The DNA Test Mistakes Every Cattle Owner Makes - With Stefanie Oppenheim, UC Davis VGL

If you've ever sent off a hair sample and wondered if you were doing it right — this episode is going to stop you in your tracks. Lori and Taylor sit down with Stefanie Oppenheim, animal scientist and senior analyst at the UC Davis Veterinary Genetics Laboratory, to break down everything Highland and mini Highland owners need to know about genetic testing — from how to pull hair correctly to what those colour results actually mean.

This is one of the most practically useful episodes the show has done. Whether you're testing for chondro, coat colour, polled, or free martin, Stefanie walks you through exactly what the lab needs, why it matters, and what mistakes people make every single day.

Key Takeaways

Always pull hair from the tail switch and never cut it — the DNA is in the root bulb, not the shaft.

Send both the MC1R and dilution tests to get a complete picture of coat colour. 

Buying semen? Ask for the bull's VGL case number before you breed anything. 

A free martin test coming back positive doesn't automatically mean the heifer is infertile — always work with your vet. 

DNA on file at VGL is a permanent fingerprint — it can identify lost animals, verify parentage and protect buyers.

Episode Highlights

Stefanie reveals the most common mistake breeders make when submitting hair samples — and it's an easy fix. 

The team digs into chondrodysplasia, brindle genetics, polled versus scurred, and what a free martin result really means. 

Taylor raises the question of DNA verification for high-value semen purchases — and Stefanie's answer is something every mini Highland breeder should hear. 

Lori admits in real time what she's been doing wrong.

Timestamps

00:01 — Welcome & intro to UC Davis VGL 

00:59 — Stefanie's background in animal science and genetics 

05:13 — How to pull and submit hair samples correctly 

07:28 — Why root bulbs matter and how many hairs to send 

09:32 — Labelling, case numbers and avoiding mix-ups 

14:45 — Why uploading a photo with your sample helps 

16:18 — DNA as a permanent identity fingerprint 

20:47 — Semen purchases, AI and DNA verification 

34:36 — Coat colour testing: MC1R, dilution and why you need both 

37:13 — Chondrodysplasia and the Dexter connection in minis 

41:21 — Brindle genetics explained 

58:28 — Polled versus horned versus scurred 

01:02:48 — Free martin testing: what the result really means

Order cattle genetic tests at vgl.ucdavis.edu

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Website: hornsnhoovesauction.com

Production Credit: Edited and produced by @the32collective_ / https://www.the32collective.co/

Transcript

SPEAKER_00 0:05

Hey everybody, welcome back to Horns and Hooves. We are so excited. We have a special guest today. We have Stephanie with UC Davis, and she's gonna share and help us understand a little bit about those tests that we're ordering and also how to order them. Because just me talking to her on a side conversation before we started this podcast, I learned quite a bit. So I learned what I'm doing wrong. That's it. Yeah, I can't tell you that. We learned what we were doing wrong. So I'm so excited to share this with you all. So, Stephanie, can you give us a little introduction into your background with UC Davis?

SPEAKER_02 0:45

Okay, well, I um grew up loving animals. I got my first horse at the age of 14. So I was already working with horses and very interested, I guess you would say, in veterinary medicine. And I went to school for animal science at Cal Poly San Luis Obispo here in California and got my undergraduate degree, went on to work in at a horse ranch. Until I got nudged by one of my mentors and professors to go back to school and do some graduate work up at UC Davis, and he hooked me up with an amazing mentor here that I worked with for 10 years, Dr. Gary Anderson. And he was doing a lot of embryo work with sheep and goats. And I decided to come up since I had been doing reproductive work at the horse ranch, that yes, my interest in reproductions escalated. And so I came up to UC Davis to do embryo work and work with sheep and goats, which is different because I had been used to working with horses. But all my animal science experience uh really helped out with all of that. And God, I stayed and did a master's as well as a PhD work with Dr. Anderson. So I was here for quite a while, did a postdoc that actually had uh was a little heavy in the genetics analysis. And so that kind of got me thinking more about genetics, but I hadn't had any clinical experience. I'd never worked in a vet hospital, and when I graduated, I really wanted to get some clinical experience. So I went here at UC Davis. We have the world-famous veterinary teaching hospital. And so I got a job there for a few years, and then the opening came up at the Veterinary Genetics Lab. And that was back in 2006. So when I joined them, they didn't have an animal scientist really working on their team. So they were looking for somebody like me to come join the team. So it was really exciting for me because I've seen the livestock testing grow to the point where it used to be me and a supervisor handling the sheep and the goats and the pigs and the cattle and the elk and the bison and the llamas and the opacas. The horses have always been a separate group at the laboratory, and the dogs and cats are a separate group. When I mean a separate group, we just have different staff and the analytical team that handles their analysis because their own little monsters, each of them. So I've been working with the livestock species now for yeah, like 20 years, and I now have like an additional four analysts working with me. And I've more moved up a little bit to more of a senior role over the year just because our numbers were growing so much. There's no way I could continue to do it, obviously, by myself. So we hired one person, then we had two people, and now we have a whole team. So it's really grown and it's exciting. And of course, the number of tests and the diagnostics just have continued to grow over that time. It's become really interesting. But it's been fun for me to see the changes in working with the genetics. For example, when you have diseases that have really bad outcomes and can be lethal, then I've seen some of those actually, I would say, I don't want to say disappear from the population, but so many people testing and getting rid of their carriers, I was actually able to see problems actually fade to the point where they're everybody is normal now. So people are only breeding normal animals, and so we don't have these problems anymore. So that's the power, one of the powers of genetics that is so beautiful when it comes to the diagnostics, when you have problem genes that you've identified that you can actually by testing your animals and doing very strict selections to get rid of your carriers, can actually eliminate a disease from a population. So that's pretty powerful. That is so awesome.

SPEAKER_00 4:18

That is so awesome. So let's talk a little bit about because I know you guys would love to educate on this part when you send in hair, pulling the hair in the correct areas, what you can put that hair into. We talked about that a little bit before we got on, but it was very interesting. Proper sampling and shit and that type of thing.

