Hello everyone and welcome to this webinar on managing a stallion in artificial breeding programme. I'm gonna be talking today first of all, about why we might want to use a stallion via artificial insemination. Then about the pre-breeding health tests which are necessary.

We're going to have a section on training the stallion for semen collection. We're gonna talk about semen assessment and how we process it to use it either fresh or chilled. And then in the final section, I'm going to go on and talk a little bit about the two most common problems which I come across with stallions in breeding programmes.

Let me talk first of all about why er we might want to use artificial insemination. A large part of it is convenience. There's no need for the stallion to be in the same place as the mayor, and that obviously opens up a much wider market for stallion owners.

For example, they can use the stallion not only nationally but also internationally, and we'll talk a little bit later on about the health tests which are required in that kind of situation. It also means that stallion owners aren't having to deal with mares coming to their premises and to take the risk of that, but they can simply have the stallion and, and send out the semen. There is a reduced risk to the stallion himself, because he's not covering mares, particularly unfamiliar mares.

But that's not no risk, and when we talk in a few minutes' time about how we collect, the semen, we'll, we'll talk more about that risk. One of the main reasons that owners and and riders bring stallions to us to include them in an artificial insemination programme is because they're competing stallions, and they prefer, that the stallions don't get in the habit of interacting with live mares and so. They managed to keep their two jobs quite separate, by using them by artificial means with a dummy mare, for the breeding programme and then they go and do their written work and and hopefully aren't too interested in the lives and that that seems to work, really quite well.

But one of the key things that as vets, we really need to remember when we're dealing with stallions in artificial insemination programmes, is that AI artificial insemination does not reduce the risk of venereal disease. In fact, as We'll see in a few moments' time, it's a fantastic way of spreading disease far and wide. So that's why it's so important, that we really concentrate on making sure that health tests are done properly and that standards are complied with.

What are the downsides of managing a stallion in an AI programme? Well, really, I think from the veterinary perspective, a lot of the downside is to do with logistics. So, often if you're involved in installions who are having shipments sent out, be that nationally or internationally.

There's just a lot of management around making sure the paperwork is correct, making sure the health tests are all in order, and just making sure that the mayor owners and whoever's dealing with the mayor from the veterinary side is, understands how much. Advanced warning you need to be able to collect the semen of the stallion's not on site and understands when the salin may or may not be available, particularly if it's off competing. And, and then there are lots of issues, logistical issues around physically getting the semen to where it needs to go to, you know, such as getting it in the post on time or not being able to send over a bank holiday weekend and and things like that.

So undoubtedly, having a stallion involved. In an artificial breeding programme does require a lot of attention to detail and quite a lot of managing. And then, as I already mentioned, the downside, a big downside from a veterinary point of view is that there's a significant potential to spread disease via an artificial insemination programme, either locally, through fomites, for example, if artificial vaginas aren't adequately disinfected between stallions.

Or, in the semen itself, and we'll talk in a few minutes' time about the various diseases which can be spread in those ways. So overall, You know, I spent my life managing stallions in in artificial breeding programmes, and obviously I'm quite an advocate of them, but I think both owners and vets need to go into it with their eyes wide open. And as the vet dealing with the stallion and with the semen collections, you really need to spend a bit of time at the outset talking to the stallion owner and whoever will be involved in bringing the stallion for the semen collections because it's not necessarily the same person.

And just making sure that everyone understands at the outset, how things are going to work, what needs to be done in advanced, who's going to deal with whom and, and what expenses are going to be involved, so. Examples of that are explaining to the stallion owners that all of the health tests which would have to be done if the stallion was working with natural cover live mares will still need to be done, and making sure that those are done in good time at the beginning of each breeding year. I'm explaining to the stallion owner if it's a novice stallion that he'll have to be trained to have the seaming collected and we'll talk in a few minutes' time about how we do that.

I'm explaining to the stallion owner that almost all stallions will need so-called flushing. Out at the beginning of each breeding season, and we'll, I'm gonna talk at the end about the so-called accumulated stallions, those who really need quite a lot of repeat collections at the beginning of a breeding season until they're ready to use and and why that is. There needs to be a clear line of communication about who is going to deal with mare owners, who is taking the bookings, who is going to coordinate with the vets who are working with the mayors to make sure that semen is ordered in a timely fashion and that you are then able to.

Deliver it when it's wanted, and it stallion owners and riders need to be very clear, about when the stallion will and won't be available so that you can notify mare owners and and vets dealing with the mayor about that kind of thing. And I'll talk in a few minutes' time about the safety concerns around semen collections, but the other thing you need to be really clear with stallion owners at about at the outset, is what the safety rules are, who is allowed to be on site when a semen collection is being done, who will be handling the stallion. And what safety protocols you have in, in place.

