other - is there any danger in grouping them and taking all this information and making an overall decision? A: Whenever you have small numbers you will have this type of a affect play a major role in your results. But our idea is to gather more

16 data so taht we have enough animals that we can cancel that affect of individual animal variation. One thing that we also do when we run this model is that we put the affect of donor cow and sire that your using in the model so we do the best that we can to cancel those factors out so we can focus on just the affects we are evaluating. We are aware that there is a huge variation from animal to animal. The First embryo i ever transferred, Darrel here watched back in 1978, and Sam Castlebarry, Holly Schneider and I published some data in theory and geneology in 1981 on 1500 donors that we collected and in my opinion it is one of the most biased piece of information there is period only because of the fact that 1 of the 3 of us mixed the FSH, Gave the Shots, Bred Those Cattle, collected those embryos, sorted and selected, put those embryos in, and palpated and determined that those cows were pregnant; you cannot find a piece of data with more information with less variation than that, it is a classic example of poor utilization of personnel because there were a lot of people who could have done these things with less training and education but I think that looking at the data they had and comparing to the data we published in 1981, was 10 embryos per donor 8 of which were fertile, 6 of which were good enough to transfer, resulting in 3 pregnancies. Q: Dr. Greiger, I had a question on your sexing semen. What % of the semen is actually damaged in the sorting process and how do you compensate for the lack of healthy cells if there is damage? A: So the % that is damaged in that process, could be as high as 40 or 50%. There is no evidence that damage to the DNA causes a mutation to the animal, the damage is usually that that sperm is not viable after going through the process after the dye and the process, so that can be relatively high and it depends on individual animals. To compensate for that...i don't have an answer. It really seems to be bull and individual specific and even breed specific. Just like if you.. if bulls come into a bull stud, gosh they look good you want calves from them and you collect their semen and it's just poor. And there is nothing we know environmentally or nutritionally to correct that. So unless technology changes some - right now we are just limited to good spermazoa, good morphology, does that answer your question?

17 Q: Well could you use more straws or semen? From the people that I've talked to that have used this process, it just seems that the pregnancy rate is significantly lower. A: That is true - I would average it to be 15% lower - 10 %-20% - you are using less sperm and it does make sense. There is an Angus breeder in Kansas and he does a lot of flushing he wants a 10million dose and they'll do some custom stuff for you. He likes a higher dose - you'll pay a little more for that but they can do that. You can compensate with higher numbers of semen. Part of that is though, sometimes we don't want technology to override a problem - say there s a fertility problem - we say we can get around that - but sometimes you don't want technology to pass the bad things on. Q: You also mentioned that there is about a 90% success rate of separating and sorting of male and female cells - does that mean that even if you used sexed semen and you select for females that you're likely to get 10% males. A: That is correct. The more you do with that bull - if you breed 100 females with that you should really expect 90 heifer calves and 10 bulls. So it's not 100% type of thing. But like i said if you just do 8 or 10 it may not come to the 9 to 1 ratio but with large numbers of the sorting process you can really bet on it. It's solid. Q: I have two questions one for embryo transfer and semen doses. Has anyone looked at administration of antibiotics to recipients at time of transfer or progesturone supplementation via CIDR at the time of transfer as far as how it affects the success rate? A: Yes we've looked at that and have certainly put CIDRs in the recipient cow the day we put the embryo in. And we pull those CIDRs anywhere from day of her normal cycle, so approximately 14 days later, and the data is there to show that you wont' increase the pregnancy rate very much if at all, the way that we use it is we resync the recips, so when you pull that CIDR out you don't give an injection and the ones that are pregnant do not show heat, and the ones that are open will show heat in about a 3 day period, so you have to do some close heat detection, then we can go back a week later and put embryos back in those recips, so you can

18 use a set of recips twice in days. Caution there: If the recip does show heat you better ultrasound that gal and make sure that she's not pregnant. Recently in the last month we have run into two recipients that certainly stood in standing heat the estrocillers were rubbed off but I ultra sounded them and they were both pregnant. So, you can't have pregnant calves showing heat. But it does offer a simply way to reuse the recips. We still don't think that its helping hold that pregnancy through maternal recognition of pregnancy. In fact at our meetings last week we were cautioned not to put in CIDRs in AI cows between days 4 and 9 - they think that the higher progesterone at that time may actually cause early embryonic death. So there is a point where you may not want to use it. Q: What about antibiotics? A: Years ago a lot of people used antibiotics but in my opinion that has a lot to do with technique and technician and I don't want to go there - my opinion on the CIDR thing is that it's a good management tool - we don't have any controls on our data - our gut feeling is that it works. It really is kind of ironic, Dr. Burgo O'malley sometime in 1970 came from Boston to Baylor and he defined progesterone created proteins in the chicken and duck that maintained resistant embryo development and we utilized that data in animal agriculture from there - in the recent history we realize that progesterone has a suppressive influence - so anytime you produce an embryo that is a foreign protein in the uterus and there is certainly some advantages to having an imuno suppressive element there. We were going to do a project in the MGA about 10 years ago and PETA got involved and we didn't do it, we were basically going to treat cows after transfer with MGA and basically... just like Darrel, we got involved in putting these CIDRs back in and I think we really did what we really talked about doing with the MGA. I think the CIDR program is a good management plan but I think that it kind of depends on who and how they are using it. Q: Semen dosage - are you talking about 10 million cells? A: Gomes: I quickly have a comment on the previous question regarding the CIDRs, there are some studies with dairy cows, not in embryo transfer but artificial insemination, and they actually used

