So a big thank you to the webinar vet for, letting us do this presentation to you today. Today we're talking all about how we analyse, and have a look at how we do the motility of, semen, whether it be fresh chilled or frozen, how we process fresh chilled and frozen semen, the packaging and how you handle frozen semen as well. So there's quite a lot to get through.

But we're gonna go use sort of tips and tricks of what you should be looking for, and, and, and so on. So, right, we come over here a little bit more. When I think on the last one we did, we showed you how you cut semen off the stallions, and I think we sort of stopped it at that stage after we did the collection.

So now we're gonna take it right the way through. So when we've got our semen sample, what are we gonna do with it and what should you be looking for? Some of the little, helping side of things, when you get this, we always weigh if you want to come in a little bit closer, we always weigh these samples.

I don't know whether you can go in a bit closer than that and see that there. So this sample's giving us 75, . mils on here and it's 75 grammes, so, it's a very easy way of finding how many mils and grammes, there are on this.

So we weigh all our samples, when we do it to make it much more accurate. Whenever you get the semen in, really, you want to look at the colour, the colour tells you an awful lot about the semen. So in other words, if it's a slightly red colour, it means it obviously got blood in it, and if it's slightly yellowy, it's got urine in it.

And you want to be seeing it's quite transparent watery. It's most we've got a lot of the seminal fluids in there as well, from that side. So I think, so it's always good to have a look at the, the, the semen when it first comes in the lounge just to make sure the colour tells you more.

Also it's quite a bit creamy colour that's more likely to be it's really dense as well, and that's how we want it to look so that can make it. So that's the first thing we wanna do. When we look at our samples, we wanna, one of the first things we wanna do is look under the microscope, so see what it's like, so what's important when we analyse semen is we get the exact same size droplet each time.

If I was to put two different size droplets on a slide when you're analysing it, you're gonna get two different results. So we want to be looking at 10 microliters is the amount that we want to be looking at. So a fix to pet it, or, or you can get a variable for better, but if you don't want one of the plastic ones where you can get a different range each time, you literally go in, get your.

10 microliters and pop it on your slide. Like that the other things you want to note, we'll try and do actually a bit of experiment where we're actually watching. I'll show you what can what can go wrong if you don't do it right.

So we will. Get your slide. Yes, .

A sample, we put 2 drops on here. And another drop Or and You get your cover slides. Now these cover slides you.

You really want to be getting. The the cover slides want to be the right size as well. So these want to be 22 by 22 mil.

If you get smaller ones that you're gonna get two, the semen's gonna be on top of each other when you're using 10 microliters. You just place that on top. But what happens if I was to pick a slide up like this.

And you actually And if you get your fingers on it and you by accidentally touch your fingers on it, it makes all the difference. It will kill off the sperm cells, that's something very easy to do. I'm slightly over exaggerating, obviously when I'm doing that.

So I'm just trying to show how. Preparing a slide it's really important, and this is gonna tell you whether you think the semen is is any good or not. So we pop a bit under.

I'll have a look. So we go up there. It's this, this is looking at a 10 microliter drop.

It's most probably a little bit. Dense. But you can see how the steaming is moving really, really nicely.

Whenever you're looking at the semen, you want to be looking at a few different places on the slide, so you want to say, is it consistent? You never want to look at the edge of the slide, cos that's usually dies off, see how it's dying off there. So it's always the middle of the slide.

But this is the one I put my mucky fingers on, and see how bad that is there. It's, it looks, it looks really, you know, you can see how, so, and that's. And that's it, make a real difference to how you can analyse it.

So it's really important to get the right size sizes and everything else. What we're looking for, yeah, if we maybe go back there again, we, we, we're looking for progressive motility on this. Pam, can you, can you dilute that out a little bit more?

Have you got one, because it's a bit too, and when we're doing semen analysis, we're trying to look at what's progressive and. Or what's going around in circles, and it's actually quite a lot of sperm cells there, so it's quite hard to distinguish, so right, we're gonna step back a little bit. So Pam's added in an extender, so we'll just do a little bit more.

The only thing that I didn't mention, which is really important, there's so many important parts of this, is this stage has got to be at 37 degrees. If we don't look at semen at the right temperature, we're not gonna get the true reading. If you just put it on a cold slide on a cold stage, it's gonna be very, very slow.

So 10 microliters on a warm stage is critically important to get the proper analysis of. Oh, no, I don't. That's better.

So we can see more individual slides now, the sperm cells, and we can identify. So what we tend to look for is, is there more or less than 50% moving there? And we'd most probably say more than 50%.

There's quite a few going round in circles, so the progressive is not so good, but I think the team's been sitting around for a bit. But you might give the semen maybe about sort of 60%. But it's basically you just break it down in, .

It's more or less than 50%, is it more or less than 75%. Now progressive, I would say there's definitely less than that, but on this slide, so a lot going around, so we want ones that are going in a nice straight line for that. Right, so we look at these other systems, Pam, can you give me a hand with it?

There are other ways that we can analyse the sperm cells. There are something called CASA systems. CASA systems stands for computer assisted sperm analysis machines.

It's, it's quite a map, and there's different various ones out there. There's, this one that's sort of a hamilton formwa where we can actually put the slide in, so I'll. It's out there and there's one also called the iceberg.