And, I'm going to run through those in a few minutes' time, but as a general rule, it is much safer to work with a team who you're familiar with, and not to have the stallion owners or riders involved unless it's something which they're used to being involved in and have been trained to be involved in. So having given that brief, kind of oversight, I want to spend some time now talking about screening for venereal pathogens because as I've already emphasised, this is a really important part of having stallion involved in an artificial breeding programme. You can see here on the screen, the main venereal pathogens which we screen for and when we talk about a venereal pathogen, obviously we're talking about something which can be spread through sexual contact, so typically in this case in the semen.

But some of these diseases, for example, equine viral arthritis, EVA, can also be spread, by other means, respiratory in the case of EVA, and others such as, CM can be spread via fomites indirectly, for example, by vets who use equipment between mares or between stallions. So, it's really important that we control these diseases, and the reason. For that is that they not only affect individual horses and obviously have an adverse effect on their health and welfare, but also that where there's an outbreak of any of these diseases, it can really bring the breeding industry grinding to a halt.

And, and that was, what happened decades ago now, when we had the first outbreak of contagious econometritis organism in this country. The regulations about how we screen for these venereal pathogens as are agreed in advance of each breeding season, and they're set out in these horse race betting levy board codes of practise, which nowadays are published online each year, and you can see an example of that at the bottom of this screen. And they set out, rules really for what samples should be taken for which horses, in the course of the breeding season and when that ought to be done, and they cover these main venereal pathogens of equine viral arteritis, equine infectious anaemia, CEM and then these particular types of leptosy pneumonia and pseudomonocerogenosa.

So for CEM, and Clebsiela and Pseudomonas, we, we swab stallions, and the protocols for doing that, are established in these codes each year, and they are, the reason they're republished each year is that the protocols sometimes change slightly according to what's happened in the previous. Breeding season in terms of disease outbreaks. So you need to familiarise yourself at the beginning of each breeding season with what exactly the protocols are for that year.

But the gist of it for stallions, is that, as you can see on the screen here, after the first of January and before any breeding activity begins, obviously. Then two sets of swabs have to be taken, and those sets have to be taken, no less than 7 days apart. This is UK rules I'm talking about at the moment.

And then they're cultured, under particular conditions, and they have to be done at particular laboratories which are, kind of registered for CEM screening. And if those swabs are negative, those two sets of swabs taken at least 7 days apart, then the Stalin is certified for that year as being free of infection and can begin breeding. If they, if they're not negative, if they come back positive, then what has to happen next is detailed in these codes, but in the case of CM, which is a notifiable disease, and that has to be in coordination with DEFRA or the APHA.

So on the lower half, on the right hand side of the screen here, you can see the sites which we take these swabs from installions, and you need to use a separate swab for each site. Obviously one should wear gloves because otherwise you have the potential to transmit disease on your hands. And the swabs have to be put in culture, media and sent back to these particular laboratories, and a list of them is published each year.

And the sites which we saw from on the top left of this image are the penile shaft and prep use. And then the next one over kind of top right of the image is the urethral fossa, and then the urethra itself. And ideally, we also take a swab from either the pre-ejaculatory fluid or the semen.

Now that's not always so easy to do in all stallions before you start breeding. So sometimes what I find in practise it's necessary to do is to do these 3 swabs. For the first set of swabs and providing they come back clear, then maybe do a flush out collection.

And as you do that, take the swab from the semen or the pre-ejaculatory fluid. How do we get the sound to extrude his penis in order to take these swabs? Well, it doesn't have to be completely erect, it just has to be extruded enough that you can.

Definitely swap these three sites, and, many stallions, in fact, when they're used to being used for breeding, will extrude the penis in reaction either to a mare or even to the dummy mare if they've been trained to use it, and that makes swapping fairly simple. Otherwise it's sometimes necessary to sedate the horse in order to get him to drop the penis. But remember, if you do that to avoid using ACP.

And then having taken these swabs, you send them back to the lab and obviously wait for the results before you begin any breeding activity. EVA is tested for installions prior to breeding by a blood test, as is EIA and again, there's a lot of detail in the codes on EVA, screening in stallions, not least because, there was an outbreak in the UK in 2019. Of EVA and that was found to have started with a respiratory route of infection.

But the problem with breeding stallions is is that they may pick up the disease via the respiratory route, for example, if they go to countries in Europe where the disease is endemic, and then come back and having acquired the. That way, then shed it in their own semen and that act as a source of infection from mares and then onwards potentially to other stallions. So it's really important that we establish that stallions and teasers, because of the potential spread of either respiratory root are are free of EVA before we use them for any kind of breeding activity.

And the way we do that, as I said, is, is a blood sample, and that's detailed, in these codes, but essentially if the blood sample gives a serial negative result, in other words, that there are no antibodies present, then we can be sure that the horse is not infected and breeding activities can begin. If it throws up a positive result, there are, EVA antibodies present in the blood of the stallion, then that can indicate one of three things. Either that he is currently actively infected, or that he's had a previous infection, or that he's been vaccinated.

