Episode 166: Shipping Semen for a Podium Performance

Dr. Julie T. Cecere, DVM, MS, DACT, teaches us about the latest research and innovations in canine reproduction techniques to maximize successful breedings.

This is an audio version of a webinar we recently held with Purina. To see the video version, click 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 Dogs mission to build a better world for our dogs and the people who love them. Hi everyone. I'm Nicole. I'm Good Dog’s community lead. Thank you again for joining us for another presentation with Purina. Today, we're going to be presenting on shipping semen for a podium performance with our guest, Dr Julie Cecere, advanced reproductive techniques for dogs have become commonplace over the last decade. Most of the procedures being performed are based on what is known about semen processing and shipping in the equine industry, which is really interesting. Dr Cecere will be discussing a review of the current literature of what is known about canine semen processing, along with highlights of new studies that will improve shift semen quality at the time of insemination will also cover standardized techniques to maximize its successful use in a breeding so those are some of the things we're going to be going over today. Just to give you a little preview, we're really excited, of course, to partner with Purina again to bring you another good breeder webinar. This is something we have not been able to cover yet in the past. So we are so excited to bring it to you. And please, again, I always say this, continue letting us know what you want to see more of in terms of canine health content, because it is so helpful for us to know what to bring you next. And as always, during the Q and A segment of this presentation, we have a bunch of previously submitted questions that we're going to prioritize, but we will try our best to get to anything from the chat as well. So feel free to ask questions throughout the entire presentation, and we will do our best to make sure it comes up in the Q and A section a little later on. And just want to share a little bit more about Good Dog, in case anyone is new here and joining us for the first time, 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, we are a secure online community created just for dog breeders. We use the power of technology to help good breeders run every aspect of their breeding program from start to finish. So posting your available litters, connecting with buyers from across the country and ultimately finding great homes for your puppies, which is what it's all about. And we offer additional breeder business resources, like webinars about taxes. I know tax season has just ended, but I feel like maybe it's time to start prepping for the next one. Already, search engine optimization and not yet a member of our community, we invite you to learn more at gooddog.com/join so you can visit that whenever and start your application to join our community. We would absolutely love to have you if you're not yet a member. So with all that out of the way, I just want to share a little bit more about our amazing speaker today, and then pass things over to her to get started. Julie Cecere completed her veterinary education at the Virginia, Maryland College of Veterinary Medicine. She worked in private veterinary practice and commercial settings before returning for residency and animal reproduction. Dr Cecere is a diplomat of the American College of Theriogenology and organization to which she has contributed in several leadership and service roles. She is a clinical associate professor of theory and genealogy at Virginia, Maryland College of Veterinary Medicine, where she has developed an internationally recognized clinical service for canine reproduction. So I'm very excited to have her here today. I don't think anyone is more qualified to be speaking to this topic. She lives in Virginia with her family on a small hobby farm and enjoys upland dog hunting with her children and riding horses in her spare time. So Dr Cecere, I'll pass things over to you to get the presentation started.

Dr. Julie T. Cecere  03:49

Thanks, Nicole. I really appreciate that my introduction makes it sound like I'm this person that's done a lot of things. I think I'm just somebody who has a passion for connecting breeders to owners, so that everybody has a fruitful life with their dog. And so certainly, I've enjoyed many dogs in my lifetime. I grew up with Miniature Schnauzers because that's what my parents had, and then have moved into more performance type things. And never in a million years would I have ever thought that I would end up with a Weimaraner, just not something that was on my radar. I met a wonderful breeder through my practice here at the vet school, and because of her, and because of what she was doing and the things that she had going in her breeding program, I actually ended up with one of her puppies, and it has opened up my world broadly to a lot of really great persons. And so it's amazing how those types of interactions, outside of what is in your day to day. Working really has opened my life up to a whole different kind of culture and made my life a lot more round. And I really do enjoy working with our performance people. So that's been really, really wonderful. 

Dr. Julie T. Cecere  04:54

So today, one of the things that I think we spend a lot of time talking to our owners about in clinical practice, and even it comes up, you know, when we're sitting around after a field trial and to kind of just chatting about what's the latest thing to do, Andrology, as far as what we know on the male side of fertility and dogs is very far behind all of our others. And so we're really trying to play catch up as fast as we can. And so that's really what today's talk is going to kind of cover, it's what we know where we're going, and how potentially, maybe you guys can help us out, as far as researchers trying to advance what we do know and how to help everyone out. And so if we're ever going to go anywhere, we need to make sure that we know where we're going. And so we're going to kind of talk a little bit about, kind of the background of the advancement of artificial insemination and where we are currently. Why would we use chilled semen in that? What's the importance of a full semen evaluation? Then we're going to talk about the comparative research that's out there, what we know about in multiple species, and how that's just been kind of the assumption in dogs, but finding out now with current studies that that assumption is not good. And then how does that relate into owner collected and shipped semen? Because certainly, there are a lot of breeders that collect their own dogs and ship them, which is great, but we need to make sure that we're standardizing that so that we really maximize that fertility when it arrives at the other end. No biologic likes to be put on ice, but if we kind of tweak things to make sure that they arrive in that box in the best shape that they possibly can, considering they've been outside of the intended spot for longer than necessary, but we really want to make sure that we get it there in good shape. 