SPEAKER_02 4:41

There you go. Okay, so basically the hair is selected because it's usually fairly easy. I mean, there's always gonna be animals that aren't gonna cooperate. So we understand that. But it's pulling hair typically is something that most people can do without a veterinarian, unlike collecting blood. Not only that, but it's much easier for us to store. So those are two reasons why hair samples typically tend to be the best because clients can send them in and because they're easy for us to store. Yeah, we're the veterinary genetics lab, believe it or not, we keep samples on file because people will add tests later on. You can test initially and then you can go back and add additional tests. So we don't just use your samples, test what you want, and then toss them. Everything is filed away, and that's why those case numbers are important. All the samples arrive, they get a case number, and that's not only for tracking the results, but it's also for storage purposes. So the hair and the thick hair is what's important, and why we recommend you pulling it from the tail switch. That is typically one of the thicker hair on the animal's body when we're talking about cattle. So when you think about horses, you have the mane, you have the tail. Well, the hair on the switch of the cattle is kind of like that same nice thick hair. And the reason why that's important, number one, is because when you pull it, you want the root bulbs to be attached. The root bulbs is where the DNA exists. There is no DNA in the shaft of the hair. In the hair, what we consider the hair itself. All the DNA is in that root bulb. So the bigger the hair, the bigger the root bulb. And there's less chances when you're pulling it that it's gonna break off. You can think about if you're pulling the fine body hair, the little root bulbs are so tiny, it's very easy for them to break off if when you're pulling the body hair. So that's why we recommend using the tail switch. And sometimes that's can be difficult in these newborn. That is something else that I wanted to talk about. People are so anxious to test these babies when they hit the ground. And granted, if you're having if you have a big farm, you have your animals closer in for calving, and then you turn them out and you don't know when you're gonna see that calf again. Of course, you're gonna want, you know, people want to take the samples from the newborns. But anyway, we digress, we can get to more about that. But the hair samples, that is why we like the hair samples because it's easy for us to store in because it usually we get good quality DNA. And if people can send enough of them, and and that was another question that you hope you wanted me to address is you know how many? The more the better. I mean, we will use your sample, we will store it, and if you send us enough, that animal might actually, if you've checked a box to allow us to use, let's say not only for research, we have control samples. We're very strict in our quality and we have control samples. Well, if people give us excess sample, that often will allow us to use those samples continuously as a particular control sample for particular test or what have you. So, anyway, having more sample is better than not enough because if we get poor results and we need to retest, or if we get a result that I feel I want to have retested, I need those extra root bulbs on file. So we ask you to just send us what I think the form says 20 or 30 hairs. That usually is sufficient. But that means well, actually 20 or 30 root bulbs. So you'll have to, when you pull them, you can kind of look. It's the little kind of whitish, grayish, waxy ends. They sometimes they look like a little hook on the end if it's really intact. But if you pull enough of them, you can usually look at them in a batch and kind of see that the ones. The biggest mistake, I'll go right into that. Biggest mistake people make is they cut the hair. They want to fit it on the form, they want to fit it in the envelope, so they cut the hair and they send us all the shafts instead of sending us the ends with all the roots. So you can cut the hair feel like you need to do, but please send us the ends that came out of the animal, closest to the animal's body, because that's where the roots are, not on the end that's the farthest from the animal's body.

SPEAKER_00 8:39

And you can put them in like like a mailing the white envelopes, you can there versus taping on.

SPEAKER_02 8:46

Yes. And I actually, depending on how people are going out, if they're going out and they're planning to collect more than one animal, you're not necessarily gonna have all of your submission forms printed out with your tape and your this and your that. So if you were to bring out paper envelopes, right, you can have those. I always recommend not having them pre-labeled, but I can understand how it would be helpful to have them pre-labeled. You go out with all your envelopes, you're looking at your envelope, okay, Betsy, I need to get hair from Betsy. You pull that hair from Betsy and you put it in Betsy's envelope. But we've often found that sometimes it can be helpful if you just bring your Sharpie and you're recording the animal that you're putting the sample in, just sometimes can help to prevent the wrong hair from going in the wrong envelope if you wait and you label them as you're taking them. So that's not totally on that. That doesn't matter. Paper envelopes are good for two reasons. Number one, it helps to keep the hair dry, it won't trap any moisture like a ziploc bag would. And the labels don't rub off. If you're using a pen or a sharpie, it's not gonna rub off the paper envelope where sometimes the labeling can get smudged or rub off of, say, a plastic bag or a ziploc. So having the paper envelopes can be handy. And then what we would recommend is just attach the staple the envelope to the form. Make sure the names match. We want the name on the form to be similar or identical to what's labeled on the envelope. For example, if you put on the envelope you wrote tag 225, and on the envelope you have 225, and on the form, you have a registered name, RPC Betsy. Well, there's no way for us to link that RTC Betsy is that tag number. So you either want to have the tag number as an alternate ID on your submission form, or you want to at least put Betsy or something that links that labeling sample to that form really helps because when we are in receiving one of the first checks and in sample preparation, they're making sure that the sample they're taking is matching the form as far as the IDs go before things get separated. Because once we take the sample out to start prepping it for testing, its name goes away. Its case number is not with it. We have other ways of tracking what samples are in what places as we're testing. But that's the first thing is that the sample need labeled sample needs to match the submission form as far as the identity.

SPEAKER_00 11:05

And I just did it the other day. I you guys had to email me yesterday or the day before because I sent some in and I put river on the form, and on the envelope it says phase heifer river Kate, and they wanted to make sure that it matched.

SPEAKER_02 11:21

Oh, see, so river and river, it did kind of match, but customers, so they're getting more strict. Kind of, but not quite. Yeah, they're getting more strict. So what ends up happening is if I understand if receiving, oh yes, our biggest thing is that we are sending the correct results for the correct animal. I mean, that's like one of our biggest quality controls and part of my job, and we can talk about that a little bit more as we go on. But yeah, so what happens if receiving catches a discrepancy, and you can imagine we get thousands of samples, things you know, oh, it doesn't matter. It gets set a small service takes the time to reach out to the client to make the confirmation and all of that. So yeah, it ends up to be puts a little hiccup in the process.

SPEAKER_00 11:59

Yeah. And then she also shared with me how important it can be to make sure that you send that picture when you're ordering the test.