And this leads us on to a kind of big problem around EVA status and the identification of it in stallions, because, as I'll explain in a few minutes' time, there is a vaccine available, but we're not able to distinguish between a sero positive result, which is due to vaccination and a sero positive result which is due to disease and therefore it's incredibly important that we keep the paperwork very well organised so that we can track that. So this slide just gives you a little bit more information about that. One of the effective ways to stop the spread of disease of EVA in, in breeding stallins is to vaccinate them against it.

But the problem is that once they have been vaccinated, they will be sero positive. So it's very important that we, are able to prove that the reason the stallin is sero positive was because he was vaccinated, not because he was exposed to the disease. And the way in which we that as detailed on this slide and in the codes, is to take a blood test from the stallion before vaccination to prove that he was sero negative at that point.

And then to vaccinate the horse, and then to retest him and prove that he is sero converted as a result of vaccination. And those are. The lab results which show that kind of train of events should be put in the horse's passport as well as kept on record at the veterinary surgeons and in the lab, so that they can follow the horse wherever they go.

And in future years that way, if anyone takes a blood test from this horse and it throws up a positive, they will be able to look back and say, oh yes, but that's because he's been vaccinated and he was negative before he was vaccinated. Now, there have been some problems over the years with the supply of this vaccine, and there is only one vaccine currently licenced against EVA in installions. There have been problems with the supply of it not always being available.

And so then that that has caused issues because horses have gone over the recommended date for when they ought to have had a booster, and then it's become a little unclear whether they were serial positive due to vaccination or because they might have been exposed to the disease in the meantime, after immunity had waned potentially. And what we can do if we have to is test for the presence of EVA in the in the semen, so. If we have a horse whose EVA status is a bit unclear, the way in which we determine whether he's currently infected or had a previous infection and is still serum positive, but is not shedding EVA in the semen, which is what's important from the point of view of transmitting it in an artificial breeding programme.

The way we can do that is to collect a semen sample and have it tested under virus isolation. But because EVA is a notifiable disease and stallions in this country, if there's any suspicion that the stallion may be sero positive, due to disease, then that process has to happen, in collaboration with DRA, the APHA. This slide shows us just a little bit about the paperwork which is necessary to organise if you're the one doing a semen collection from a stallion and sending the semen out elsewhere.

So on the left hand side of the screen here, I've just put up an example. Of the paperwork which we typically send out when we're sending out semen from stallions, which we're collecting from at my practise. And you can see that in this paperwork, it just simply names the stallion.

It makes it clear that this semen is for use within the UK only because it hasn't been collected under the conditions necessary for export. And then it states that it has been tested free of these various venereal diseases and that we have those test results on file. And then we go on to give some details about the semen itself in terms of when it was collected and what the progressive motivity was and how much the dose rates and so on and so forth, which I'll come back to in a few minutes' time.

So, that's the kind of form you need if you're sending semen within the UK. You just need to enclose something with the shipment, which the person receiving the sea. And at the other end of wanting to put it into the mare can look with look at with confidence and say, yes, OK, I can be sure that this stallion has had the necessary health tests done and that the semen isn't a disease risk to the mayor in terms of venereal disease.

Now, if you get involved with the stallion who's having semen exported, it all becomes much more complicated. And I'm not going to go into that in detail now, except to refer you to this APHA website, which you can see on the bottom right hand side of the screen here, and on the top right hand side you can see the various testing programmes which have to be gone through according to where the semen is going to be exported to. And of course those rules and regulations change a bit over time.

And indeed at the moment, we are in a, a somewhat unknown situation because they will almost certainly change after Brexit, but we don't yet know how they're going to change. So, the important thing is really to keep an eye on the DEFRA websites and the APHA websites. And if you do get involved with a stallion whose semen, the owners want to export, then to make sure that you speak to someone at one of those organisations in order to understand very clearly what needs to be done, when and do that well in advance of when you actually need to be doing the cement collections.

So having talked through how we test for for venereal diseases and EIA like EVA is tested for by a simple blood test, I want to spend a few minutes now talking about how we train the stallion for semen collection. It's important to emphasise the need to consider safety in all of this. Remember that as the vet, you're the one who's kind of in charge of the situation and ultimately responsible for it.

And there are likely to be a number of people involved in the semen collection at a minimum, even if you don't have a mare there and you're just using a dummy mare, you're gonna have one person handling the stallion and one person. Handling the artificial vagina, and it's really important to keep those people safe. And obviously, you know, when we're dealing with a stallion, particularly if it's a largeish warm blood, stallion, you know, we've got 500 kilogrammes, so, of, of animal, which we need to control because otherwise it becomes dangerous for everyone, not only for us but also for the horse himself.