Dr. Julie T. Cecere  06:32

So that's kind of what we're going to talk about, and a few other things just about general health that we need to talk about, just related to the stud dog as well, because if he's not healthy, then he's not going to be producing healthy sperm cells to be able to breed in the long term. So certainly, the demand for artificial insemination has become quite high, certainly even in my tenure, just in reproduction, as we become more global in our exchange of genetic material, ship chilled semen results in higher pregnancy rates and larger litter sizes than does frozen semen in the majority of the time. And why, some people will say doing frozen semen is more convenient. Frozen semen is never more convenient, ever in any species. We'll just put that out there. It takes a lot more time to get it in a frozen state, thought from a frozen state, and prepare the female to be able to put it in that frozen, thawed state. And so again, ship chilled semen, for something that has to travel a long distance, it's going to be much preferred over frozen semen. While no theory genealogist, or I think most breeders, are going to argue that the best is going to have the male and the female within the same room, either with Natural Cover or side by side. That really is kind of going to get us our best pregnancy rates and butter sizes. There are a lot of logistics around shipped cool that, I think, is not something that we always think about. And so one of the things that we teach our students, our veterinary students, and we talk to our breeders about is one of the biggest things that you need to think about is what days can we collect and ship based on what shipping mechanism we're using, and so unless you're doing a lot of shipping, and you have what's called counter to counter status, most semen has to go overnight, and you were limited by which days that you can ship. So Monday through Friday, we can ship something out and get it there. Tuesday through Saturday, but we can't routinely ship anything on a Sunday. So we can't collect something on a Saturday, have it delivered on a Sunday, or collect something on Sunday, have it picked up and delivered on a Monday. So we're talking about logistics. That's one thing as a breeder that we kind of have to keep in the back of our minds. And hopefully the other half, which is the female side, is going to be aware of that as well. Because what we usually talk to our owners about as we're timing them is that we really need to make sure that if we look like a female is going to straddle a weekend, we're going to be playing with that. Oh my gosh, I can't get anything on Sunday and I can't get anything on Monday. Is it better to put semen in early? The answer is yes, and then we reinforce on the late, rather than playing the odds game on waiting too late, and if she goes super, super fast over the weekend, you may end up with nothing or a very small litter because you waited until the very last minute. But and so again, logistics for that shut cool when you can have it shipped is going to be really, really important. Compare pregnancy rates, again, equal rates if timed appropriately when we're talking about natural side by side and ship chilled, if all parameters are considered equal. So fertile female, fertile male. And we are following some of the guidelines that we're talking about. And really, quite honestly, most of the time, when we get referred things here at our practice for infertility, I kind of use that term loosely. Most of the time it's a timing issue. And so as a stud dog owner, specifically, the use of progesterone timing and accurate progesterone timing so that your stud dog doesn't get blamed for a poor timing problem is really, really important. And there are a lot of people that we work with actually put it in their contracts that the females must have progesterone timing performed to make sure that the semen goes in at the right time. So again, the male is not blamed for a mis-breeding when it probably was timing. Now the other thing that we need to talk about is standardization of breeding soundness exams. And this is something that we're working on, both as a college of Theriogenology, the Society for Theriogenology, which is our partner, general practice, type of organization we're really trying to come together to kind of standardize, if somebody says they're doing a breeding soundness exam on their dog, what does that constitute, and what parameters should they be doing that we can all make educated guesses and best practices for that individual dog, and certainly in this concerns for pregnancy outcomes, the female needs a follow up on their side as well. 

Dr. Julie T. Cecere  10:42

We can time the female we can put semen in them, but if they're waiting until the whelping date to see if she's pregnant or not, it's really difficult for us to say, hey, if we ultrasounded her at day 21 or 28 post ovulation and she got pregnant, your male did the job. He delivered the semen, he got her pregnant. But if she wasn't pregnant, at the end of her pregnancy, she lost the pregnancy. And so again, it's not the fertility of your stud dog that failed. It was the female that failed. And so again, making sure that we kind of have this overall arching view where the two halves kind of have to work together and hold up their end of the bargain to make this work for everyone involved. And so what we're going to do now is we're going to talk about kind of like pre breeding exam, things that we need to talk about for a general health of the individual, any dog to be able to produce normal sperm cells in a high enough number that is going to give us great outcomes, needs to be healthy. There's a reason that the testes are located outside of the body. They cannot work at high temperatures, not even at body temperatures. They're usually five to 10 degrees below what the body temperature is, and that's why they're contained outside of the body and not within the body. And so making sure that our dogs are up to date on their vaccine status, because if they get kennel cough, or they get parvo virus, or they get canine influenza virus, anything that's going to cause an inflammatory event within the body is going to impede the ability of those testicles to be able to produce normal sperm cells. And we don't think about that in day to day conversations, but it's absolutely true. If your dog is not feeling well for a week. We expect to see that in his sperm cells 60 days from now. It is pretty profound, especially in some of our hard working performance dogs, who we are looking at on a regular basis with their sperm evaluations, we can say, Well, what happened 60 days ago today, if we had a bad evaluation, and somebody will say, Oh, that was such and such nationals. Yeah, it was 80 degrees outside, and I was running my dog for four hours. And so again, those types of things are really, really important for us to pay attention to. 