SPEAKER_02 12:09

Oh, we had we talked a little bit about you have the option when you're creating your test request to upload a photo. And it's not necessary, but it's really helpful for a few reasons. Number one, if you're submitting for coat color testing, which we'll talk about a little bit more, the analysts will look at the picture and make sure that the results of the sample actually match the description or the visual, what we call the phenotype of the animal. We want to make sure before we're sending those results, or it gives us a little more confidence if the genetics for the animal, its genotype, matches its phenotype. Oh, and the color that you submit. If you list the animal as black and then the picture is of a red and our results are black, we're gonna immediately think, oh, okay, the sample is right, but the picture appears wrong, right? Because the sample is matching the description, or which means kind of like which one of these is right or wrong. And so that can be helpful in that situation. So for co-color, it can be especially the more complicated our tests are getting and with the dilutions and all of this. And if people are sending in multiple samples, it's not hard to get a couple samples mixed up. And we understand that. And that's why we have all these quality checks and controls in place. The other thing to point out is these genotypes, if you send it not just for diagnostics, but if you send it for genetic marker testing with or without parentage, that DNA result is a fingerprint on file for that animal. It's its identity. So just like we have a criminal database that we talk about where you have DNA on file for all criminals and you catch some your perpetrator and you want to prove that that is the person that should be guilty or whatever, trying to link somebody to a crime. And we do have a forensics department. That's another really interesting thing about our laboratory. Yeah. Where we do animal forensics when animals are involved in the case. But anyway, so being able to identify, so for example, if you know you guys have are getting really diverse in your herds, but let's say we're talking about just black dexters. You have a herd of black dexters, right? Somebody loses their tag, there's no markings. You think it's so and so. Well, if you pull a hair and you can send it as with a request to match it to the genotype if that animal has already been tested, and we'll let you know, yes, it does match Betsy. You think it's Betsy, yes, it does. That is Betsy. Or we'll say, no, this is not a match. But we might find someone else on your account that it does match. And so whether you send it who who you think it is, and then we can tell you yes, it matches if it doesn't, if that animal is already on file, we can let you know by matching the genotype, the new sample, to the old sample. The DNA doesn't change, it's a finger, just like your fingerprint. So that's how it can be used to identify animals. And that can be not only for lost tags, but sometimes people, oh, it happens more with smaller livestock, like the sheep and the goats. Well, you'll loan out your ram or your buck. I know you usually you don't, you know, you'll ship semen now. You don't usually loan out your bull, unless it's maybe for a neighbor or somebody. Or if you're trying to, yeah, prove that your calf was bred by the bull next door that got over the fence and topped back over, which we've had those cases, you know, we can sort those things out because the genotypes are already on file, right? Now, some of that was will be done with parent verification as well. But yeah, being able to, so there was a story, I'll just go off a little bit, that a person had loaned a buck out for breeding for a neighbor, and they swear that when the animal was returned, it was not the same buck that they had loaned out. And if their standard markings and colors, let's say it's a savannah or something that's all white, I mean, how do you prove that except with DNA? And so they were able to prove, yeah, that the animal they returned because their buck, of course, was already on file. So these are just some interesting things that you can do. But most people are doing the genetic markers because they're also being required by the registries to prove the pedigree. You can't just register an animal anymore and say it's a son of so-and-so.

SPEAKER_00 16:02

You have to prove I love that with uh sign of the registry because then you don't have people making it fraudulent claim and mocking up papers and things like this.

SPEAKER_02 16:12

Yes, and it would be very easy to do if it wasn't done that. Yes, and for so long it wasn't. But the herd books responsibility the registries are responsible for keeping the herd books. Well, the herd books are the pedigrees, and so now, and when they started doing it, they kind of grandfathered in up to a certain amount of generations, but a lot of them went back and they made breeders test their older breeding stock to kind of match it up back through the generation. Now we're moved forward, but that's how the pedigrees are established and can be relied on because they're proving it with the genetics, yeah.

SPEAKER_01 16:42

All right, I'm gonna ask this real quick. So, like that's all on the registered side of things in the non-registered, like in the miniature world. If we were to say buy semen from someone else, we're using that semen on that calf, and then we're in turn gonna advertise that calf as this really well-known bull, it's that it's sire, whatever. Do we have to like if I wanted to prove that, say on their paper, so I have to like get with that breeder and they have to release that to be able to say, yes, that is the sun or heifer of it.

SPEAKER_02 17:08

Well, that though, that's a really good question. So when it comes with what we call assisted reproduction, okay, so that's AI and embryo transfer is what we would consider assisted reproduction, where there's the whole human element, the whole things have been collected, things have been labeled. There are, I can go on and on about stories of mislabeled straws or embryos that got stuck on the glass and flushed out from a previous donor that got mixed in with another don. I mean, this is where the power of where we can use the DNA to help resolve a lot of those problems that come up. But a lot of people, if you're spending a lot of money to buy a lot, and I say a lot, not as in many, but a group, a batch, a lot of semen, you might want to start by confirming that that semen is from the bull that it should be from. And chances are that animal has already been tested. You would ask the person you purchased it from or the bull owner for the VGL case number for that bull. And that's all that you would need is to use that case number to confirm, you know, list that case number as the sire for comparison and a parent verification request, right? So you can do that, and if it's matching, that's a pretty good chance. But you could also, if you wanted to do it before breeding any of your animals, you could submit a straw of semen and then ask send it for a match comparison to the case number given by the owner of the bull. So you can match up the semen that you got, matches the already the DNA fingerprint on file for that bull. So you can do that before you even start any breeding, right? So that's another way to do it. You would use it also. But I wanted to also, for people that aren't required for parent verification, but for the exact reason that you were stating, Taylor, where you were saying, you know, it's a big well-known and you're selling this calf. Well, if you can hand that person purchasing your calf a document that proves through DNA bred by that bull and out of such a cow, that should allow you to ask for the price that you're asking for, because you have proof now that it is of the pedigree that you're selling it for. And often the price is related to the pedigree. And so being able to prove it, and I tell people, you can absorb the cost of that extra testing in your sale price because you're providing. I mean, how much more? I mean, to me, I would do that if I was selling animals, regardless if it was a high price. I want to prove the pedigree. Here, this is the pedigree of the animal that you're buying, and you can pass on a copy of your DNA report. The other thing that makes that very helpful, assuming you've already tested your calf, now they have the case number for that bull calf. So when they start breeding him, they can just use, they don't have to retest that bull calf because he's already on file.