So it's important before you start getting involved in this kind of programme, to have a think about the safety protocols, to establish them with everyone and make sure that everyone understands what they are and where they should go if something unexpected happens. So, this was, someone who came as a student to my practise and is just modelling for us here, the kind of protocols we have in place. So, as an absolute rule, everyone involved has to have a hard hat on.

We modified our rules to everyone involved wearing a back protector, after I actually started wearing one at the suggestion of a stallion owner, and very shortly thereafter got kicked by a stallion we were collecting from, and, and really. The back protector saved me, from a nasty accident. So we then inst introduced a rule that everyone who's in the breeding barn also wears a back protector.

And it's actually important also to wear, sturdy shoes, so that if you should, mistakenly get trodden on by the stallion, your feet are adequately protected. And then, as you can see here, obviously, disposable gloves, if you're going to be involved in, in handling the stallion and particularly in in washing his penis, which we'll talk about in a few minutes' time. The other rule I have in my practise, is that all stallions in the breeding barn have to wear a chiffney bridle.

You know, whatever the owner thinks about the way in which they're normally handled at home, I say to all owners, look, you know, I'm the one who's responsible for everyone's safety, your horses, as well as my personnel's, and this is the way in which we Make everyone most safe by having a Tiffany bridle on the stallion. Of course, we don't use it severely. It's just to have there as a safety measure.

And make sure that when you attach the lead rope, it's pointing backwards, as you can see in this photo, so that should the pressure go onto it as as a horse mounts to the dummy, it doesn't come open. And also if you have a Tiffany bridle which doesn't have a throat latch, then hook it through the head collar as you can see we've done here, because that avoids the whole thing coming off over the horse's ears once he rears up to get onto the dummy. So now we've got our safety protocols in place.

How are we actually going to collect the semen? Well, we've essentially got three options. Either we could use a real mare, or we can use a dummy mare, or we can use what's known as a ground collection, which is one of the salin doesn't actually mount anything.

And I'm gonna talk a little bit about each of those. If we're using a real mare, you could use either a mare as an estress or a mayor who's been ovarectomized and it has been treated, with estrogenol. Some of them stand well enough, even if they haven't been treated.

Of course there are interesting ethical issues around ovarectomizing a mare. For that reason, and it would be more normal to use a mayor who's an estress, but if you do get involved in using a real mayor, which I'm about to counsel you against, then make sure it's a mayor who you're familiar with and whose behaviour is as predictable as it, as it can be. This is a a slide taken from the University of Pennsylvania's website, and, you can see they are extremely well equipped in in terms of safety.

They've got their hard hats and their back protected on, and so on and so forth, and they're using a real mare, and she has also got protective equipment on. But really, I just wanted to put this slide up to make the point to you that in my view, using a real mare for cement collections is it's kind of the worst of both worlds. Because, the stallion is still at some risk from, the mayor kicking him.

The mayor is still at some risk from the stallion biting her, and the person with the artificial vagina is really in a bad position because they're between the mayor's back legs and the stallion's front legs. So, this really is a situation which I try to avoid, if I possibly can. Some people advocate collecting semen from stallions standing on the ground, and, and I've given you a reference here.

For a very good article by Fawnia McDonald about this. And there are some occasions on which that is appropriate. For example, if you've got a, a trained stallion who has an orthopaedic or a back injury or, or some neurological problem which makes it impossible for him to mount a dummy mare.

But generally speaking, it's not something which I'd advise. It, again, it's, it's just not very safe on, you know, the stallions tend to kind of stagger forwards rather, if it's not a particular. Well behaved style and it may kick out at you.

And whereas when it's on a dummy mare or even a real mare, it's much harder for them to kick with their hind legs because their front legs are elevated. When they're standing on the ground, it's much easier for them to do so. So, really, this is, is something I would only use if you absolutely have to.

I definitely would not use it, as a choice, rather than using a dummy mare, for example. And if you do find yourself in that situation, then I would recommend, this reference. This is my preferred method of collecting semen, which is collecting semen using a dummy mare, and this is in my own practise.

And you can see the advantage of this system is, there's no real mare involved, so we're not kind of squashed between the mayor and the stallion, and the stallion is not at risk from the mayor, and obviously the mayor isn't at risk from the stallion. So it's all just a much more controlled situation. Whichever way we're going about collecting the semen, we always need to use some kind of artificial, vagina.

And there are a couple of models available. This is the one which I use, which is called a Missouri type of AV. All AVs are essentially like a hot water bottle.

They have a kind of double layer which you fill with water and you want the lumen, where the penis goes to be at about body temperature at about 38 degrees. Or so and so what that normally means is filling it at about 45 °C or so. And then it has another part which the bottle attaches to, which isn't included within the area with the water in.

So that part is not heated. And then it has a cover, in this case, the leather cover, which is wrapped around it with a handle on, which enables us to hold on to the artificial vagina. And stallions are quite variable in terms of how hot they like the artificial vaginas and how tight they like them.