Dr. Julie T. Cecere  12:49

Tick borne disease. If you're in a high tick borne area and or heartworm status. Again, anything that's going to cause inflammation or cause a systemic disease is going to affect their ability to produce normal sperm cells. Certainly, as breeders, we all are trying to produce better and so making sure that genetic testing is done as they come out, and how we interpret those tests, depending on what they're testing for. We're going to talk about that a little bit more, diet. A good, well balanced diet is very important. There are some studies out there. And again, we'll talk about this just a little bit that may help but not hurt, but there is not anything that's tried and true to definitively say, Yes, this is going to make their sperm quality better. So that's really, really important. You need to have them on a good diet that is balanced, but there isn't going to be a magic pill that they can take that's going to make their fertility that much better. If you're in an area that has a lot of tick borne disease, flea and tick it's going to be really, really important. Standard deworming medication on a regular basis that we know is safe for breeding animals. It's going to be extremely important. There is some concern out there on some drug classes, on whether or not, we routinely think that they're safe to put our breeding animals on. We certainly have some problems in our area with topicals working and have some resistance. But the feed through products we also sometimes have an issue with, as far as poor fertility quality, and so generally, what we try to tell our breeders is any isoxazoline drug class that is out there. Those feed through flea, tick and deworming medications. Be very careful about what you're giving your individual animal, because we certainly have seen some problems with those for the females more than the males. But sometimes people discuss this cytology and cultures. There's only one accessory effect claim that we have to worry about in dogs, and it's the prostate most of the time in our breeding animals, we know that it's there. It contributes to the fluid fraction of an ejaculate, and it doesn't tend to cause any problems. If it does cause problems, the first syndrome that we see is what's called benign prostatic hyperplasia, and that in itself. Health is something that is a normal process as they age, and they're under the influence of testosterone, if it goes a little bit out of control, what ends up happening is we get some blood contamination, then blood is a wonderful culture medium, and we go from just straightforward BPH right into prostatitis, because the bacterial component is evolved. That is the time at which cytology and cultures is going to be very important, because we need to make sure that we are confirming that they do have this infectious process and that accessory sex land, and that we need to make sure that we clear them prior to moving on with anything else beyond that, unless we are having some serious infertility problems that have been documented by veterinarians over weeks to months and or several misbreedings, and we have ruled the female side out of things. There are a few bacterial organisms that we know have been implicated in causing problems, but the issue is that those bacterial organisms are also normal commensals. They are expected to be in the reproductive tract, so just because you grow them doesn't necessarily mean that they're causing a problem. So we kind of have to be a little careful on how we use and interpret those things. And that's why that question mark is there because really, this should be something we can certainly culture everything under the sun, but if we don't have signs of inflammation or repeated infertility, that we've ruled out anything else that we interpret those very carefully. 

Dr. Julie T. Cecere  16:29

The Brucella Canis is not something that is only sexually transmitted, and so we need to make sure that we are testing any actively breeding fed dog every three to six months. If he's not breeding that often, then at least before he's being bred. And again, that is something that every stud dog should have current because his semen is going into all of the females, even if he's not going to touch her. And so again, every three to six months, the asterisk is there, because if they're not breeding that often, then before they're bred, and we should have a bench top test that was just validated by our group here. Paper is pending right now, but that should be available again for everybody to be able to use the day of, rather than having to do these send outs like we've been doing for the last probably year, year and a half. Okay, for those of you guys that have a breed club that works with the OFA. This is one really nice central location for veterinarians and breeders to be able to say, okay, based on our breed, these are the type of things that we need to have tested for on individual breeds. And so again, making sure that these animals that have been chosen for breeding stock have these types of tests, we know that they have a heritable component to them, and that to the detriment of the breed, that we're not glassing over these types of tests, we need to make sure that we are hoping not to propagate these types of features, or we're working within the means that we can, especially if it's a polygenic trait that we are trying to circumnavigate safely. How to preserve our genetics and our genetic diversity, but at the same time, we're not propagating any heritable disease that could be at the detriment of our breed. And so that kind of segues into my next slide, that's talking about genetic tests. And so when we're talking about genetic tests, we think there are very different levels of understanding of what these genetic tests are and how they're supposed to be used single gene traits. So something like PRA PRCD and multiple breeds where we actually have a single gene mutation that we have identified in various breeds that if they have that mutation, we know that that dog will develop that disease down the line, and they will become blind. The problem is that blindness doesn't show up until after this dog has been bred several times, because they're much older when this affects them. And so single gene traits are wonderful, because it's a very yes and no, it's kind of a black and white. And we know if it's 100% penetrance, we know that animals that have the affected gene mutation a double copy of that affected gene mutation, that those animals probably should not be bred. We have very few of those in our veterinary population. Most of what we have is a polygenic trait, okay? And polygenic traits are something that, yes, part of it is genetic, but part of it is environmental, part of it is dietary, and part of it is what combination of genes you have with that. And so with polygenic traits that we're a little bit more complicated, and having somebody who can work with you, as far as interpreting these genetic tests is going to be very helpful. 

Dr. Julie T. Cecere  19:40

As a breeder, we spend a lot of time sitting down and talking with our breeders over what tests they've had. What does it mean? Let's say they didn't get a great Pennhip score on this individual, and then they bring us three separate individuals that are potential pairs, which one would be the best to breed to. And so again, those types of things should be taken. And with an individual basis, with somebody who actually understands and taken with a grain of salt on not every genetic test is black and white. If you throw any dog that's affected by anything out of the gene pool, you just lost that dog's genetic diversity. If it had the disease or the potential to carry a trait of a disease, but it is not lethal and it is not something that is painful and affects the life of the dog significantly, then let's have a conversation. We may be able to kind of work and massage our way around that very carefully, but again, everybody has to have full disclosure at the same time, and so this is really important for us as veterinarians to rely on breeders and breed clubs to make sure that we're up to date on the most common things that are up and coming because genetic things are cropping up. The more we know and the more things that we see, we really, really need to make sure that we are helping you guys achieve the best that you can. And so the way that I try to tell people about genetic testing is the screening process. It's not a process of elimination. It's here for us to say, Okay, what do we know that may be a problem in this breed? And if it is something that this animal is carrying two copies of the gene or one copy of the gene, how do we circumnavigate or how do we handle the situation with this individual? That's right there. And then again, how often are genetic tests? It's a one and done. Okay, that being said, if we don't have an actual DNA based test for something that we know is genetic, we need to make sure that we're doing those tests on a regular basis. So I'm gonna get off my soapbox on that one, because that's super important, and we're going to move into more specifically about what we do with the stud dog. And so whenever you show up to have an initial examination with a full spermiogram, then the thing that should be happening first, and the way that I teach this to my students, doing a physical exam on a happy go lucky dog is probably not going to be that much of a libido killer. But if you have a very soft dog that's a little bit nervous, going and doing a full physical exam, to include a per rectal prostate examination, is probably not going to make him want you to give you a seam and sample. So typically, what we do is we get them in the room, we talk to the owners, we make sure the dogs are comfortable with us. We may go outside, but we're going to collect them first, and I'm looking at their libido, okay? Response, the procedure, is he willing? Is he unsure? Is he really unsure? Is he painful? And so that's the first thing that we're going to do, is we're going to try to collect them and assess that libido. 

