Episode 209: The Spin on AI

Julie Cecere, DVM, MS, DACT discusses the current research related to types of artificial insemination and semen processing for better breeding outcomes. Can differences in the route of insemination, types of semen, and owner versus veterinary handling impact pregnancy rates, litter sizes and overall outcomes?

Watch the video version of this presentation here.

Transcript:

Nicole Engelman  00:04

Welcome to the Good Dog Pod. Join us every other Wednesday when we discuss all things dogs, from health and veterinary care to training and behavior science, as well as the ins and outs of Good Dog and how our platform can help you successfully run your breeding program. Follow us and join Good Dog’s mission to build a better world for our dogs and the people who love them. 

Thank you everyone for joining us for another Good Breeder webinar. Today, we will be presenting on the spin on artificial insemination with our guest, Dr. Julie Cecere, who we are so excited to welcome back this year. Dr. Julie Cecere joins us to talk about the current research related to types of artificial insemination and semen processing for the maximal outcome. So lots of great questions and things to dive into today. But before we get into all of that, I just want to thank Purina again for helping us bring this webinar to life. We have hosted so many webinars this year already with them. And for anyone who's new here joining us for the first time, we host two live webinars per month with Purina. So this is the first of April. But like I said, we've been doing this for a while now, so we also have a whole library of recordings for you to take a look at whenever you'd like. During the Q&A section of this presentation, we're just going to prioritize some previously asked questions. And for anyone who is new here and joining us for the first time, I just want to share a little bit more about Good Dog before I talk about today's host. Good Dog is on a mission to build a better world for our dogs and the people who love them by advocating for dog breeders like yourselves, educating the public and promoting canine health and responsible dog ownership through events just like this. We’re also a secure online community that's created just for responsible dog breeders to securely place their puppies with applicants from all across the country. We help breeders run all aspects of their breeding programs from start to finish—things like getting your litters listed, marketed, connecting with the right applicants, getting to know those applicants, accepting payments securely, and all of those other steps that happen in between a litter of puppies being born and going off to their new homes. Good Dog is here to support you every step of the way through that. And we also have a number of free educational resources and health related discounts to help your programs thrive in other ways too. So if you're not yet part of our community, we would absolutely love to have you join us, and you can apply at GoodDog.com/join. I think that's it for my little Good Dog spiel, and I am going to pass it over to her so I don't butcher her background.

Dr. Julie T. Cecere  02:44

So yeah, I graduated from V-MCVM, went out into general practice, and then did a brief stint in both mixed animal practice, as well as ER and industry, and then got called back to do a residency. About two years into my residency, my mentor decided to retire, and so I was fortunate enough to be able to step into her position here at V-MCVM, where I've been since. When we were talking about what topics needed to be done this coming year, everybody kind of agreed that this was something that we probably needed to talk about. I will forewarn you guys, this is going to be very kind of research heavy, but I think that's really, really important, because we as a community like to make jumps very quickly when it may just be coincidental and it's not something that actually is cause and effect. And so those are a couple of the things that we wanted to address in this talk and see what are best practices moving forward, now that we are working on a global scale, as far as transferring our genetic candidates. How do we best do that on a global scale? We no longer really always can get the animals in the same zip code, per se.

So for an outline, we're going to do boys first. And my daughter, who is becoming quite the feminist, would say, “Well, why do they get to go first?” And we're going to do that just because they're the 50% of the equation that we can constantly evaluate very easily. And so proper semen evaluation is important. And we're going to spend some time talking about that. And then what we're going to do is we're going to talk about the two Hallmark processing studies that we actually did here with our former resident, Dr. Sugai, who's up in Michigan now, and why we did those studies. There was a lot of stuff that had happened in other species. There were some studies that were in dogs. But we really wanted to kind of boil it down and figure out if we could come up with a standard processing to maximize semen when it arrives to wherever it was being sent. 

Then we're going to bring it back around and talk about the girls. And so 50% of the equation is the semen that we're using. The over 50% of the equation is: who is it going in, how is it going in, and what can or cannot negatively affect the outcome of that? And so we're going to talk about the studies that are out there. There were actually some pretty big studies that were done that have very, very good data. And then how can we positively or negatively affect the AI again, kind of circling back around and intertwining the boys versus the girls, and how that interaction happens. 