19 100s of cows on these experiments, and they used CIDRs from 0-7 then from artificial insemination not embryo transfer - different story but some of the principals do apply; and what they did find was there was a negative affect with the use of CIDRs at 30 days. What they have found is that the embryo needs to be synchronized with the uterus; if you put too much progesterone at that time you will have different stages in the uterus and in the embryo and that ends up causing pregnancy loss. A: Your question about the dosing for semen and what those doses were...i think they label them approx: 5million sorted, 2.1 million in a lower straw, it can be 10% less than a conventional semen straw but once they do the sorting process that's what they are gauging that for - to get 5 million it takes a little longer and that's why you're paying a little more for the viable semen in that straw Q: Is it just total sperm? A: That 5 million would be total sperm cells not motility - all the ones that survived the process. So when they start with a bull with high motility and they sort him they try to maintain that through. But yeah that's total sperm and they have not checked all of them for motility. Q: On your same question on that 5 million, are you talking live sperm? 5 million female? A: There s a total 5 million in there - you take the term CSS - it means that there should be 10million viable season post thaw. They may have 30 million in there to get 10million but the sexing boys, because the overwork on these flow-through cytometers they're putting exactly 5 million in and they are losing on that freezing and thawing you are going to lose sperm. I would guess that the 5 is only giving you 2.5. Some things that might work that the AI doesn't like to talk about, when it started we could see better results in heifers than cows with sexed semen, because the reproductive tract is smaller... it's harder to find that egg. I still believe that if your horn breeding you're going to have an advantage there.singer and I did quite a bit of work on that, the industry didn't want to change because they felt that there was going to be too much problem in puncturing the uterus if technicians were going to change from the side of depositing the semen just

20 passed the cervix to make sure you put half in each horn. I still believe in that technology but it's not accepted by the industry. A: Darrel, I'd like to make a comment from the question with donor cows. At some recent meetings it is reiterated to us that about 20% of your donors will produce about 80% of your embryos. You really have to look at your donor cows. If you look at 100 cows at random, 35% will give you nothing, 35% approximately will give you below average, and 35% will be the winners, they will give you above average, and that's where that comes out to that 6. As Gabriel said, he showed it back 10 years, we could go back years and it hasn't changed one embryo per flush through the ITS a crossed Canada, US, South America, Europe so forth. In all the stats that we get we have yet to improve it much more than 1 embryo - maybe in some instances, slightly more. One of the speakers a few years ago stood up in front of all of us ET Veterans and he said all of us would like to raise that average to 10. I'm here to tell you how to raise the average number of embryos per flush to 10. You get rid of every cow that doesn't give you more than 10. And it's immediate. Come next Thursday your average will be 10,11, or 12. But at the present time more people are using the cows that are new in the breed, pedigrees, and so forth. Another little comment we are recently working with a new veterinarian who is getting in to the ET business. We were discussing with her over a 4 or 5 day period about donor cows, those that work those that don't, what do we have to do to change things. Amy listened to us for a while and said, I've got one conclusion for this, just because she has a uterus does mean that she's a donor cow. After 43 years of this business I think I just heard the comment that is most crucial when we are picking out donor cows. If we can cull those cows that are doing a better job for us, our averages naturally go up. But overall in the industry it is that number - between 6-7 Q: This is for Darrel - you work all over the word and all breeds and have done more Wagyu cattle than anybody else, can you tell us anything about Wagyu cattle that standout compared to your industry? A: Darrel: I was just at the Embryo transplant meetings and I interviewed with 5 veterinarians there that do embryo transfer in Wagyu. I said specifically give me some ideas about the breed not

21 particularly in a certain clinic. I came out of there with two or 3 things that we could all agree on. Do not get the donors to fat, fat donors don't work. We know some reasons why, it goes back to insulin and egg quality, the ova and ovary can be damaged in some instances, and fat donors decline in embryo production. Another one was be careful with the amount of FSH given to these younger donors, particularly the virgin heifers. Keep it on a lower level that we might with other breeds. I heard one more thing, that they think that the IVF results are slightly less in Wagyu than they are in other breeds that he works with. He sees a slightly higher early embryonic death, we don't know why, but maybe as much as 10 or 12%. Early embryonic death is real - we know it happens anywhere between fertilization clear through to probably declines after 50 or 60 days of pregnancy - but it is very rapid between the area between days where we see embryos dying on us. We also see that if you pregnancy test a cow with ultra-sound at 28 days and go back and check her at 60 days and the fetus is gone - that's an early embryonic death. The point is that those kinds of things are what we need to look at, and the IVF particularly with Wagyu. Other than that, I think we treat the mature cows much like we would a mature angus or Herford in relation to their age, weight, and whether they have a calf and so on. A couple of breeders now are waiting till they ween the cow before they are used for a donor.