Dr. Julie T. Cecere  22:34

Response, I'm looking at why we're collecting them. If we're successful, do I have any lesions on his previous peanuts that I need to worry about or that we need to address, and then I'm watching the color and the clarity of each fraction. So dogs actually give us three fractions of their ejaculate. So the first fraction comes from the urethra and the prostate. Think of it kind of like a clean out. It may have some urine in there if they weren't allowed to act or they didn't have time to empty their bladder fully before you collected them. The second fraction is the sperm rich fraction. As a reproductive veterinarian and as a breeder, that's really the only fraction you should care about. Is that second fraction? That's the one that's going to have the bulk of the sperm cells in it, and that's the most important for breeding purposes. And then lastly, the third fraction is purely the prostatic fraction, and that's perfect, that that third fraction is in a natural breeding, it increases the pressure within the vagina, which drives that sperm rich fraction up through the cervix and into the uterus. So when we're collecting a dog artificially, we don't need that third fraction. And so most dogs, if you're watching the color and clarity, you'll see that fraction come through so it's clear, white, clear. Once we've switched to that second clear fraction, we can stop collecting that we are not getting any benefit from that third fraction. And that's really, really important. Once we've collected that that seeming could kind of hang out for just a little while, while we complete our physical examination and again, things that we're looking for, any systemic, genital or heritable traits that we might need to discuss, making sure that the heart sounds great, that we don't have any abnormal rhythms we're looking in their eyes, making sure that we don't see anything that's there again, that might need to be addressed. And then pain or reduced range of motion, hips, joints, dermatologic conditions, all those types of things are really, really important, and so making sure these things are happening on a regular basis, then your stud dog is going to be extremely helpful. And so the reason that I'd say that we look at these dogs and sometimes we're relying on owners to call up and tell us when things crop up. So these two pictures that I just put up here on the screen. This is a condition called a prolapse urethra. This is something that we do in certain types of breeds. Is heritable, but it's where the urethra actually kind of Everts itself outside of the end of the penis. It can be painful, it can be extremely bloody, and something that can happen. And so again, we're looking as we're doing these types of things, not just collecting the semen for collecting semen sake, we're looking for any abnormalities that we may need to discuss. They're going to affect your stud dog's career.  

Dr. Julie T. Cecere  25:02

Now, once I've gotten a semen sample, this is where standardization of technique is going to have to happen. And your full spermiogram is going to consist of all of these things, the color and clarity of what you collected, because that's really, really important. The second part is motility. If it's not moving, it can't swim to where it needs to go, which is through the uterus, up through the uterotubal junction, and into where it fertilizes the egg in that uterine tube. So we're going to say the overall motility, and we're going to say something about its progressive motility. Those two things should be reported morphology, that is the makeup of these sperm cells. And so we've got a head, a midpiece than a towel, there are certain abnormalities that we know are going to have a definite effect on their ability to fertilize and oocyte an egg. And so that should be a major part of any spermiogram that you have done BSD with looking at the sperm cell. And then lastly, concentration. If I don't know how many sperm cells there are, I can't tell you what a breeding does. And that is really, really important, because a breeding dose needs to be standardized, and we need to maximize the number of cells that are provided by a female again, so that we are making sure that your stud dog is not blamed for a missed breathing okay. There are ancillary tests that are out there. There are viability, hypo osmotic swelling tests. These are all advanced things that we may do as a theory of genealogists that may not be available at your general practitioner that are doing these types of things, but color, clarity, motility, morphology and concentration, everybody should be able to do that if they're collecting dogs and charging for a BSE. So we talk about that color and clarity. So we've got what is considered here on the far right, a normal collection. So this dog did not empty out their bladder completely. This is the first fraction, and it's got a yellow color to it, and that's just because it's a little urine tinged. Not that big of a deal. But if we're shipping semen, we need to pay attention to that. The second fraction here is the sperm rich fraction. Okay, you can see how it's cloudy, and that's because of the sperm cells that are contained within it. And then this last tube is the third fraction of the prosthetic, and it's very clear. You could read newspapers through it, and that's what it should look like in disease processes. You get something like this. So these both are contaminated grossly with blood. If we are getting these types of colors, this is an immediate you go to your veterinarian and you have them evaluated. Okay, it could be coming from the surface of the penis. It could be coming from within the penis. And again, the first thing that we would think about is his prostate, and we need to get that under control. The nice thing is, is a breeder that we actually know in dogs and horses that it can be up to 20% blood contaminated before it actually affects its fertility in its natural state, not in a ship state, and definitely not in frozen but in its natural state, it will get diluted out in the uterus pretty quickly, but you certainly cannot or would not and should not Ship semen that is grossly blood contaminated. 