So the first thing that we're going to talk about is the spermiogram, and this is the evaluation that happens after we've collected semen. And this should be done every single time. The only time that I may not do this is if we are collecting a dog multiple times in a row, this is the only animal that we're going to use, and I've already done a full morphology on him 48 hours apart. I may not do it a third time, but that being said, if I'm not looking, I don't know. And so we should always be commenting on the color and the clarity. We should also be looking at total motility, as well as progressive motility, if it's spinning in circles, it's technically moving, but it can't get to where it needs to go if it's only going in circles. Michael Phelps would not be the swimmer that he is today if he could only swim to the left. Morphology is how the actual sperm cell is made up. There are definitely morphologic abnormalities and sperm cells that we know negatively impact fertility, and those are the ones that we definitely want to pay attention to. That being said, if I had a dime for every time I said this to an owner: Just because I called it abnormal doesn't necessarily mean it's negatively going to impact its fertility, and we will typically break those down. And so regardless, we like to see the morphology number be extremely high. That being said, I have my own stud dog who usually runs about 40% morphologically normal, and he has litters of 10 to 13 puppies. So again, just because it's not pristine, I can't call it normal, but it may not negatively impact their fertilization capacity. Concentration is something that really is very, very important and how that concentration is determined is also important. So volume does not always equal high sperm numbers. So you can collect 15 mils of a semen collection—that does not necessarily mean that that 15 mils is going to have more sperm cells in it than the one or one and a half mils that I collected from this other dog. And certainly, if it looks “cloudy,” that doesn't necessarily mean that those are sperm cells. Those could be inflammatory cells. So again, concentration is really, really important, and how you're determining that concentration is important. And there's some other ancillary tests that you can do, especially if we're having some fertility issues. They're not very common in general practice. But that being said, things like a swum up test or a host test certainly can be things that we can do without any problems. And again, that's going to be done in more of a specialty clinic, and most of the time, we're reaching for those if we have an animal that is struggling a little bit, and we're trying to figure out how we can help them achieve the pregnancies that they really, really want. 

So again, here's the slide to kind of hit home that color and that clarity. So in a dog, they fractionate their semen when they actually ejaculate, and so the first fraction, which is this one here that looks slightly yellow tinged, because he did urinate a little bit in it, but you can still see the background. So you should be able to hold these up, and you should be able to read through them if they're normal. Sperm rich fraction is this white, opaque white fraction here. So that's fraction number two. And then fraction number three should be purely prostatic fluid. And again, I should be able to hold this up, put a piece of print behind it, and I should be able to read through it. If it is not that color, then we should be concerned. These two here are blood contaminated. So we certainly have blood in here because it has that red tinge. This is also blood contaminated, but maybe not as much as this one here. When we receive collections like this, we get concerned. While gross blood contamination, why it stays under a 20% amount doesn't have a negative impact on fresh semen, it will have an impact on shipped chilled semen, and we cannot freeze this. So if, for some reason, that you are around and somebody did a collection on your dog, and they went to freeze it, and you saw that there was blood contamination in it, do not let them freeze it, because when you thaw that semen, the rupture of the red blood cells actually is toxic to the sperm cells. If you get this color, which is kind of more of a muddy pond color, this is a dog that has severe prostatitis, and so again, we should be looking for this nice, clear prosthetic fluid. And if we're getting any of these three colors here, then we should definitely have some pause. The second thing that we talk about is motility. And so all animals should be evaluated for motility. There's gross, where we're looking at it under a microscope, and we're looking at not only are they swimming, but are they swimming in a progressive manner? So are they just spinning in circles? Are they actually going where they need to go? We're also looking at hyper motility. So are these cells, I try to explain it to my students, are they out on a Sunday drive, or are they getting it? Like, do they actually have somewhere to go and they're not going to stop to ask for directions? And so that makes a big difference. One way to take the subjective motility out of the hands is to use this system that's called a CASA system. And so the CASA system is great. It's fantastic for studies. It's fantastic for doing an objective measure of both total motility and progressive motility. It also gives us all these other parameters that we can use in scientific studies. That being said, in a trained operator, okay, a trained operator, between gross evaluation and CASA are usually within 10% of each other as far as total and progressive motility goes. So again, just because you don't have this very expensive piece of equipment doesn't mean that you cannot evaluate motility. But again, if we get a sample, and all of them are technically moving, but they're just swirling around in circles, that's not going to be able to deliver the DNA material to affect a puppy. So motility is very important.

10:56

Morphologic defects—this is something that you have to be trained at, and this is something that takes months, if not years, to perfect. Most of our residents, when they come in fresh to us, unless they've done a lot of morphologic evaluations prior to, it usually takes them about six months to really kind of get somewhat close to what we as senior clinicians are calling. And all of the sperm cells represented here are abnormal, and all of them have fairly subtle abnormalities in them. And so we're going to go through. This one has got what's called the swollen mid piece here, and then you can kind of see this halo that is here. That's a detached acrosome. That is going to render the sperm cell not able to actually fertilize an egg; because that acrosome is already detached, the sperm cell is going to die before it reaches the egg. In this second one in the middle, this is a reflex mid piece, so you can actually see how the tail is flipped back up on itself. And obviously this one is going to be swimming very erratically, and not in a straight line. If we move over to this third top one, we've got a detached head up top, okay, and below it, we have a proximal droplet. This is also known to have a negative impact on fertility, because that proximal droplet in that space right there prevents the sperm from binding to the oocyte. This tightly coiled tail on the bottom left is also going to negatively impact motility. This is something that we see very commonly with heat stress. So animals that are in a southern hemisphere and are working very hard, certainly this can happen. Animals that are coated breeds, that are going to dog shows and being blow dry quite often, are going to have a negative impact. So again, there's a lot of things that we can see there. 