And to a certain extent that's just a matter of trial and error each time you train a new stallion. So this just is a series of slides showing how we prepare, the AV so you can see here we're pouring, the warm water into it, putting the case around it. We can use within the AV's disposable liners, which are commercially, available, or in fact, some people even just use rectal gloves and, and cut the hand part off.

And the advantage of that, of course, is because they're disposable, it reduces the risk of disease transmission. Some stallions don't seem to like them very much, in which case an alternative is simply to have a separate artificial vagina for each stallion you're dealing with. They should always anyway be disinfected, obviously, between use and, and we typically disinfect them by scrubbing them with chlorhexidine and then rinsing them and then soaking them in surgical spirit for half an hour or so and hanging them then to dry.

And then this just shows the bottle, which is attached to the AV. Again, you can, there are obviously various types of bottle commercially available. The AV I first used when I went to practise actually had a glass bottle, which I wouldn't recommend, because obviously it's a risk of, of getting broken in the course of the process, particularly by the stallion's front feet.

So, plastic is better, but obviously, you need to make sure it's not spermicidal plastic. And then this particular model comes. With a very useful connector, which goes on the top of the bottle and enables us just to connect that to the latex liner of the artificial vagina nice and easily.

And you can see here in my hand, I've got a, just a beer bottle, cooler actually, but we're using it as a warmer to help insulate the bottle. And there can be a lot of change in the temperature of the AV, according to the environmental temperature, and that's something you need to guard against. So having got the AV ready, we need then to get the stallion ready, and it's important to wash the stallion's penis because otherwise the whole ejaculate can become quite contaminated with kind of debris.

No ejaculate is ever, ever sterile. We will also have always have some bacterial contamination and some small amount of debt, but we try to minimise that by washing the stallion before we begin, and most stallions, once they've been trained and got used to this, using warm water, accept it perfectly, well. You can see here, that we're washing the stallion's penis, obviously got disposable gloves on so as not to spread disease.

As I said, use warm water, but don't put anything in the water, because if you start washing the penis, with soap, for example, what inevitably happens is that you wash away all the commensal bacteria and end up with some nasty pathogenic bacteria like pseudomonas. So we should just use plain water. And it's easiest, to start at the free distal end of the penis and work towards the body, of the horse.

That way you end up with the part which is gonna go into the AV, cleanest. Don't put paper towel back in the bucket, you. You can have it in the bucket to start with, so it's kind of got wet and and so forth, but then you use it, you wash up and you put it on the ground because the risk if you put it back in the bucket, is that the bucket itself becomes contaminated, with venereal pathogens or, or bacteria generally.

This is just a video of a semen collection. You can see the horses walking in. Jumps onto the dummy.

I've stepped out of the way, the person using the AV is facing the stallion so that she knows where his front legs are and doesn't get kicked. And in a minute, you'll see the tail starts to flag. Just there, and then as he finishes ejaculating, she'll gently lower the AV and enables the horse to dismount from it.

So how do we train the stallion to undertake the semen collection using the dummy in that way? Well, the way in which we do it is that we start off with a mare in Easter on the far side of the dummy, and obviously this is a mare who we're very familiar with who we know will stand quietly, and won't kick out, and so on and so forth, and we allow the stallion to tease that mare, kind of leaning across the dummy. And eventually the stallion will start to get a bit frustrated and and start to lean towards the mare and, and try to kind of go towards her to mount her, and will climb onto the dummy.

Now, that can take some time, and I'll talk at the end of this webinar about how we deal with the tins with quite reduced libido. But I, what we found over the years, it is much better to do, fairly short training sessions, maybe a couple a day, but not to kind of hang around in the breeding barn for a prolonged period of time if the Stalin isn't really getting the idea, because then they just get frustrated and start misbehaving. So, you know, when you're training a stallion, allow him to come in, allow him to get used to the surroundings.

Some of them find the dummy. A bit disconcerting because it makes a bit of a noise, as you just heard in that video when they mount it, and that can sometimes scare them a little. So they just take a while to get used to the whole thing.

But each time they behave in the way in which you want them to, obviously, reward them with kind words and a and a bit of a pat, . And eventually they do build up courage, and and the time it takes to do that is very variable. What we found over the years is that the young stallions, generally pretty enthusiastic, normally get the hang of it within a day or two, whereas the older stallions, particularly those who have been used for natural cover, take a lot longer to persuade and and to understand what we're wanting of them.

Generally speaking, once they're on top of the dummy and you manage to kind of slot the penis into the artificial vagina, and they've ejaculated once, then from then on, they have got the hang of it and we will do it relatively easily each day. And, and what we find in our training programme is we start off with the male on the far side of the dummy, as I said, in our setup, once the sals got the hang of that. We then move the mare into the stocks, which in our setup are actually just in front and one side of the dummy.