Dr. Julie T. Cecere  28:02

So again, if you get any of these color aberrations, other than urine contamination, because you can even see, in this urine contamination, I can still see through it, it's just yellow. You cannot see through these. If you get this, you need to have your dog examined right away. That being said, motility is something that can be done grossly or with a machine. And so here is a video of sperm cells moving. And again, we're looking at the total motility as well as progressive fertility. And if we use this machine, which a lot of people who are doing reproduction use, it's called a Catha, it's a computer assisted sperm analysis machine, very good at analyzing motility, but if you take an experienced practitioner and pit them against the machine, we're usually within 10% of that machine in any given time. So the machine is nice because it's taking the human factor out of it. It is not necessary to still get an accurate example of how those sperm cells are moving, and it's really important to know how they're moving. The other thing is with morphological defects, and I said, if there is one thing that gets skipped over most often, it's going to be this step. And the reason that this step is important is because of all of these abnormalities that you're seeing on here, not a single one of these sperm cells is normal, and all of these are going to affect fertility. So this sperm cell actually has what's called a detaching acrosome, and so it's this little I call it, like a toboggan on top of the head that have to bind with the egg to be able to have to come off and be able to bind to the egg to be able to fertilize it. If it's already detached at the time of ejaculation, this sperm cell is going to die before it even reaches that point. This is what's called a reflex mid piece. This one wouldn't be moving. The ones that have these flagella that are flipped over, they're going to be spinning in circles. Can't go anywhere if you're spinning in circles. These two abnormalities with Reflex mid pieces and tightly coil tails are you. Usually associated with heat damage. So are we blow drying the dog too much? Did he run too hard in the hot weather? What exactly happened in something that we need to address right away. There's what's called a distal droplet in the bottom middle, and then proximal droplets on both of those firm cells on the far right hand side. Those proximal droplets we know have an inability to be able to fertilize oocytes. So these proximal droplets, they could be moving, and he could be 90% modal and look very progressive, but if these proximal droplets are there, they can't fertilize an egg. So morphology is extremely important, because we can tell you exactly what you have and if you have problems. 

Dr. Julie T. Cecere  30:40

Okay. Now that being said in our production species, we say that 70% or higher normal morphology is considered excellent. In our dog species, we are not seeing dogs that are that high normal anymore. I used to see them 10 or 15 years ago. Now we're looking kind of more on the 50 to 60% normal is considered really good. My own stud dog runs about 40 to 50% normal morphology, and he's still having litters of 10 and 12. So that being said, morphology is important. But if somebody tells you that your dog is 90 or 95% normal, the likelihood of that actually being true is probably pretty low. So just be aware that morphology is really, really important, and depending on what morphologic defects they have that certainly can affect their fertility. Now, concentration is something that I said everybody should be able to do. Those calculations are extremely important. So more is always better. We have to maximize those doses of total normal modal sperm cells. So again, people joke, oh, it only takes one to fertilize. No, it actually takes the concerted effort of all those sperm cells that are all trying to penetrate the oocyte at once. So the less numbers you provide that make it to that oocyte, the less likely that you're going to be able to fertilize those oocytes. So again, regardless of what you're doing, evaluation of every sample to know what you're sending is going to be extremely important. And really standardization of that breeding dose of total normal modal sperm cells. Okay, not just total sperm cells. I want ones that are modal and normal. Toy and small breed dogs are going to be 100 to 150 million total normal modal cells. That's a breeding, medium to large breed dogs, it's 200 million total normal modal sperm cells. And again, always think of it this way. More is always better. We cannot split ejaculates in dogs, like we can in horses, they can't produce as many sperm cells as a horse can. Therefore we can't split ejaculate, and we shouldn't be doing that. And dogs have difficulty, sometimes covering multiple females at one time again, because they're not producing as many sperm cells. They don't have as much tissue to be able to produce those sperm cells as our larger species can. And then consideration of viability, just because you send that many there, if it'll get there alive, you're not doing them any good. 

Dr. Julie T. Cecere  33:05

And so again, we're going to talk about that a little bit more in our semen processing. And moving into that we need to talk about it. So semen processing for chilled shipment had to be processed for multiple reasons, urine contamination, certain that we're worried about volume increase or a volume decrease. So if somebody collects 15 or 20 mils, they hang on and collect a lot of that third fraction, which they don't need. I can't adequately extend the semen to be able to go to the female end and have them within reason, put that in her uterus. So if you send somebody 20 or 30 mils of semen to put in a dog, that's not going to happen. Really. The uterus itself can hold between three and four mils of semen if you put it in vaginally. You know, we really shouldn't be putting in more than 10 and so again, you're just going to be pouring maybe good sperm cells right onto the ground, just depending on the volume that is sent. What we know about processing mainly was based on what we know from equine This is a huge industry in horses and previous canine studies, and those are few and far between. But the things that really, really affect those sperm cells is something that is really, really important. And what we're looking at is a reactive oxygen species. These are chemicals that are released in a negative growth and death. So as the sperm cells become compromised and they die, we get higher reactive oxygen species, which is toxic to the other cells. We're trying to minimize that best that we can seminal plasma to a certain point is beneficial, but over that point is actually detrimental and actually contributes to reactive oxygen species. So we have to be really careful about how much of that seminal plasma we leave in there, aka, be very careful about how much of that third fraction you collect extender type. Some dogs prefer certain types of extenders over others. Types of extenders and duration of cooling is also going to affect that. So just because minitube has what they call a 10 day extender, that doesn't necessarily mean that you should be putting your dog semen in an extender for 10 days and expecting it to be quality. And we're going to walk through some studies to kind of prove that to you, historical studies that have been done in canine were using pooled samples, and so they were collecting multiple dogs for the study. They were putting all that semen together, and then they were studying that semen as a pooled sample. In the more recent studies that we've done here, we actually used individual samples, and the reason that we did that was because pooled samples, there might be some inter aggression between the sperm cells and mixing the plasma fractions from those dogs, but we also can't say how that individual animal compares to himself, and so that's why we decided to do individual samples, because while pooled is nice, because you get more numbers and you have more volume to work with, you really can't individualize that stallions and canine What we have done in the past was based on science. We're finding that, oh, right, they're different species. They behave very differently. And then so again, trying to pull from one species to another. Sometimes it's a good idea in the conceptual phase, but then we actually have to follow through with studies to compare the two, and then reduction of abnormal or dead sperm cells from the final product. We can do this very easily in stallions, because they will give us 30 to 60 mils of an ejaculate, and we could put it through a cushion and be able to reduce those numbers. I mean, they give us two, 3 billion sperm cells and an ejaculate, and we can get you down to 500 million, which is considered a breeding dose really, really easily. We don't even start with those types of numbers in most dogs. 