SPEAKER_01 19:49

Right. There's no way that they like are able to, okay, just like a clarification, because I know that has been out there, right? So I have the calf. I bought the semen. There's no way for me to lie and say it come back looking like I'm being dishonest and saying this calf isn't out of this bull. It's gonna be flat honest as long as that person who has that bull is willing to give that case number, right? So in future references, I think that we should just as a whole in this little mini world, because it's not on the regist side, is if we're gonna be buying Strausa semen, we should say, I need this case number in order so that when I have these calves, I don't have to come to you and say, Hey, I need to DNA test because I bought three different bowls of your semen and I need to make sure I put this semen in this cow, just a DNA test to verify that. Like that shouldn't be a big deal, but maybe that's something that I don't know, in other world that is not registered, and that is a thing when you're paying for high dollar semen to get these high dollar bowls is just to have, okay, well, if I'm buying the semen from you, I need your case number. Wouldn't you say that would be like an important thing nowadays to do?

SPEAKER_02 20:52

I would say, yes. That's assuming that the bull's been tested. Maybe it's had all the diagnostics and all the color. Maybe they never got gent, you know, genetic testing. So it would be something that you should certainly ask when you're purchasing semen. Can I, has this animal been DNA tested? Can I have copies of all his reports? That way you're gonna know is he a carrier of this? What's his color genetic? You know, all those things. You have copies of his paperwork. So you have his case number, you have his results, right? Because you're gonna want to decide what cows you're gonna breed them to based on his genetics. Right. And what happens if they don't, we'll not get to that. Right. Let's say the bull's not been tested and you just, I don't know, whatever your reason for getting the semen, right? You decide you want to breed to this animal, would just submit a sample from that lot of semen yourself for all the testing you want, and then you have all your answers and you would wait to get those results before you started using the semen. But I would say that nowadays most people are gonna have if they're marketing a bull, they're marketing it for his genetics. Right. And so they're gonna have the testing. But yes, I think it's very fair for anybody purchasing not even. High value. It doesn't, it could just be your standard. If there are genetic tests that have been done on that breeding animal, why not ask for copies of those results?

SPEAKER_01 22:09

I think that's a great note.

SPEAKER_00 22:10

Do you feel like with bowls, when you're sending in bowls, should you send in a little bit more hair? Like last year, it was the year before. I had four or five bowls in the same pasture doing my cleanup for me. And so I had to send everyone in and say that matchup, who's the father? Who's your daddy? I was playing Maury Povish.

SPEAKER_02 22:28

Oh, the extra hair. I say send in as much as you can collect. You don't have to go overboard, but yeah. Because what's really interesting is once the testing is done and it's on file, we don't usually need to retest. There are a couple reasons why we test routinely. Almost every genotype for a breeding animal has been tested twice and independently. And the reason for that is if you were to ever get a parentage report that excludes the dam or the sire as a correct parent, we never send those exclusions without confirming the results by retesting. So, in other words, everything comes through, the genetic marker, the profiles go out. Everything looks good. I have all my results. Then I get the case back and it's now listed as a sire, but it's excluded to the as a possible sire in the as the offspring because they don't share alleles, it becomes pretty clear talking about the alleles and how parentage works and analysis. That's kind of another conversation or another question to answer. But if you would expect that offspring's genotype is a combination of the mother's alleles and the father's alleles, I mean, that's how we match the parentage. And if that bull's genotype has alleles that are not present in the offspring, then they're not a match. And so, in order for me to, I look and I say, huh, this animal was genotype. It's only been tested once. I'm gonna test that sample again to confirm that the results we have on file are the correct ones for that sample. I don't know what bull it came from, right? That's the other thing people to remember. We can we have case numbers. I mean, it's labeled as this, but who knows who that sample came from? Only you that's pulled it. So we retest to confirm that our genotypes are all so once they're tested twice, and I can look in the history, I'll look, oh, that bull's already been verified. The types have been verified. I can exclude him, but I'll retest the offspring to make sure its types are correct. So all exclusions have been retested to verify all of their results before exclusions come out. If the parentage qualifies and everything looks good in the primary panel that we test, those results can go out because the mother and father work, the chances of it being wrong are very slim. I can say that, but I say that with the caveat of saying, if you're running a closed herd or an inbred herd, sometimes results do change, okay? Because I might report and then later on someone goes back and goes, you know, that's really strange. I submitted that bull because I thought he was in that pasture, but he wasn't. It was his son. So then maybe a new parentage request will come in for that same animal that's already parent verified, and they'll ask us to compare this bull with the mother. Oh, he also qualifies in the primary panel. It's like, how can this be? There's not two fathers. I will run more markers. I will eventually pick up an incompatibility somewhere. The more markers I run. Okay, right now I'm looking, I was trying, there's a gentleman that acquired a herd. It's been a nightmare because he was given it without paperwork. He has the herd. He's spoken to a previous breeder that did test them. He's tried to match up his new tags to the animals we already have on file to try to work out the genetics. And he has some of the older animals. And there's one that I've run up to 44 markers. These two bulls are both qualifying in all 44. I mean, this is not common. Wow. And we don't have the dam because it's an older cow from a herd that he acquired, and we don't have the dam. And so that's become a little problem. And I think I can run up to like 64. So we could run more, but based on some other paperwork and things he's been working through, he thinks that this bull and not that bull. And the one of them, the other bull is the son of the mother. And so, anyway, it makes sense on why they could be, but still, with that many markers, you would expect to pick up some differences. So, and sometimes I'll just get a single incompatibility, and I'm not gonna exclude a bull with just a single incompatibility necessarily. So, I'll also run more markers, and again, this is why we need the extra sample. We need, if we're gonna be retesting and verifying our results, and it's all about the quality controls and us having the utmost confidence in our results when we send them. And that's one of the things we really pride ourselves, the VGL really does, is in our quality of our work and our customer service. Those are the two things that we really stand behind. And our mission for outreach, because we're part of the University of California, which is a public institution non-for-profit. So people don't realize that either. Being part of the University of California, we're a non-for-profit. All of the profit that we make, which isn't a lot considering the cost of everything these days, and all of the new equipment and the new technology that we're moving into the SNP testing. You guys might hear that word thrown around a little bit, SNP testing. That's an again another question we could talk about. But there's new technology, these machinery. Oh my god, they're hundreds of thousands of dollars. All new reagents and all new, I mean, the training, and it's just so there's a lot of expenses that we have anyway. It goes into paying our salaries, and anything left over goes back to the school of veterinary medicine. So it gets put into their pool for what they do in supporting the school of veterinary medicine. So, yeah, so we're kind of unique in those compared to a lot of the big public laboratories that are trying to get the most bang for the book.