And with the stallions who we're collecting from frequently like this one you just saw, in fact, we no longer need a mare in there at all. They're just trained to come to the breeding barn, they know what they're there for. They'll extrude the penis so that we can wash it and then they'll just walk in, as you just saw, and have a semen collection done.

So I'm gonna talk now a little bit about how we process and use the semen if we're using it either fresh or chilled. As soon as we've collected the semen, we need to philtre it, and you can buy inline philtres, as you can see on the screen here, or you can simply use milk philtres and philtre it out of the bottle into a warmed receptacle afterwards. Personally, I prefer to use the milk philtres because I find that the inline philtres it sometimes takes it a long while to come through, in from the AV into the bottle, and the reason we philtre it is to get rid of every, any debris and also to get rid of the gel fraction, of the ejaculate because that is spermicidal.

We need to extend the semen, and the reason we need to do that is to provide some osmotic buffer. Sperm cells are extremely, highly specialised cells and have a high metabolic rate, and therefore produce a lot of, of metabolic waste products very quickly. And so they need to be buffered against that whilst it's being preserved or in transit.

And the easiest way to do that is really to use a commercial. Stallion tenor, and you can see some examples of that, on the screen here. When you're first training a stallion, it's worth spending, a couple of sessions just testing his semen with different extenders because different stallion semen works better, with different extenders.

So try it in a few extenders, keep it chilled overnight and have a look at it the next day and see how it's looking in each of them. There are some fundamental principles of semen handling which you always need to be aware of. Most importantly, avoid sudden changes in temperature.

So try to keep everything at about body temperature, 38 degrees or so, all of the vessels you're using and and so forth should be. An incubator warm to that temperature before you start. And then it's OK to let the semen cool down gradually, but what you don't want to do is to kind of let it cool down, heat it up, let it cool down, heat it up.

So after it's been collected, either keep it at 38 or let it gradually cool to room temperature. Avoid contamination with toxic substances like water and rubber. Label all semen containers as you go along.

That's really important, or if you're anything like me, you'll get confused and not be able to remember what's extended and what's semen. And then just adopt a routine and stick to it and that way you'll avoid. Mistakes.

Important to keep records of each assessment of each ejaculate, not only because it then provides a kind of quality control, should you get any queries about the semen which you've sent out, but also because it provides you with a retrospective control of individual style in fertility, so that if In a few weeks' time, it becomes evident that pregnancy rates have fallen. For example, you can look back through those records and say, oh yeah, well, actually we can see that at that point in time, the semen quality wasn't good, and in fact, maybe we can link that with the cell in not having been well some time before that. And that once you can work out that process, then you can try and figure out what's happened and it gives you confidence that things will come right again, once the salin has recovered, because remember it takes about 6 weeks for the whole smatogenesis process to be complete.

So we've collected our semen, we've filtered it out of the bottle, and, and then we need to assess it, and we measure the total volume. That's hugely variable installions. And in fact, the volume itself is somewhat inconsequential in the sense that you can have a very small volume, which is very highly concentrated, or a very large volume, with low concentration.

So the volume itself doesn't really tell you much about the quality. The motility is really important, and when we're talking about motility, we're talking about sperm cells which are moving at all, whereas when we're talking. About progressive motility, we're talking about those which are moving in a fairly kind of determined way across the screen of the slide from one side of it to the other.

So, a sperm cell could be motile, but if it was going around and around in circles, it wouldn't be progressively motile. And the relevance of that is that it is assumed that only those which are progressively motile are capable of getting to the site of fertilisation, through the utero tubal junction to the oviduct. We look at the morphology, in other words, the confirmation of the sperm cells, and I'll show you some slides of that in a moment, and we, I look at the concentration and I'll talk you through that as well.

Remember that these are the components of semen. It has pre-ejaculatory fluid, the seminal plasma of the sperm, the gel, which you already mentioned, and then inevitably some foreign bodies and and bacteria. And the seminal plasma is important because it's the non sperm rich fraction.

It comes from the. Sex glands, it serves to kind of flush the combined pathway for urine and semen of the urethra. In some salines it can be quite spermatoxic, but it also has a dampening effect on inflammation in the mare's tract.

So, There used to be a fashion for a centrifuging most of the seminal plasma out, but I think it's generally accepted nowadays that for most sciences we don't do that because we understand that it performs the usual function inside the mare's reproductive tract. When we're looking at motility, it's very important to assess motility at 38 degrees because it's otherwise an unfair assessment of how motor semen really is. So you need a warm stage for your microscope or as a minimum, some kind of plate warmer, and as I already said, avoid letting it cool and then warm it up again.

And, and you're looking for total motility and progressive motility. And we would expect progressive molatility to be a minimum of 50 spent it ought to be much higher in a normal stallion. So assess the raw semen, assess it once you've extended it one part semen to one part extender, and you do that under a low power with a cover slip.