Dr. Julie T. Cecere  36:45

And so again, there are a lot of things that have been done historically in horses that we were trying to pull over to Canine, but they're not really that applicable. And so the current studies that we have in canine talk about seminal plasma. Okay, so again, I said, at certain amounts, it is beneficial. But over that, we actually get a detriment to the sperm cells, centrifugation when we're processing so how fast do we spend these sperm cells to be able to get maximal recovery of the sperm cells, reduce the amount of seminal plasma, urine, whatever it's contaminated with, but without compromising the quality of those sperm cells the concentration. So what concentration do they want to be shipped at? What's best for them, and then ultimately, the longevity of those firm cells? So again, I mean, we can kind of walk through there's papers out there if you ever want to go looking for things, but certainly a lot of this stuff has been pulled over from what we know of Equine and we're just now starting to kind of tip of the iceberg work through with canine and so the most recent papers have actually been Dr bollocks stereo resident here at Virginia Tech. And so what she really looked at was first looking at the optimal range of centrifugation for canine semen processing. Again, horses can actually withstand very high centrifugation rates. And so since they go at a very high centrifugation rate, they can actually do it for less time, and have great sperm recovery without a lot of damage. And so we were kind of interested in looking at what is that maximal centrifugation rate that we can do with great recovery of the numbers, but without damaging the sperm cells. And then once we determine that the second part of our project was okay, you know, in horses, we have a standardization of 25 to 50 million sperm cells per mil, is the concentration that they like to be shipped at for ship chilled statements. That's what we were using, kind of, as a gage for canine Well, nobody really, kind of looked at that. And so we were taking that to look at different concentrations and say, What do dogs really like? Because we know that horses like this, but horses and dogs are not the same thing. And so what we did was we had a total of eight to 14 dogs, depending on which study we're talking about. And the reason that we have larger dogs here and not smaller dogs was because, again, we have a volume issue. And so as much as I would like to collect Pugs and have an ease in New York keys with as small as they are, we didn't have enough volume, so we had to go for bigger dogs. And so we had these dogs. They were within their maximum fertility, two to six years. They had negative Brucella Canis. They were healthy On physical exam, and they were included in the study if they had that normal morphology greater than 40% their total motility was greater than 70% and we had a concentration that was greater than 75 million per mil. And the majority of these dogs were proven at the time of collection, so that we knew that they had puppies on the ground and that they were actively breeding. We weren't using babies and we weren't using old stud dogs. We were going for the middle of the row. And so the first part of this was that centrifugation study. And so I'm going to go through some data here so that you guys can actually look at what we looked at and what we decided was the best. And so these dogs were spun anywhere from 420 times g all the way up to 900 times g for five or 10 minutes. And what we found was the. Recovery rate across all of these study groups was applicable. We didn't really get a loss if we were spending on them slower or if we were spending them higher, which was good. That sperm recovery rate was very, very good. And so we found out that it really, in these healthy dogs, didn't make that much of a difference. But what we did was we took these after centrifuging them, we diluted them just as if we were going to ship them, and we did them at a standard dilution rate, and then we looked at them once every 24 hours for two days. And so your time one, which is your very first column here, is, is what their viability was at the time that we collected them and had resuspended them after centrifugation. Time two is at 24 hours, and at time three, that's at 48 hours. And so what you're looking at is a decrease in viability, which is what we would expect, right? We don't expect these sperm cells to last an extender for really, any longer than 24 to 48 hours and still be able to do their jobs. And so the viability doesn't look too, too bad. We're looking at, you know, maybe a 10 to 20% loss of our viability. But when we start talking about our progressive motility and our total motility, this is the progressive motility. You see how much drastic drop we have from 24 hours to 48 hours. These sperm cells really just don't like being an extender. And this was a 10 day extender. It's the best one that's out there on the market. They really just don't like being an extender any longer than that. And so again, we're looking at morphology. We had an extreme drop in normal morphology from time one, again, this is the time that they were put in the box, to the 24 hour check, and then an even further drop from 24 to 48 hours. So because of the reaction of oxygen species and all of the things that are happening in that box, we didn't do anything to them. This was them just sitting in the box. We certainly are causing some damage, okay, and so something to really kind of take home. And so what we decided from here was anywhere from 400 to 900 depending on the dog and maybe individual. For that individual, the centrifugation speed really didn't matter too much. There was no real significant difference. But what we do know is they do not like being in a box for 48 hours, and that comes into play in shipping. 