SPEAKER_00 27:41

You are. So let's talk. I'm gonna say that 99% of the time, what people are really looking for is the color of their animal, the chondro. Let's and sometimes pull the horse's horn. But let's talk about the color because you really should get it's the I should have this. When you're doing the color, you need both of those, right?

SPEAKER_02 28:03

One of your questions, it was relating to the color. It had to do with why is genetic more reliable than just your visual appearance? That's how you pose the question. Why genetic testing and also what are the genes are typically included in the standard color panel? Okay, so we can go off of that. Typically, the MC1R testing, it's called extension, that is a base black or red or wild type. Okay, so that is like a base color that the other color genes modify. Okay, so there's the base color, and then you have the other genes are having an effect that modifies that base color. So, for example, when we talk about the dilutions, right? If you have a black base and you have, let's say, when you're looking at your dilution for the Highland dilution, the one that you guys test for a lot, you're going to be looking at one copy of the dilution gene versus two copies of dilution gene. And they're what they have a dose effect. So if the animal is black and it has one color of dilution, it's gonna come up as a gray shade. If you think of mixing black paint and putting drips of white in it, it's gonna slowly lighten it and lighten it and lighten it. Well, if you have two copies of the dilution, you're gonna dilute that base black color out even more so that it can be a really light gray or a silver or sometimes appear even white, where the black is completely washed out, right? And we're seeing some of these animals that have just the black ears and the black little marks on the hooves and the black nose versus the reds and stuff like this. And again, those colors are based on that MC1R result, right? So you have the dose relating and you have that. But there's other now, is your minis, how are you getting the small stature? Are you breeding to dexters or how are typically Yeah, I think most of them have been bred to Dexter somewhere in the in the line. Well, and that is important because especially it must have been, because otherwise you wouldn't be testing for the Dexter Bulldog, or you wouldn't be seeing carriers of the Dexter Bulldog. Right. That is proof that animal somewhere in its lineage was crossbred to a dexterity because that BD1 and that BD2, those bulldogs, otherwise known as chondro dysplasia. Chondro dysplasia is the technical scientific term for the bulldog condition. And the bulldog condition was given just by the way that they look when they're born. And yeah, it's not pretty. And the reason why people are testing for this is because they want the small stature that they can get from a carrier, but they two will be lethal, right? So we're not seeing unlike the PHA, we're not seeing the disappearance of this condition because people are using it to help with the smaller stature, right? Right. This is where it becomes really important when you talk about breeding carriers to other carriers, right? Yeah, that test is very important. This and we get into the heterozygote and the homozy, it gets into a hole. Add a coat color, so we'll save that explanation.

SPEAKER_00 31:04

So when you get a color, let's just say with some my color test, they will say that they have maybe one copy of black and one copy of red or wild. But then when you look at their or not, no, it'll be red. But when you look at their delusion, they could be have the wild and should have put that paper in front of me. Not that no, the not the delusion. Not the delusion.

SPEAKER_02 31:25

Yeah. So the wild type, I think, in the MC1R is the un, let's call it an unadulterated gene. They call it. That was the wild type. Then there were mutations in that wild type version of the gene turned it into black expression of the black pigmentation. Or a different mutation that created the red pigmentation. So that's why you have the three. Okay. But the black is dominant. So one copy of the black. That's why it's a big D. It's dominant. It's gonna override any other allele, the red or the wild type that is associated with it. Black is dominant. Okay. Black is dominant. Black is dominant. Both wild type and the little E, the red and little E, are recessive. So if you have two copies of the little E, the animal's gonna be a true red. And depending on the breed, like with the Dexters, when you look at a homozygous two copies of the red versus two copies of the wild type, which is also can give a reddish color, they're usually pretty different in the shades. The wild type is usually more of a rusty brownish kind of hues, where the reds are a true, almost red, rich, orangey red type of color. And so you see that in the dexters. Now, you guys are breeding, everything is getting all kind of mixed up. And you can have a, it's an interesting color if you have a little lee and a wild type. They're both contributing. So it'll be a form of red. But you won't be able necessarily to tell it from a homozygous wild two copies of the wild type, but you can usually tell it from a homozygous recessive red animal that is a true red.

SPEAKER_00 33:05

I know the conversation gets brought up a lot. So we have brindle. When you're getting into brindle, does wild necessarily mean it's brindle?

SPEAKER_02 33:14

No. But, and unfortunately, there is no test that I know of for the brindle gene. So we can't directly test for it, okay? But if you want to confirm if your animal's color type or the category you're gonna describe it as brindle, that MC1R must have at least one copy of the wild type. You will not have a brindle D D homozygous black animal, and you won't have a brindle if you have an EE, a homozygous recessive red animal. It has to have at least one copy of the wild type because the brindle is associated to that wild type form at the MC1R.

SPEAKER_00 33:52

And do you know how you know if that animal does have brindle at all?

SPEAKER_02 33:56

Is it just by Usually it's phenotypically evident? Yeah, you see the banding of the colors. It's almost like that kind of a stripe type of appearance. And we can see it, guy, you see it now a lot, a lot in dogs. You're seeing more and more brindle and all kinds, even little Frenchies and things are getting pretty fancy.

SPEAKER_00 34:13

I feel like with my cattle, I can see it on their nose. And if you really get down into their hair, because it will show up. Doesn't it show up on the skin, the brindle? Or no? Not on the skin.

SPEAKER_02 34:23

No, I've never heard of it showing up on the skin or on the nose. But I'm glad you brought up the nose because you also asked when we were talking back about pictures. If you want to send in a picture, what's a good type of picture? Basically, one shot of the animal from the side, preferably looking at you so that you can see the face, or at least so that you can see the color of the nose. Because we had somebody send in a Highland. I think this was through the American Highland Association of an animal that they thought was Brindle. And when we were testing back for it and there was a question about the base color, anyway, I asked to see it's a picture where I could see its nose. And I forget the whole scenario, but that was very helpful in me determining that it wasn't coming up. I know it wasn't coming up as Brindle. So the first thing I thought about, and this gets a little more complicated, I'm not sure if we want to go there, but is the animal what we call a chimera? Is it a combination because it had this brindle appearance? And if it wasn't brindle, like how do you explain what it is? So when I say I told them to the red hairs look now, once I saw the nose was black, I said the red hairs should have pink skin. The dark hairs will have black skin. So if you can see differences or striping in the red hairs versus the black hairs, and the red hairs are on pink skin, and the black hairs are on the black skin, that could be indicative. I can't promise that's how the brindle is, though. I have to admit that I don't know that. But it could be evidence of a really interesting animal, I'll just say mixed genetics.