Here's another reflex mid piece. This thing's just going to spin in circles. 

Here we've got a distal droplet. This is not normal. Okay, so this is a proximal droplet, as well as this one, too. But proximal droplet, this is a distal droplet. Distally, the droplet is not a problem. It's not going to negatively impact but it's not normal. And so a lot of times when we start having fertility issues, we will see dogs that will have distal droplets and that will soon turn into proximal droplets. So again, making sure that we're paying attention. And then lastly, this is a proximal droplet, and what we call a microcephalic head. This head is just really, really small. And so morphologic defects based on morphology: if they are really good or they're really bad, that's where morphology really shines. The ones that fall in the middle, again—let's say your dog is between 40 and 60%, that does not mean that they are not going to be able to affect a litter. It just means that they don't have 100% normal sperm cells. It's the type of morphologic abnormality that’s going to negatively impact, potentially, your ability to get a litter at the very end. Now, concentration, again—super important. I want to know what I am sending to that female owner, so that they know what they are getting, hopefully before it arrives. And the bottom line is: more is always better. This holds true for everything. Unless there are a lot of dead cells. Am I putting up a lot of dead sperm cells into a bitch's uterus? Then we may be causing some inflammation there. So again, the rule most of the time, if it's more, is always better. That applies to live sperm cells. We need to maximize the doses of total normal motile. We do not have dogs that should be split between two bitches at the same time, and so this is very true of older individuals. This is very true of small breed dogs. They just don't produce the sperm cell numbers because their testes are smaller than, say, horses and bulls, to be able to really adequately split doses. But that being said, we should be doing a concentration evaluation no matter what. This tells us how many numbers that we have and how many total normal motile we have. Again, the end that is doing the insemination, knows what they're putting in most of the time. This is based on the size of the animal. So toy and small breed, we say 100 to 150 million total normal motile per dose. In medium to large breed, it's 200 million total normal motile. And then we have the ability at our facility to be able to use a machine that will give us membrane integrity. And so it kind of gives us the viability. If the membrane is not intact, those cells are probably going to die before they reach their destination. And so that's something that's a nice added value to have when you're doing an evaluation, but is not 100% necessary if you're doing that. 

So when we're kind of talking about these dose calculations in the spermiogram, this number here, when we talked about, like dosages, if we're calling doses and we're putting them in, started our conversation about doing studies. We were just like, “There's no real standard.” Some people you talk to, it's 100 million for any size dog. Other people you talk to, you are going to be saying 150 million. So we wanted to try to standardize, if at all possible, and so we're going to kind of walk through some of the stuff that has been done over the years, because we know what's normal for a dog based on kilogram weight, what they can produce. Any male should be able to produce at least 300 million sperm cells in an ejaculate. Beyond that, usually it's about 10 to 20 million per kilogram. That being said, I'm always going to go back to more is always better. I don't want just 100 million sent to me. I want the whole collection sent to me, and then that way we're accounting for any loss that happens, potentially in transit, and that includes transit through the uterus as well. 

So current studies that existed before we kind of launched into our study: there are studies about seminal plasma, how it has a positive or a negative effect, and how much we should leave in the sample, if any at all. Centrifugation—certainly, taking a biologic and putting it in a centrifuge and exposing it to G forces is going to cause some damage. And so we had some studies out there about centrifugation, but all of this was based off of stallions. Concentration—again, same thing. What is the standard dose? Is there a standardized dose? What is best? And so we'll talk about that a little bit. And then, how long does the semen last? And really, how should we use it to the best of our ability that we can? 

So again, I'm just going to kind of like go through some of these studies. Here's the one on cryo preservation and seminal plasma. And they were actually looking at the chromatin structure, you know, in this nice review paper by Dr. Colster, looking at canine sperm and the management of semen disorders. There was a nice review of what literature was out there. Again, Dr. Randall did the semen evaluation protocols and pitfalls for clinical therio and presented that at our conference. And then that led to this very first study that we're going to talk about, that we did here at the V-MCBM. And so what we really wanted to find out is what's the optimal range of centrifugation when we're processing? So let's say that you are going to collect a dog for insemination, either on site or at a distant site. The uterus of any size dog, for the most part, can really only handle between two and three mils at a time. And so we cannot expect to put in 15 mils of semen, or even eight mils of semen, and not expect to get some of that backflow. We can't have them have their back end elevated for 15 or 20 minutes at a time and expect to still have really good rates. And so what we're trying to figure out is: how can we maximize our recovery without damaging the sperm cells? And so that was kind of the thought process behind this. And what we did was we would collect a sample, we would split it into six equal volume aliquots, and we spun it at 400, 720, and 900 Gs for either five or 10 minutes, and then we actually evaluated the sperm over time. And so what we found: this is the change in viability, so that membrane integrity that we were talking about. We've got these here are the five minute spins. These are the 10 Minute spins. And we've got 400, 720, 900—it's going to be the same for the next several and what we found is there's kind of a generalized trend downwards over 24 hours. That being said, at 48 hours, we have a downward trend as well, in the viability.