Dr. Julie T. Cecere  42:18

No, you can't collect your dog on Saturday and expect them to arrive on Monday in really good shape. That's just not a good practice to do. And so what we did was we took that centrifugation and we kind of moved this into concentration. And so what we decided was the control that we used was just collecting the dog, just the first and second fraction, no third fraction whatsoever, and we diluted that at a one to three ratio, which is what an owner would do if they were owner collecting. But you have to be very careful not to collect that third fraction, versus actually processing it with a centrifuge, pulling off that seminal plasma and then putting them back in a concentration of 25 million per mil, 50 million per mil, 100 million per mil, or 200 million per mil, we were trying to figure out, are you more like horses, or do you like to do something else? And what we found is, quite honestly, the one to three dilution performed very, very well in viability. Again. Time one is at the time that we actually put it in the box. Time two is at 24 and time three is at that kind of 48 hour time frame. And so you can see a very drastic drop from 24 to 48 hours. But the one to three performed very, very well. And for people who are collecting a lot of that prostatic fluid you are actually getting once you extend it very close to that 25 million per mil, and again, they do not perform as well as everybody else does. If we look at the total motility, same type of thing we've got looks really, really good at 200 million per mil and that one to three dilution, as long as you're only collecting that first and second fraction, not doing super great at 25 million or 50 million per mil, and again, 24 to 48 hours, we continue to see decline. Same thing with progressive motility. I mean, there's kind of a trend here. We were expecting to see some things, but I was very pleasantly surprised and shocked at other things. And so again, progressive motility, we saw that trend. And so now that I've bored you with all of this data, what does that mean? So current recommendations when you go to have a BSE done on your dog, which you should be doing if you're standing your dog at stud, you should be doing this several times a year if he's actively breeding, or at least before the breeding is coming up, within 60 days of when that bitch is supposed to come into season, you really need to have that Full spermiogram, motility, concentration, morphology, and then discussion of doses, and then physical examination. Right now, we usually tell people anywhere from 400 to 900 is okay, but we standardize ours at 700 or 50 for five to 10 minutes, depending on the dog, and then we actually take off the seminal plasma that's there at the end of the centrifuge. Information to only 10 to 20% of the original volume, because, again, they like to have some of that still present, but they don't like to have all of it there. 

Dr. Julie T. Cecere  45:07

And based on our study, dogs much prefer to be highly concentrated when they're shipped, versus highly diluted. And so if you have somebody that is doing this work for you, make sure that they know the dogs really prefer to be 100 to 200 million per mil. That the concentration they'd like to be shipped at, or if you're doing it as yourself, is making sure that you're collecting only that first and second fraction. You're diluting it at a one to three volume to volume ratio, and you're not collecting a lot of that third fraction, because I don't want ancillary third prosthetic fraction contributing to that volume, I want the extender contributing to that volume, and then again, adjustments based on the quality of the scene in that day. So if you have a dog that his fertility, he's getting older, and his fertility is dropping, being very forward with people who are calling about his services and saying, I can only do one female at a time. And then you kind of just choose wisely and or he's no longer shipping very well anymore, you need to bring your female to him. Those are okay. You stand your stunt dog. You need to advocate for him. You want to make sure that his services, you're not going to get a bad rap for services if he's not shipping very well. And then the bottom line is that semen has got to be used within 24 hours of collection. It should not be sitting in a box. It should not be sitting in somebody's refrigerator. You need to collect it, and it needs to go in the next day. Good Dog semen can last if you take it out of the box or out of the dog and you put it in a female, can last up to seven days or more, but that's in the female that's not sitting on the camera. Okay? So really need to use that within 24 hours in the future. And this is my little plug for breeders to help us. We're addressing things like standardization of these protocols, the types of extenders used in horses like skim milk based extenders. Dogs don't like that animal protein, and so looking at what types of extenders and additives may be beneficial filtration that's practical. Like I said, these filters that they use in stallions, again, they give us so many numbers. It doesn't matter at the reduction of the numbers that they get on the back end. But for canines, we start with so few numbers but the filtration is awesome, because we can actually filter out some of those dead and dying cells, it reduces our numbers so much that they're not practical. And then again, optimization with in vivo fertility parameters, I can take semen from a cow or a horse and put it in a cow or a horse and I can say, Oh, yep, it made a pregnancy in 14 days. I can terminate that pregnancy right away, without any issue, and say that it was proven. I can't do that in a dog. So we're working on an out of the dog process. 