SPEAKER_01 35:56

There you go. I have a question that goes with this brindle. Okay, so this is like just a small number. I mean, people have a lot more cattle than I do. But one thing that I have noticed, because you don't see it present in a black as you would see in a like black brindle versus red brindle, right? So I have a cow that is black and like her tips of her hair are very red. She has thrown me brindle calves.

SPEAKER_02 36:17

The red tips, that's a different gene. That could be more like what we call a goody. There are so many coat color is so complicated. And I like working with cattle because we don't have a lot of tests. So that came simple for me. No, I'm serious. Yeah. When it comes to the cats and the dogs, there are so many coat color genes, and each one has its own effect. It the unique phenotype is based on all those possible combinations of all those different coat colors, all making their effect.

SPEAKER_01 36:45

So since that cow throws so like the Brindle is a gene that gets passed through to get that color. So if that cow consistently has Brindle calves, or vice versa. So I have two Brindle cows now that have both calves, and both of those calves came out as black, and then like, so I have their test right here. So it came out dominant black with a carrier of wild. Could that correlate that they carry that gene to throw Brindle calves? Like, could that mean something? Yeah, I mean, especially if the cow herself has some indication of the strike.

SPEAKER_02 37:16

If she's giving it's one of the two, it's either the bull or the cow. You can't have brindle babies without one of the parents having the brindle. But the fact that she has the wild type makes it possible.

SPEAKER_01 37:26

That the calf would have it from getting it from the mom. Since the mom's moms are red brindled.

SPEAKER_02 37:30

Do the bulls also have a wild type? Because I'm wondering if these ones that are born, and it's spanking brindle. I mean, there's no it's striped. I want it, and I don't know if it's dose related. So I'm getting a little bit out of my expertise here to think if it would be a more dominant type of appearance with the brindle if it had two copies versus one. I don't know that. But that would be interesting.

SPEAKER_00 37:54

Sometimes if you can't just my brindles, like it's blatantly obvious they've got but that's on like your reds, right?

SPEAKER_01 38:01

Like your reds with the black stripes.

SPEAKER_00 38:03

My black ones turn really from almost a dun to or dark done to a black, and then they have the color left over until they shed it out. And sometimes those all turn black. Right.

SPEAKER_02 38:16

So if they shed it out, if that's a totally different thing, and I don't know enough about the co-color genetics where the colors change when the hair is shed, but that's another question as far as people testing animals when they're just born and deciding, you know what they are. Now the genetics isn't gonna change, but the appearance of the calf sometimes changes when they shed out, and that's where we see sometimes a discrepancy in the color listed for the animal and the picture. We will look at the age, right? And you can see, especially if there's not a picture, and they list it, let's say they list it brown, right? And it comes, we test it and it's black. And we see, oh, it was taken when it was three weeks old. It had its baby fuzz. It probably looked brown, right? But its genetics say it's a black cat. And when it sheds out, it will probably be more of a black appearance. So, yeah, so that's sometimes where the discrepancies come up and where the pictures can come up, and then we use the data birth. So we pull all of this together, and sometimes I'll actually can email and say, you know, and sometimes I have to ask this, and they didn't request coca. But we have a lot of our testing, we run a bunch of tests and then we send the results for what the person had asked for. So sometimes I have more data than than what they asked for, but that helps us with our quality control, right? Because I have all those results to match up to make sure that I'm sending those Chondro results. Some people are just testing Chondro, I have co-color that conflicts with what you described. I'm a little worried about saying that this animal is NN or this animal is a carrier when I'm concerned that maybe this sample is not from the calf you're describing. So sometimes I would email and just say, hey, some of our testing is indicating that this animal is a color, other than just confirm the color of the, you know, and we go through all of that. Oftentimes the say very rarely is the sample wrong. It's just the way people describe them is not genetically accurate. So you always have to kind of, you know, it's what does it look like.

SPEAKER_00 40:17

So basically, black brindle is really hard to figure out if it's truly brindle or not.

SPEAKER_02 40:23

I would say that it, especially if there's the one copy of the brindle, it could be the darkness of the MC1R, the black base kind of mutes, let's just say it mutes the Brindle appearance a little bit more than in a redwood. That seems totally possible. Yeah.

SPEAKER_00 40:38

So I feel like it's super important that they choose both of those markers because we always have people that throw into groups and say, What color is this animal? But they don't have the the MC the MC1R as well as the dilution, yeah. Dilution. Yes, both of them. If they don't have both of them, it's very hard to consider what color it is because we've got seven colors that they could be.

SPEAKER_02 41:03

Yeah, it just you don't have all the information. So you gotta start with the base color because that's gonna tell you how that dilution will kind of an effect. Is it gonna be a black turning gray or is it gonna be a red turning cream or peach? Those are the two shades that you're gonna go. So sometimes you can figure out what the MC1R. You know if it's a reddish or if it's black, right? If it's black or gray, you know that it's gonna have at least one copy, but you don't know if it's homozygous. And again, the importance of having the homozygous or the heterozygous results only becomes important when you're making breeding decisions because the breeding animal, the bull or their cow, is gonna have, right? We see two alleles. There's two possibilities at every marker. Okay, that reflects what's on one set of chromosomes, what's on the other set of chromosomes. Okay, now without getting into a lot of biology here, each parent passes on one of their two alleles. So if their homozygous black, it doesn't really matter. All their calves, a bull a DD black bull will only pass on black. All of its calves are gonna get at least one D. And the D is dominant, right? The black is dominant color over whatever that second allele is. Okay. Yeah, so that becomes important. So yeah, to have the most information, to make your breeding decisions, you should have both. And then you have to think about how far back was the dexterous. There's a dexter dunt, and I don't know that y'all test for the dexter dun, and I'm not doing a lot of the reporting of the color these days, so I don't even know if we're picking up that dexter dunt. It only plays now that has to have two copies. That's an interesting one. But anyway, getting back to the Highlands, now that you have that dilution, I mean, that really exploded the interest. And now these minis and they have the colors in the mini, so we've gotten a whole lot busier with you all want to understand, and and it's really been fun to see the colors. And that was another reason why the pictures. Oh my god, you have no idea when you're looking at letters and numbers all day, and then you come across a record that has this adorable picture or a beautiful bull, it's just like wow, jaw-dropping. And even if they're not, at least I get to see who am I analyzing. Who, yeah, it's kind of fun. Right. But if we come across something that's really extraordinary, we actually share the picture with the other analysts. We'll say, hey, look at this one, you know. And it's a lot of fun. It kind of makes us smile and it keeps our day kind of a little bit lighter when we have some pictures to look at.