It's better always to err on the side of being critical because that way, you will provide enough semen per mare. This slide which you can look at and read through for yourselves tells you a little bit about the morphological assessment of semen. We don't necessarily do this every time we collect semen, we would do it at the beginning of a breeding season when we're working with a new stallion.

If we run into any problems, but as I say, not necessary for every semen collection we might normally just assess the motility. And this shows you how to make, semen, slice, you just get a, a drop of semen and much as when you prepare a blood smear, make a smear this way and then stain it typically with neosinigrocin. *** in is in Spain.

Remember, this is the anatomy of a normal stallion sperm cells. So we've got the head, the midpiece with all the mitochondria and the tail. And this is what that looks like under a microscope.

Again, you can see the head, the, the bright midpiece where all the mitochondria are and the less bright tail. In this slide, you can see that there's a problem with the acrosome. So it's all become concentrated at one point of the head, that bright little point, and that would not be capable of normal fertilisation, nor would these because they've obviously got a disrupted midpiece, quite probably due to some osmolar problem, which we've caused.

Another type of midpiece abnormality is a remnants of cytoplasmic droplets. That is because the sperm cells not properly mature. Something's gone wrong with the process as it comes through the testes and the epididymis.

When the proximal droplets like this, in other words, the droplet is right up next to the head, you can see the round. Area of cytoplasm next to the head. That's abnormal.

In most species, but not in the stallion. It's also abnormal to have a droplet, as you can see here, at the junction of the midpiece and the tail. But in stallions, that isn't necessarily an indicator of a problem with fertility, but it can be an indication that the stallion is being overworked, because what's happened here is that the sperm are being effectively kind of rushed through the epididymis and coming out before they've had a chance to mature completely.

And so if you see this starting to occur in a stallion, it's an indication that it should ease up on how much is being, bred from or collected from. Some stallions actually just have distal droplets all the time though it doesn't really seem to affect their fertility. Bent tails, as you can see in these images here, are, are due very often to something we've done to the semen, so either to a heat shock or to a problem with the osmolarity of the extender.

So if you see semen that looks like this, you need to go back and think about what you've done that has caused it to be this way, and these will typically be going around in circles when you look at the motility. Assessment of semen is important because it enables us to know how much semen to send out to each mare. And you can see on this slide here and read for yourselves the description of how to fill a hemocytometer, which is one way of assessing concentration of semen.

We do nowadays have a number of computers available to us which can also do it, for example, the handheld spermacu computer. If using a hemocytometer, then we count the number of sperm cells in 5 large squares, as you can see on the right hand side of the slide here. And you need to decide for yourself that you're not going to count on two sides of each square.

So I typically say, OK, I won't count anything which has landed on the top or the right hand side of a square. You can see those ones with an X through here. But we will count anything that's landed on the left in the bottom of the square.

And the reason for that is it enables us statistically to deal with sperm cells which have landed between two squares. So we count the number of sperm cells in 5 large squares of the hemocytometer, and that enables us to work out how much we need to dilute the semen by because that will tell us how many million sperm per mL we've got in our raw semen. And then we work out how to dilute it by.

So again, the way in which you do that is shown on the slide here. And in order to make that simple for myself in practise, I just have a table up on the wall, because what we're aiming to do is to provide each mare with a minimum insemination dose of 500 million progressively motile morphologically normal sperm. Because that will give them a reasonable chance of conceiving.

So we worked that out by calculating the concentration of the raw semen, and then by extending it, and I do that using this simple table here. So, I know that. If I dilute according to this table, then once it's been extended, each mL of extended semen will contain 50 million sperm per mL.

So, for example, if when I counted the concentration of the sperm, it was 100 million per mL, then I can read across here and say, OK, in that case I need to extend it with 3 parts extender to 1 part semen. Typically that would be 20 mLs of cement and 60 mLs of extender, and then I can divide that up to ship it, and to work out how much I actually need to send to each ma, I multiply, The answer to how much the concentration is by the percent of progressively motile sperm to give us the number of progressively motile sperm per mal, and then we multiply that again by the percentage of morphologically normal sperm, if we've looked at that as well. And that will give us the number of Progressively most are morphologically normal sperm per mL, and then we obviously provide the number of mils needed to give 500 million of those.

And then you can be generous and say, OK, well, some of it's going to die off in transit, so why not send double the amount and that way we can be sure each mare gets as much as she needs. How do we send it? Well, there are a variety of seamanship is available to us.

These are the disposable kind, important always to label the test tubes or the centrifuge tubes that you're putting the semen in. Or you can send it in one of these larger blue equittainers. The advantage of these is that they tend to hold their temperature much better under difficult environmental circumstances, the disadvantages, they're heavier in terms of shipment costs, and they tend to go missing unless you keep good track of them.