Dr. Julie T. Cecere  47:48

Couple of things just to do here, maximizing dog fertility. The best thing you can do is select for good fertility. Really give them the best shot. If you have animals that are having fertility problems at the age of two. Don't propagate that line. Our producers do really good job of this. We don't, unfortunately, BSC is often, again, peak fertility that is two to five years of age, but making sure that you're keeping track of what's happening if there's a mis-breeding, making sure he's getting evaluated right away, a quality diet. But again, we can't really say that there's something that's 100% is going to help them. We do know things that are going to be detrimental excess heat. So people who are blow drying their dogs show dogs, make sure that you're using force they are not hot back there and or avoiding work during hot days, or if you are working during hot days, realizing that 60 days from those hot time frames you may or have poor quality semen just because of the heat. And then supplementation. I'm not a huge supplementation person, but things that we know that may help aren't going to hurt. Glucosamine is never going to hurt them. Are high fatty acid, the three NPF, use zinc, selenium, vitamin E and folic acid, those types of things in small amounts are not going to hurt anything, but at the same time, just realize that it's kind of conflicting data whether they actually help. So again, as long as they're on a good diet, we really shouldn't have any issues. Biggest things for breeder tips is plan ahead. Plan ahead. Plan ahead. Make sure that you're telling female owners when you're available when you're not available, and realizing that female breeders are going to have to understand that life gets in the way sometimes, but since these females only cycle once every four months to 12 months a year, really planning ahead is going to make things a lot more helpful. Collections limited to only that second fraction or the first or second fraction, because again, we don't want excess fluid, especially if you're collecting yourself to contribute to that final concentration and dilution, and again, evaluations on a routine basis, so that you keep up with your dog's fertility, and then processing for animals that may have a higher number abnormalities, we really are going to help them if we can get them to an optimized concentration between that. 100 to 200 million cells per milliliter when we send them. And then, if there's nothing else that you take from the talk, please, please, please, semon needs to be put in the female within 24 hours of being collected. That's the biggest thing that we saw. We knew it going into the study, but we really wanted to put some scientific data to it. It didn't matter what we did to those firm cells after 24 hours, they looked terrible. So again, sometimes packages get delayed. That's outside of your realm, but to the best of your ability, make sure that these are going 24 hours in the female after they've been collected at this time, I'd be more than happy to take any questions. 

Nicole Engelman  50:39

That was amazing. Thank you so much for going through all of that. That was an incredible presentation, but I want to be conscious of your time and all of our viewers, so I think we should have time for two or three questions. If that sounds okay to you. Oh yeah, sure, awesome. So I know you briefly touched on this in some of your last slides, but we got a lot of questions about diet, so one of our community members is wondering if there are specific foods to avoid or offer to enhance semen production and quality. 

Dr. Julie T. Cecere  51:08

That is a great question. So certainly, I know a lot of people are raw food feeders out there. Barf diets carry the high risk of contamination with Salmonellosis, Campylobacter, toxoplasma. Like there's a lot of things that we can talk about, and really, having any one of those things cause disease in our dog is going to affect their ability to produce normal sperm cells. And so avoiding those types of things are really, really important. The bottom line is, the very well balanced commercial diet is something that you need to feed your dogs. A lot of our performance animals require a lot more calories. We need to watch some of our Labradors out there, not our field labs, but our show labs, just because they think about food and get fat, but again, making sure that we're keeping them in a lean and mean condition on a commercial diet. It's fine, be very careful about any supplements. There are a lot of supplements out there that offer and claim to give you better fertility in your dog. Yet, if you actually look at the ingredient list, there are things in there that we know are detrimental for fertility. So just because the label claims that that's true, it's a nutraceutical, it's not regulated, and those claims are unfounded, and again, to date, there's really no scientific study outside of a few things like glucosamine that will actually potentially help their sperm cells, and we don't want to do anything detrimental.

Nicole Engelman  52:33

Amazing. So this is a bit of a follow up to that question. One of our community members asked if there's any truth to blacks, peas and legumes affecting canine fertility.

Dr. Julie T. Cecere  52:45

So what we're worrying about in the legumes is a high estrogen level. Okay, so they're phytoestrogens, and so you would have to be feeding your dog, probably quite a bit of those to have that type of effect on fertility. That being said, I think everything in moderation, we all should probably pay attention. I know I should for my own diet. Everything in moderation is a good thing. Don't overdo it. I don't think that you have to avoid it. If you want to give your dog some peas every once in a while, or have a diet that contains peas. Again, if you're buying a commercialized diet, they are formulating that to balance for the individual animal that you have, and not overdoing it with excess phytoestrogens, you would certainly have to feed a lot of edamame to get enough estrogen to have an effect.

Nicole Engelman  53:33

Amazing. I think that's good to know. Okay, I think we have time for one more question. This is from Tim. Tim is curious, what factors you think have caused the large drop in normal sperm cells from what you were seeing a decade or two ago.

Dr. Julie T. Cecere  53:44

Loaded question, okay, if we could choose our breeding stock like producers were, we would be better off. And why don't we do that? There's a reason that I see a less drop in fertility in my performance dogs than I do in my confirmation dogs. So confirmation trends as far as what they look like in the ring, coat type and body type, versus I'm choosing for an animal that is out doing their job. And I would say that specifically for my coon dog breeders, they breed their dogs to be able to produce litters. They get pregnant, they carry the term, and they raise their babies, and they will not keep a female and they will not keep a male that can't do that, we kind of give a little more leeway to some of our other types of dogs, and I think that genetically, the first thing that you see when you're seeing a loss in genetic diversity is your fertility drops, and if we don't pay attention to that, then we are going to walk ourselves right into a corner. And so again, sometimes we have to make the unpopular decision to go out to a left desirable mate so that we get genetic diversity back in our lines. And that's something that we all need to talk about a little bit more.

Nicole Engelman  55:01

Amazing. I think we also have some resources on Good Dog about genetic diversity that we will look for and drop in the chat, in case anyone's curious. But definitely, very, very important, okay, I think that is unfortunately all the time we have questions. But thank you so much for this amazing presentation. I think everyone learned so much, myself included.

Dr. Julie T. Cecere  55:22

Thank you so much, Nicole, and I hope everybody has a great rest of their afternoon.

Nicole Engelman  55:26

Thank you again for being here, and we loved covering this topic. Thank you again to Purina for helping us put on this event. Bye, everyone. Have a great rest of your day.

Dr. Julie T. Cecere  55:37

Thank you. Bye.

Nicole Engelman  55:41

Thank you for listening to the Good Dog Pod. We'll be back in two weeks with another episode, so be sure to subscribe to the Good Dog Pod on your favorite podcast platform.