This is progressive motility. The diamonds mean that there's significant difference from one point A to point B, but if you're looking at when we're getting out to 48 hours, we're seeing a lot of drop in progressive motility, and that means that's going to negatively impact where it's going. And so certainly something to watch really, really carefully. When we look at morphology, we knew that we caused damage, but what we're seeing is we're actually seeing a lot more acrosomal damage. And if we damage the acrosome and we make it worse, then we know that those sperm cells are going to have reduced fertilizing capacity. So again, look at time A, time B, and time C, and so we see a drastic drop, and we need to be really, really careful about what we're doing. So where do we go from here? We said, “Okay, well, we think we have a timeframe that we think that we can spin semen without causing a lot of damage and being able to concentrate what we need, get off of seminal plasma that may have a negative impact on it, and have it arrive in fairly good shape at the other end. 

So that led us into our next study, which was: Okay, that's great, but what concentration do we need to extend that semen so that again, it arrives in good shape? Nobody to date had looked at actual concentration and what we thought was horses. We were extrapolating from horses. When they ship horse semen, they like to see it at 50 million to 100 million per mil. That's where horses like to stay. So we were like, “Okay, we will try to see if we can find an optimum concentration that will optimize, again, that arrival.” So what we did was our control group was a one to three volume semen to extender. That being said, we are very careful to only collect just the sperm rich fraction, or the first and second fraction. We do not collect any of the third fraction that contributes a large amount of the volume. So we were starting out with small volumes. Then what we did was we diluted. We took our aliquot for control, then we diluted at 25 million per mil, 50 million per mil, 100 million per mil, and 200 million per mil. Those were our study groups all the way through. And so if you look here, concentration is this lighter blue at the top? Okay, so this was after we centrifuged it and prepared it. This is at 24 hours, and this is at 48 hours, so time three is 48, and you can see concentration 25 did not fare super well. It did not like being that dilute. 50 did okay, and 100 did okay. But by and far, our dogs really like to be concentrated. And so this one to three dilution was the best. But if we have to concentrate it, really putting in a 200 million per mil was winning at least as far as viability went. 

Now, progressive motility—we were seeing the exact same trends. So again, these are all the same, and you can see where our groups were. But we really saw these top two lines being the blue ones, which is the control, okay, where we just diluted it one to three, and this other one, where we made it 200 million per mil. And so again, we're seeing an outward trend. So what does that mean for the stallion side of things? Full spermiogram after a physical examination is something that has to be done every single time. So if you were going somewhere to have the animal collected, and you're paying for that, you should be getting a full semen analysis. This should be giving you a concentration with actual numbers. You should be getting a motility with actual numbers. You should be getting a morphology with actual percentages as well as the abnormalities that were being seen. What we landed on was centrifugation at, you know, 700-750 for five or 10 minutes. There's really not that big of a difference. The range—there wasn't a statistically significant difference in that first study that we had. And so this is just kind of what we landed on. 

The seminal plasma—it does do better if you can reduce that prior to shipping it out. And that's going to require centrifugation. And then the final dilution concentration going is 100 to 200 million per mil, if you can get to that, or one to three, volume to volume rescue. The one to three has to be if you're only collecting the first and second fraction, or just the second fraction, only, because if you do a one to three and you collected 10 mils of semen, you're sending so much semen that we're going to have to process it at the back end to be able to put it in the female. And that actually causes more damage than if we just processed it at the front end. So again, the good thing about the male is that we can make adjustments real time. If we see that there's something going on, then we can try to adjust it right then. If we are doing multiple shipments in the same week to the same bitch, we can find out how he arrived in one extender, and if he didn't arrive super great, I can pivot very quickly and switch extenders and see if he arrives better in a different extender. The bottom line is, is, what we also figured out in both of these studies is that semen needs to be put in the bench within 24 hours of collection. If you leave it in the box, all of the semen is negatively impacted, and it's almost exponential. And so the only time that we would potentially put something in that had been in a box longer than 24 hours, is if for some reason we didn't get it in time and it was still alive and doing okay 48-72 hours later. But we also tell the owners that that is probably going to have a very negative impact on the semen quality. So don't be surprised if we end up with a reduced pregnancy rate or reduced litter size. So again, this is really, important, and take into account when we're doing this final dilution concentration, if somebody is planning on doing a TCI at the other end, that we don't want any more than three mils being sent to us for a TCI, because I don't want to have to process it when it arrives at my clinic, because I'm going to cause more damage after the sperm cells have been chilled by centrifuging it and then getting it down to a smaller volume. It's better if you process it at the front end, put it in the extender at the appropriate concentration, and then it arrives with us, and we just put it directly in the female.