So for the last few minutes, I just wanted to talk you through the two most common problems I run into when I'm using stallions in an AI programme or when we're training them to use in an AI programme. I think the most frustrating problem we come up against, and probably the most common problem I come up against, is a lack of libido on the part of the stallion. And that sometimes has a real physiological cause to it, for example, in neurological cause.

But it's much more commonly, I think, due either just to the stallion. Being unfamiliar with the process, or just being a bit nervous about the whole thing, being what we call a slow starter, a novice stallion, particularly if these are stallions who've spent a lot of time in their competition careers being told off for showing an interest in mares, it sometimes takes them quite a while to kind of pluck up courage to show signs of sexual interest. And certainly, not having the person who normally competes or handles them there, can help with overcoming that inhibition.

And sometimes something's happened to the selling like it's been kicked, and then, is, is no longer interested and that actually is the image that's on the screen here of selling I dealt with years ago, who had had a very nasty kick from a mare and, and thereafter had real problems with psychological inhibition, when it came to breeding. The other thing that can cause a lack of lobido is pain. And again, this is something we see not uncommonly in older competition horses who've spent a decade competing at a high level and had then come to be trained for semen collection to be used at stud.

You know, sometimes they're carrying old orthopinic injuries, particularly in their hind legs, which just makes it quite uncomfortable for them to be up on their hind legs on top of the dummy having to thrust into the AV, and ejaculate. And so, again, that's something we need to take into account. And typically those stallions, unlike the novice stallions who just stand there looking a little bit clueless, not really getting on with it at all, typically the stallions who are suffering from pain in the process of the collection routine have Normally quite good lobido, or at least sometimes have good lobido, and will mount the dummy, fairly happily, and then either fail to ejaculate or not seem to be able to get comfortable on top of the dummy or get off again, rather quickly.

And those are the typical signs of the stallion being in pain. And then of course you have to have a discussion with those who know the stallion and try and work out what's going on and if need be, get that diagnosed and, and treated. But a lot of these problems can be overcome, either if it is an orthopaedic problem, obviously we need to call this treat the source of that, and sometimes that involves use of non-steroidal inflammatories, for example, to make the cell uncomfortable when he's being collected from.

But if it's problems more with the routine, then the way to overcome that is simply patience and reassurance and just establishing what's expected of the cell and rewarding him when he does it correctly. So that stage really, I just, this slide really just runs through that. A careful diagnosis of trying to work out what's really going on.

Sometimes there is an underlying physical problem, as I said, like neurological problems. Retraining, however, is often appropriate because the problems quite often, psychological rather than physiological. The pharmacological manipulation is difficult.

There are no licence. I already mentioned that non-steroidals can help where pain is a cause of lack of libido. And there are a couple of very good articles here by Sue McDonald, who's done a lot of the work on this, which you might want to look at if you find yourself in a situation when you think, that you're needing to treat a tin pharmacologically for lack of lobido.

I just wanted finally to talk about this problem of accumulated cells, which I referred to at the very beginning. So, we know that the cell's reproductive tract has the full complement of accessory glands, and it has this rather long pathway, if you look at the diagram on the right, from the tail of the emis through the vas deferrings round to where that enters the urethra, and when ejaculation occurs, obviously the sperm have to come all of the way around. From the teledives through that pathway and get ejaculated out.

Now, in the non-breeding season, if stallions aren't ejaculating frequently, they can effectively store old sperm cells in the vas deferns in that kind of long pathway. And that can be a problem when we first start collecting from them each year, or if you're training a stallion for the first time and it's not been used before. Many stallins actually ejaculate spontaneously, and and that used to be something which was thought of as a vice in which we tried to find a cure from, but we now understand it's actually a really good thing because it avoids this problem of them accumulating dead and old sperm cells.

But in salins who don't, then when we First click from them we may typically see an image like this one at the bottom of the slide here, where we've got a lot of nonmotile dead sperm cells. They've taken up the stain because they're dead. And you can see that a lot of them have got detached heads, the heads and the tails are detached from one another.

Typically, as you can see from the table, these collections will also have a really high concentration. So when you see this picture, a high concentration, but a lot of dead immotile cells with dead and and detached heads, this is a real indication that you've got a stallion who has accumulated dead and dying sperm cells through not having ejaculated and that. Needs flushing out.

And the good news about this is that although it's not an uncommon problem, it's normally very easily solved one. You simply need to keep collecting from the stallion until you've got rid of that accumulation. So, and you just look at the semen each day, and you should see it improving, each day.

But in some sals that can be quite a protracted process. And can take up to about 10 days of of daily collection. So that's another reason why you need to get the sals in early at the beginning of each breeding season to do at least one flush out collection to see how the semen's looking and if need be, do more and to explain to the owners the need for that because obviously you don't want to be sending this kind of semen out for mares to be inseminated with.

Thank you very much indeed.