SPEAKER_00 43:30

You get bored today. I did send in, I'm hoping we'll see if they get back in time. But we have an auction this weekend, but I sent in all my pictures after we talked to go with my samples.

SPEAKER_02 43:41

Yes, because you can go in. I think even after the record is, even after the samples have been received, I believe you can go in and still upload a picture to your record. So even if you don't have your picture when you're submitting your testing, the next time you happen to be out with your phone and you can take snap a shot, you can. Always go back later and upload your photo. And if you ever need assistance with that, just give our customer service team a call. They'll happy to walk you, walk you through it.

SPEAKER_00 44:08

Sometimes if I'm doing it with my phone, I found out the other night because it was after we talked, I went back and put all the pictures. It says file too large, but if you clear that, because sometimes if you're on a roll and you're going or you're going down each one, I don't know if that has anything to do with it, but if you hit clear in that picture box because it says file too large, go out of that the site and go back in, you usually they'll it usually takes that picture after that, like accepts it.

SPEAKER_01 44:32

Yeah. I screenshot and edit it and make it so the files.

SPEAKER_02 44:37

I go way. You store so much information you can imagine. So those files get a little bit if they're you sometimes I just have to reduce the quality and they still look fine when we're looking at them in little thumbnails, even when I can click on them and make them larger. But I'll tell you what, there are times when we're so we have our other analyst team, and so if there are discrepancies or there are exclusions or there are questions about the results, no matter how little, those get bumped to me. So I'm the one that is reviewing all of these discrepancies, right? Because the regular my other analyst team, they're trying to just everything looks good, they send it out, everything looks good. So they don't have time to start the nuances of the investigating and the contacting the client and doing all of the extra checks. They do an initial check and they send all of and they kind of post a review case, is what they call it. I go in. Do you guys test for the Rhone gene? No. In cattle, I don't even know that that is available for testing. Like that it's been identified enough for us to say, you know, yeah.

SPEAKER_00 45:38

It's just, I think there's just I didn't know with the short with short horns. If you did anything with short horns, if it was one or two copies, not a big deal.

SPEAKER_02 45:46

Yeah, we don't, but we they do we could do the base color, so you know if it's a red round or a dark, you know, a black round or great. Okay. Yeah.

SPEAKER_00 45:52

So the base color definitely helps with that. All right. We're getting close on time. So I want to talk real quick. Also, there's two things, but the pulled gene, because now we have a lot of people, white parks that they used to be back in the day, horn, but now most of them are pulled, to Highlands or whatnot. So we're sending in for pulled or horn. Now I had a one-off case where it said it was pulled, but I shipped him to the people, like he sold. They wanted a pulled. They're like, he's growing horns. I'm like, oh. So I brought him back here. But feeling those horns, I shook them and they're very loose. So they're most likely scurs. Is that correct?

SPEAKER_02 46:35

Well, that's what I yes, a horn is not gonna be movable around. And I don't know a lot about the genetics of scurs, but I do know we don't have a test for it. Okay.

SPEAKER_00 46:44

Okay, I had one case where they put SCRD on it. So if you get those tests back and they're SCRUD, it may say polled because it says 50%, 50, 50, which is usually right. That's why we're sending them in 50% that they could have pulled offspring. Is that right? Pulled is dominant.

SPEAKER_02 47:01

So you only need a copy of a pulled gene. We test three different olgies. So the results that we report are based on three different markers for the pulled, okay? And different breeds have different types of mutations, but the pulled is dominant. Okay, so there's no reason to test a horned animal. It's not pulled. Now, if you have an animal that has scurs, that is interesting if it comes back. I would only expect one, but one pulled, if it comes back, is pulled and it has scurge. You know what that tells us? That tells us the scur gene overrides the pole gene.

SPEAKER_00 47:38

I had a little bull. He had scurs, and some of his tests were just a little bit different because he had scurs, so that kind of passed down just a few. Kind of a personal question. Are you choosing to keep him a bull? Is that something that He was a bull when I got and he was actually beautiful.

SPEAKER_02 47:54

No, but I mean keeping him breeding versus cast breeding him because he had scurs. In other words, that's something now that you're passing on into your herd or you're selling, you know, so that scur is not a one only. Okay. It's like any other gene, it's gonna be passed on. Now I can't say that all of his offspring are gonna have scurs because again, he could pass on the pole, he could pass on the scur if he's got one of each, and there it could be again, we're not testing for the scur, so we don't know if he has two scurs or one scur. Is scurs bad or it's just uh that's no. When I say bad, just like with horns, there's no nothing that is bad for the animal to have horns. People just choose for management purposes to dehorn. And since now they're realizing that they can do that genetically instead of having to assault the animal with those processes, uh, then I think people are certainly wanting to do that. It's a little hard in the Highland because in my mind they're horn. You know, it's like a horn breed. Right. It's a horned breed. So when you start seeing them without it, it's kind of a whole new era.

SPEAKER_00 48:56

Yeah, that's for sure. So the another one I wanted to bring up is now Free Martin. And a lot of people have a misconception about Fremart. They do.

SPEAKER_02 49:06

Now, do you guys do how do your minis or do your Highlands have a twinning rate that's even we do?

SPEAKER_01 49:11

We can I have a set of twins right now. I have the test paid for for you guys. I just haven't drawn her blood. I had a bowl and a heifer. And so I have it all ready. I just need to get her blood drawn and sent over to you, paid for everything. So I have that.