So we talked about the first half, which is the easy half to keep track of, because they're easy for us to be able to get samples from. The other half is the females. And so it would go without saying that we need to make sure that we realize maximum fecundity, which is their fertility, is going to be between two and five years of age. And so certainly we are going to see reduced litter sizes and pregnancy rates if they're over five years of age or they're under two years of age. And some of this is going to play with genetics, but most of the time, we tell people really, you should be breeding your bitches during this timeframe, because that's when they're most fecund. Health testing, very important, trying to test for those things that we know potentially can be genetic, or we know are genetic, so that we're breeding appropriately. And they need to be in good physical shape. Certainly obesity in humans as well as other species out there who are working on the study right now in bitches—it negatively impacts fertility. And so I'm going to pick on the Labradors a little bit here. You know, the way that they want them in the show ring is obese. Most of those dogs have to come back and lose 15 to 30 pounds before they're ready to be bred. And certainly it can make a big difference, not just with their fertility, but how they carry that pregnancy in the very end, and the dystocias and whelpings.

Core vaccines are not only important for the health of the bitch, but it also boosts what IGG they're going to pass to the puppies through their placenta and through their colostrum. And so rabies is the only titer that we actually have, hard and fast. We know if that's a protective titer for the bitch, but none of the others, we have no idea. So not distemper, not Parvo. So why titers are great for the individual dog—it's not telling us what she's going to be able to pass on to the puppy. So what we try to have a conversation with our owners is, is that if we're breeding them, we need to make sure that they're updated with their core vaccine so that we can actually boost that immunity, so that those puppies are receiving adequate immunity. 

And supplements, flea and tick, heartworm preventatives—which of those things are safe, which of them are not safe? When in doubt, don't use it. And so certainly we have those conversations on a fairly regular basis with our clients. But when in doubt, just commercial dog food. You can certainly do fish oil. You could do Dasaquin or Glucosamine supplements. But be very careful about any pharmaceuticals, especially feed-through pharmaceuticals that you're putting in their mouth. 

And then lastly, timing is everything. A lot of the animals that we get that are having “fertility issues” or having issues with what's going on, the problem is, is that we get them in here, we do proper timing, and they get pregnant. So timing is everything. So that leads us into: what do we use? People are talking about vaginal cytologies. People are talking about progesterone levels. People are talking about LHs. There are things that are all over the place. The modern gold standard is progesterone at the moment, and this is for multiple reasons. Vaginal cytology only tells us two things. Are they under the influence of estrogen, or are they not? Do we have inflammation or not? It doesn't tell us about where they are in their cycle. It doesn't tell us about ovulation. It does have its utility clinically, but it doesn't really tell us much. LH, on the opposite hand, is a very labile substrate. We do have those kits, which are semi quantitative, ELISA kits, but there has been a recent study in the last five years where they actually attenuated. So the dogs did not have an LH peak, and they went ahead and ovulated anyway. So whether or not we actually use that as our gold standard is coming into question. Those of us that do a lot of breeding usually just use progesterone. And so again, it's accurate timing, and kind of gives us that idea of most fertile days. It also gives us an accurate due date on the back end.

All machines are not created equal. I'm just going to leave that there. We got to be really careful about these in house machines. You're going to pay a lot of money for them, and if you're doing a frozen semen breeding, it may not be the most accurate. It depends on what machine you're using, or if you're timing on your home machine, but then you end up doing the very last part of it at another place, technology bounces are going to give you different numbers, and you cannot treat them equally. Quantitative Methods are absolutely preferred, so they actually physically give you a number, not a color change. RAA is hard to find these days. It's the gold standard. We use that when we're doing scientific studies, but chemiluminescence is the gold standard that we use in the day to day practice, and I think we've validated it enough for this that really it's replaced RIA to be for sure. 

So here's a nice diagram to remind you guys of the fact that bitches like to be different. And why would they give us oocytes that could be fertilized right away? That makes life both a little easier and a little harder. And it's a little easier in the fact that we don't have to panic, but it's also a little harder because, well, how do we know when that oocyte is mature and can be fertilized? And so there are a couple of big things on here that we need to talk about. 

Primary oocytes are the ones that cannot be fertilized, and that's what's released from the very beginning of ovulation. Secondary oocytes are the ones that have gone through their two meiotic divisions and can be fertilized. And so those are the ones that we want. Also realize that ovulation happens in a bell shaped curve. It's not an all or none principle, and so we have these bell shapes of what is happening. Total lifespan of fresh or ship chilled semen can be five to seven days, so at least they give that to us. We've got more timeframe than we do, say, in bulls, small ruminants and horses, to be able to cover a larger period of these nice bell shaped curves that we have. And so what do we do? Most of the time, we're waiting for those secondary oocytes to be ready. We're talking about fresh and this is also side by side, in or chilled semen, because what we're doing is we're maximizing the numbers that we're putting up there over that oocyte maturation and fertilization period. Where it gets real sticky is frozen semen, because frozen semen really only lasts maybe 12 hours. And so this is where this gets really, really important, that we are making sure that we are kind of in the very peak of that upper bell shaped curve when we put frozen semen in. But look here, I don't have fresh semen and chilled semen all the way over here. Don't wait until last minute, because if you have to adjust based on poor semen quality, you waited until the very last minute, you don't have any way of being able to adjust if you have to, real time.

So how do we simplify this? Most of the time we do progesterone testing around five to seven days of the cycle. Cytology, as needed, again, it only tells us two things, estrogen or not, or inflammation or not. Vaginoscopy, obviously, that will help us out a little bit, but that's directly linked to the estrogen levels as well. 

Breeding based on your scenario. Best pregnancy rates are still going to be natural. We do every other day as long as she's standing. You're going to maximize your litter size numbers up in the female, and that entire bell shaped curve.

Ship chilled. Please, please, please err on the early and not the late. Again, most semen will last five to seven days, if not longer put in the female. It is up there. It is waiting for the first oocytes to come down. And there will be numbers, if you continue to breed them, there will be numbers to get that entire bell shaped curve.

Frozen is where it gets real sticky, and so progesterone levels, most of the time we're using ovulation dates. It's most fertile day is going to be three to four post-ovulation. LH, peak. If we're using that off of a progesterone, we're going to be doing it at five to seven days. But realize: semen only lasts 12 to 24 hours, if you're lucky. So again, we have to be very careful about our timing with frozen. We have to be very careful about what motileity we're using for frozen and making sure that we're maximizing that. And then lastly, remember what days UPS, FedEx, DHL can ship or receive. So I can technically collect a dog on Saturday, but it's not going to arrive until Monday, and it's already sat in that box for 48 hours. That's not ideal. But I can get semen collected on Friday and sent to you on a Saturday, and then we can reinforce with a Monday collection that's inseminated on Tuesday. So again, keep that in the back of your mind.

One of the things that I wanted to talk about, and then we're going to roll into the studies that they have here in a couple of slides—I need to remind you guys. And the reason that I put all of these pictures up here is that the dog cervix sits underneath their lumbar vertebrae, and the opening to that cervix sits ventrally. And so when we're talking about AI types, we need to talk about the size of the pipettes that we're using. So down here on the bottom, this is our TCI scope. And the reason it is so long is because we have to traverse that entire vagina to be able to get up to where her cervix is. Now in a toy poodle, we may be only up to this area, but in an Irish Wolfhound, I might be all the way up here. So again, sides does matter when it comes to the AI pipette. These are all pipettes and/or catheters that we use to do things up near the cervix. So realize this is not just a five inch pipette that we're using. We're actually using a very, very long pipette. We've got three different flavors of artificial insemination. We have vaginal AI. It's appropriate for fresh or shift till, no big deal. And we're putting that semen as close to the cervix in the cranial vagina. Super easy. Has fairly good success rates. Transcervical AI is when we're actually using an endoscope. And I have a video here of us to put semen directly into the uterus. We actually catheterize the cervix, and we're placing the semen directly into the uterus. This is the gold standard for frozen semen nowadays. A lot of people choose to do TCIs, all things considered. TCI versus vaginal AI depends on the paper you read. They're almost equivalent in pregnancy rates. Surgical AI is something that has been used in the past, before we got transcervical; it is a laparotomy. We're going into the abdomen of the animal. We're finding the uterus. We're injecting the semen directly into the uterus. Nowadays, most of the surgical AIs are only reserved for cases of infertility, when we actually want to get our hands on the uterus and to maybe take other samples. It's invasive and it's not necessary, as TCI gives us the same or lower rates compared to surgical AI. Surgical just causes a lot of inflammation outside of what the uterus is already going through. So here we have a picture of us doing a TCI, and you can kind of see here, I'm going to traverse up this vagina at the very top part here. This is the dorsal vaginal fold, and you'll see us get to the cervix, but the cervix is hanging down here. And what you'll see is we'll go directly up to the cervix. It gets cobblestone-like, okay, everyone's a little bit different. We'll look for the cervical os, which is right here. You can see how ventral it is, and then I'm going to take that catheter, I'm going to pass it directly into her uterus, and then we're going to inseminate. I'm going to pause this here, because if you missed it at the very beginning, I want you guys to see what we see on transcervical. This dog has a very large, what we call dorsal ventral band. This is going to preclude this dog from probably whelping naturally or breeding naturally, because this band of tissue here is splitting her vagina in two. And if you watch the video, it's a fairly large band. It keeps going. It keeps going, and then it ends right there. So again, transcervical does give us the ability to be able to be able to look up there and see if there are any problems or things that might be surprises when we go to whelp naturally. So where is the evidence? That's what you guys have been waiting for. And rather than going through, you know, all of the literature that's out there, I kind of just made a chart, and we're going to talk about these papers. So one of the very first papers that we had about fresh AI, natural and frozen AI was from 1975. It was Seguir et al. And what they found was, with fresh semen, they have a 65% whelping rate, but with natural, they had a 78% whelping rate. So again, that kind of shows that, you know natural, you know, we're pretty good at our job, but I don't care if you have a good stud dog or not, he's going to put more numbers naturally up in a female than if you were to collect them and then put it in the female. 