SPEAKER_02 49:23

So the misconception, and I can go into a lengthy conversation teaching about this, but just to keep it in the basics and the time that we have remaining. Number one, it only blood samples are submitted because that is the only place that we will detect a Y chromosome in that female. Okay. And that is why you are testing that animal. When you have, think about the uterus of a cow is really designed to carry a single calf. So when she double ovulates and they both get fertilized and you end up with a set of twins, you're now sharing that single fetus space with two fetuses. She can do it, obviously. We have successful twins all the time. But you have to understand that those fetuses are going to be more confined. And what ends up happening as they grow is that the placentas of the two fetuses become directly opposed. Not only do they become directly opposed, but they start to fuse to one another because they're tissues that are growing and are highly vascular. And what ends up happening when those, because it's the placenta that carries all of the vessels that go up through the umbilicus to nourish and provide the blood for the fetus and the nutrients and everything from the mother. So when you have the circulation of the male and the female fuse and they start exchanging blood, the hormones and the chemical factors that are in the male fetus start drifting into the circulation of the female fetus. Okay? So we use the test. We don't look for those hormones, we look for presence of a white chromosome because the cells that are the blood cells, the nucleated blood cells, the blood cells that are crossing over are going to be shared. And some of them, when the development of the fetus, the cells they go into the actually seed into the bone marrow of the fetus, so that all her life she's going to be producing blood. Some of them were the original hematopoietic progenitors that came from the male fetus. Okay, so she doesn't necessarily will have those all her life. Would you be able to detect it? But it depends on when that fusion occurred, because how much was if it was very early on before the actual sexual differentiation occurred, really early on in the fetuses, when their maturity of the genetics of reproductive organs, the gonads, are starting to develop. If they've already fused at that point, his hormones are gonna affect how her genitalia is formed. Because the females are usually by default because they don't have the hormones. So when they're exposed to the hormones, it can affect the development of that heifer. However, if it happens later in the development, and this is where the misconception comes, and that fusion and the mixing of the blood happens after sexual differentiation, she will still have those male cells in her blood, but she could be reproductively normal. And that's why if it's a valuable heifer and it tests positive, I tell people, well, first of all, if she looks abnormal externally, chances are probably pretty good that things are different and not as they should be on the inside, the the ovary and the uterus and such. But if it looks normal on the outside, you can actually have, I mean, and she's not bully, there's no male characteristics, she just looks like a nice heifer and she looks normal on the outside. You can work with your veterinarian to do some physical examination to see if her reproductive tract appear normal. Even though we detected that Y chromosome in her blood, if she's extremely valuable and she looks otherwise normal, I'd wait and see if she cycles. But I would also have a veterinarian examine her, try to pass a speculum, do things to make sure her canals are all open and passing, because just because her ovaries are ovulating and she has the hormonal thing situation, she's producing the hormones to make her cycle and come in heat or esteris or let other cows mount, you know, heifers mount her or whatever, doesn't mean that she's uh reproductively sound in her internal organs, all of the canals and connections and that everything will go normally. Okay. So again, there's a few things that you can do with there, but it is a misconception to say any number one, any heifer born with a bull calf is a free martin. That's the first misnomer, because it's not always true. It's usual, again, unlike sheep and goats that have twins, right? They typically have twins or triplets or quadruplets. Now, when you have quadruplets and you have triplets, things are a little more squished. And if you have a male and a female in the same horn, you probably there's a high chance you could have a Free Martin little doe. But if you have twins and a goat and the male's on one horn, because they have horns in their uterus, because they're litter bearing, they have more room, and the and the goat fetus, the female fetus, is on the other side, there's a good chance that she's not. So it's unlike cattle where they're usually only have single fetuses and it gets more squished in there. Same with horses and humans and everything else.

SPEAKER_01 54:28

So if that test comes back negative, you're good. Like that heifer is good to go. You should, yes.

SPEAKER_02 54:34

If that comes back, we're and what our tests, and we have three gender markers. We're looking for that presence of the Y chromosome. That's all that it's telling us is that yes, there was a mixing of the blood. That's all that it tells you. You have to rely on behavior and you have to rely on her phenotypic appearance of the external genitalia to help put it all together on whether or not you should have a concern about her fertility moving forward or not. So it really is all three of those things.

SPEAKER_01 55:02

Are you guys the only one that are testing for Free Martin like that? So I had talked to someone in the Angus world and he's like, There's a test for that? You can test for that. I was like, I I paid for it.

SPEAKER_00 55:11

So yeah, I'm getting Angus world people. They have twins, they go to the sale barn. They don't even test for it.

SPEAKER_02 55:18

Well, unless they're two females or two males, right?

SPEAKER_00 55:21

Well, yeah, that's what I meant. If they have a male and a female, they go to the sale barn.

SPEAKER_02 55:24

Yeah. In a commercial situation, it's it's probably it's not worth it because you have 50 other heifers that were born that year or whatever. So it's not worth the time. And again, that's why I when I talk to people, it's like, what's the value? Or are you a homesteader that really she otherwise looks normal and you don't want to get rid of her if you don't have to? If you can afford to feed her for six months or however long in nine months until she starts cycling or showing some appearance, then that would be good. But again, I really encourage everybody in those situations, please, please, please work with your veterinarians. Don't make these decisions on your own. Oh, she looks fine, let's go ahead and breed her. You could end up with dystosia problems like times 10. Don't like it doesn't always go well. Or she just might get pregnant but not carry it to term and she's continually aborting or absorbing, and just you really want to work with your veterinarians. If you choose to keep a heifer that tested Free Martin positive, meaning we picked up the Y chromosome in her blood. If you choose to keep her because she otherwise looks normal, please work with your veterinarian before you even consider breeding her. That's great advice. Yes, that is great advice.

SPEAKER_00 56:32

Oh my gosh, we could probably keep going on forever, but we are running out of time. And I know Stephanie has more things to do today. I just want to thank you so much for being here with us and sharing this information.

SPEAKER_01 56:45

Yes, thank you so much.

SPEAKER_00 56:47

So much education in this, what we talked about today, and I'm very thankful that you said yes to join us.

SPEAKER_02 56:53

Absolutely. It was my pleasure, and part of the mission of our laboratory is education and outreach. And so doing these types of things, been invited to come to speak, you know, and a lot of times now they'll do little Zoom calls or whatever back in COVID, and you know, we were doing the Zoom calls, but to speak to, yeah, to be able to educate the people that are the clients. I mean, the registries come, the clubs come, and they just say, hey, people are investing money, but they have a lot of questions. Can you come and just share with us? And it's one of the things about my job that I really enjoy doing. So thank you so much for the invitation. We there's a lot of questions on that list that we didn't get to. So if you decide you want to do another edition, a follow up, I'd be happy to do that. Okay. Oh, thank you so much.

SPEAKER_00 57:34

Awesome, thank you. You're very welcome. Thank you. Hey guys, we will see you next week on another episode of Horns and Hooves. We have another special guest coming in.