So again, one of the big things they were looking at in this paper was can frozen semen be inseminated vaginally? While they did get pregnancies, it is not recommended to do so because of the short life span and what we put those sperm cells through during frozen. We prefer putting it directly into the uterus. I'm going to jump down to this 2014 paper here. This is Mason et al. And so they were looking at TCI versus surgical AI with frozen only, and it was over 118 inseminations. So again, here surgical AI, they only got a 45% whelping rate; in TCI, they got a 65% whelping rate. And so certainly, one of the things that they took away from this was you had to at least have greater than 100 million total normal sperm cells to affect a pregnancy. And they did not see any difference. And they actually saw or lowered success rate if they did two TCIs versus one. So certainly sometimes, especially with frozen semen, more is not better, because we are going to have dead sperm cells, we are causing inflammation. And so one well-timed TCI is going to be much preferred to a surgical or two TCIs. Okay, so then we're going to jump to Fiona Hollinshead and her group that did this huge study, and it's got, you know, I put lots of data on here. You should see the amount of data that's on here. They did fresh versus frozen, and the type, and it was over 1,203 breedings that they retroactively—well, they proactively studied them. But then evaluated them afterwards, and what they found was AI and TCI, pregnancy rates were equal, okay? And litter size was equal. And so again, it's kind of one of those things where we're finding that as our technology has gotten better, that we can overcome some barriers, but still well-timed, and making sure we're putting good quality semen up there kind of negates those. So one of the biggest takeaways from the study was progressive motility was predictive of their whelping rate, and so making sure that we have good motile sperm, and so I'm going to give you some of their statistics as far as the motility, if the motility is less than 30% they were getting a 37% whelping rate. If their motility was between 30 and 65% progressively motile, it was a 67% whelping rate. And then if it was greater than 65% we jumped to 79% whelping rate. So again, the more numbers we have, the more motile semen that we have, the better off that you're going to be. And so again, that was really, really important. And then in 2023 there was another one that was done at 267 inseminations. They used fresh, chilled and frozen TCI. And so fresh whelping rates were 83%, chilled were at 67%, and frozen were 66%. They had 6.8 puppies per litter with fresh, about a puppy less—you know, we can't really have like 0.25 of a puppy—so that being said, about a puppy less for your ship chilled, and another puppy less for frozen. And so the takeaway from this is they did see a 15% increase rate in pregnancy rates with two TCIs was chilled, as long as they were using a minimum of the 100 billion progressively motile cells. So I'm not talking frozen semen here. I'm talking ship chilled, they saw an increase of 15% with two ship chilled shipments. So again, putting more numbers up there over a more broad fertilization window is certainly going to make a big difference. So where does this leave us? You're like, “Oh my gosh, you just threw a bunch of numbers at us.” TCI, AI, surgical AI. Natural is always going to be best. Next is going to be fresh or ship chilled. So that's either going to be a side by side or it's going to be ship chilled. The bottom line is: tell your owners, put it in your contracts, at least two well-timed inseminations. Sometimes that doesn't always work out. I understand that, but more is always better. We have the data to support that. Make sure that you're doing one on the front end, and then they're reinforcing a little bit later. Frozen: do one well-timed TCI. We are seeing decreased rates, probably because we're putting more dead up there, just because of the inflammation that we cause. So again, frozen, it's a single well-timed TCI, but natural, you know, you're the same zip code. Let them go to town. Fresh or chilled, two inseminations are really going to be best. Remember, we want them to have a motility greater than hopefully 50% and we want at least 100 million total progressive motile sperm cells to be able to do their job. The other thing is, is semen matters. Okay? It's 50% of the equation. Everybody wants to blame the girl, or everybody wants to blame the boy. It takes both. So progressively motile, the better. 70%—you're doing great. That is awesome. If you're receiving 70% motile semen, the more the better. Greater than 100 million total normal motile—awesome. For me, I want 200 million. And I want 200 million times two or three inseminations if I can get it. And then lastly, veterinary involvement allows for real time adjustments prior to the insemination. So I know there are a lot of people out there that collect their own semen, but if they don't have access to be able to do all of these parameters, then we don't know until we have a missed pregnancy, or we're getting lower litter sizes that something's going wrong. And so making sure that we're having fairly regular spermiograms performed on these animals is going to make a big difference in using your stud dog for the longevity of his breeding career. And with that, I'll be happy to take any questions.

Nicole Engelman  44:14

Amazing. Thank you so much. Dr. Cecere. We have quite a few questions, so I will do my best to either consolidate them, or just make sure they all somewhat get asked. We have one question someone's asking: Can someone at home, using a microscope, get a sperm count, and would it be accurate if so?

Dr. Julie T. Cecere  44:32

So there are ways, if you've been trained, to use what's called a hemocytometer, but you would have to have access to formalin and you would have to have access to pipettors to be able to dilute it very, very specifically. So concentration in a home environment without a laboratory is going to be really, really difficult, and something to be considered if you're collecting your own dogs.

Nicole Engelman  44:54

Amazing, and if you're doing side by side AI, how much of the fractions are recommended to collect?

Dr. Julie T. Cecere  45:01

So again, we really only want the sperm rich fraction, and that's the second fraction. That being said, we routinely will collect the first and second fraction here, but once they stop giving any white fluid, then you should stop. The more of that prostatic fraction that you collect, the potential negative impact that can happen unless you need a pushable volume. But we cannot put 10 mils of semen, even vaginally. A lot of that is going to come out. And so again, I call it a pushable volume. Most people are going to want anywhere from two to four mils for a pushable volume, so that you can actually get it in there without having any waste in the pipette, but anything beyond that is too much.

Nicole Engelman  45:46

Great. Thank you for answering that one. Someone was curious about nomograph studies being done at the University of Wisconsin. I'm not sure if you're familiar with Dr. Schultz's program. They were just curious if you could talk a little bit about that.

Dr. Julie T. Cecere  45:58

Yeah. And that could be a whole different talk. The thing is, is what they're measuring is maternally derived antibodies, and that's why I'm saying boosting moms is going to boost what “titers” they're going to have. My problem with titers is, is we don't have studies to say, “If you have this titer and I infect you with parvo virus, I know that you're going to be protected and you're not going to succumb to the disease, or you're not going to have severe disease,” if that makes sense. So even doing titers on the puppies, you're measuring what mom passed on; there's lots of variability in each individual puppy in the same litter. And so again, we kind of take it as a herd health, and we say, if we can boost mom's immunity, then we know we're going to be boosting, hopefully, the quality that they're getting in the colostrum and through the placenta.

Nicole Engelman  46:48

Awesome. This question is maybe a bit more existential, but someone who is wondering if two dogs can just be, frankly, incompatible, and if there is just nothing you can do.

Dr. Julie T. Cecere  46:59

Frankly incompatible . . . if they can expand on what they mean by incompatible?

Nicole Engelman  47:04

So I think this person did TCI twice with shipped semen, and they just really did not get the results that they wanted. And they even said that they astounded their repro vet, and she suggested that they might just be incompatible. So I was curious if that could be the case, or if there are just more steps that you would need to take to actually make that determination?

Dr. Julie T. Cecere  47:29

I feel like that would be something that I would say I would have to do a telehealth, to get all the information, because that seems like there could be a lot of different things that could have gone wrong. Was it the semen quality? Was it the timing? You know, usually there's not an incompatibility genetically, where we're not going to get what we want, if that makes sense. But yeah, I think that's a little bit more loaded than I would be able to answer right now.

Nicole Engelman  47:53

I did say it was existential, so . . . Well, we’ll end things, I think, with a very straightforward question. But I really like this one. What guidelines do you recommend for locating and evaluating reputable AI semen databases, particularly in breeds with limited availability and ensuring confidence in semen quality and shipping viability?

Dr. Julie T. Cecere  48:14

Oh, that is a very, very loaded question. There is no reason why a general practitioner would not be able to offer these services. There's also no reason why—I know if you go to dog shows, we've got several services that are offered for them to be able to evaluate. I think you need to be aware, as the owner, of what evaluation parameters you are given. And if you are asking them, “How are you doing the evaluation? Motility?” Sometimes you'll get a “good.” Well, “good” doesn't mean much. Like, is that good to you? Is that good to me? Is that good to someone else? And so again, I think being an informed owner and asking for as much objective data as you possibly can is going to give you a better idea of what you actually are receiving as a product, if that makes sense. Hopefully that answered the question.

Nicole Engelman  49:02

Yeah. I think that was great. I think that was actually a perfect note to kind of conclude the Q&A with. But thank you so much, Dr. Cecere, for doing this and for being here. We love having you, and we hope we'll have you back here again soon. And thank you all for joining us today. If you're not yet a member of our community, we would love to have you join us at GoodDog.com/join so you can stay up to date on all of our future webinars and events. And I think that's it for us. Thank you again for being here. Thank you again, Dr. Cecere, and we will see you at our next webinar. Bye, everyone. Thank you.