I'll show you how these both work and how they can actually then. So you come here. Excellent.

It's like. So you can actually see. The sperm cells there.

In there and actually it's looking, it's looking actually quite good on this slide. So we will just do a scan. And this will count hundreds of sperm cells in a fraction of a second.

In fact, it'll count 390, and it's giving it actually quite a high reading, but the progressive, as you can see, isn't, isn't so good there. So, and then we try that on the. OK.

And you put a drop on there, and this way we just put a drop on here, so these are just different . Ways that you can analyse semen if you, if you're not using microscope, there are other devices out there that you can use, and if you can see with this one, you can actually go in. And you can actually analyse even on the iPad itself.

And you can zoom in. And you can zoom out so you can get a a bit of a OK. So sea analysis is really important.

I really recommend before obviously as soon as you get a, a clutch notwithstanding, it's analysing either under microscope or with one of these other, devices, you can. If you're doing chilled semen, really it's nice to warm the semen up, a small drop of it before you AI the air. With frozen semen, we normally recommend that you actually check the sea after insemination because once it's thawed out, it should go through them in.

You want to make sure that it's in a, it's, it's properly thawed out and put straight in the mare, you're not hanging around while you're analyse it. Right, so the other thing that we've got to look at, we'll come back to viability and things like that after, is centification. So with fresh demon, you can get the sample, we can have a look at it in it's raw state and if you're gonna AI it raw, it needs to be AI straight away.

It needs to be kept at 37 degrees and it really needs to be seminated within a half an hour. The best way is to add an extender to it to keep it alive, and there's lots of extenders on the market, we're not. He try to say anyone's better than others.

The stallion will pick which extender you should use and there's lots out there, so. There's one called Quality and Ira and Survival, there's a new one called come out from called Beyond Bota Creo. So there's lots to choose from, and certain stallions will prefer certain extenders and you've got to find out.

Extended there is to produce the longevity of the semen. It's either usually got milk or egg-based in it. It's got proteins in there, it's got antibiotics in there.

It's got sugars in there to feed and look after that semen. So what we'd normally recommend is when you collect it, we are gonna put it in fresh AI. We'd normally recommend you get your sample, mix it 1 to 1 with extender, and that quite often be quite happy for several hours and then you might go and inseminate, say 20 mLs of that quite easily, depending on the concentration of the semen.

We'll come back to concentrations in a minute. So, with chilled semen, . We've always got to put extender in there, because the chill is usually 24 hours, sometimes 48, but the best results are, are usually up to 24 hours.

Remember, not all standings will chill. Now one of the reasons that a lot of sans won't chill is because some, a lot of people won't centrifuge. So most people do, but some people won't, so we'd always try and recommend the centrifuge of semen.

If the semen arrives with you it doesn't look very good. I think you can always ask that question. Has the semen been centrifuge?

so with the centrifugation, what we normally recommend. You you come a bit closer, yeah, it's. If you can go up a bit and just see it into there at all, just, that's it, you've got it.

So we'd normally recommend swing out rotors. So when we put these, these, 50 mil falcon tubes, you want the rotors to swing out. And when you get these 50 mil falcon tubes, we normally do, say 1 to 1 initially we dilute it out, and then we'll take the semen down so usually after centrigation we take off all the top stuff and take it down to 10.

10 mils. The speeds that are the big thing with this, so if we're in Chile, we normally centrifuge around about 5 or 600 G. Another little tip, some of these centrifuges are measured in RPM revs per minute, so what is that in centrifugation cos you've got to work it back.

And there's a very easy calculation that you can do on the internet. If you put RPM versus G, it just tells you to measure the radius of, of this, and, that'll give you, it will soon work it out for you depending on the size of your centriation. Alright, so.

600g for frozen semen we tend to do 1,000g but we spin onto a cushion, for that, but we maybe can do a whole new webinar on on frozen semen, so that's chill scene and we normally just do it for 10 minutes as well. The frozen semen we do 1000g for 20 minutes for chill, we tend to do 5 and 600 for 10 minutes. So, and say we take off the top, supernatants which we can show you in a minute.

So, and with that, so, and then you can use a cushion, so yeah, so basically all we do, but. Makes you extend it up. But then Yeah Yes.

Right. And obviously with centuries you wanna balance it and and it and away you go. Once it's finished with the with the chilli, we leave it in there for 10 minutes and take it out.

And we will then this way is if you have. We literally, once it's finished, You just take off the top. I would just take it down.

And that And then you resuspend that, in an extender that you've already tested on your standing or if you're doing an extended tests, you split that between a few. So you just take it down, and then just resuspend that and extend yourself so that could. That's with chilling.

I think I've missed out on that, huh? Chill's fine. So, so just on the, on the dilution rates for chills, we want to be taking down, we, we want to really resuspend it from about 50 to 100 million sperm per meal, so we need to work out what the concentration is here and then re-suspend it on, on that.

Oh yes, that's a very good point. Thank you. So, so it's the little crib sheet here and we can get that on there.

So, this is quite often, you know, if you're, depending on what the concentration is, how much you should dilute it out for. If you're not centrifuging, you've really got to stick to this. If you are centrifuging, you can actually make it a little bit denser.

But this, because it's got no seminal fluids in there. Those seminal fluids can be really toxic to the semen, so that's where it's really important to dilute. Those, those samples out as the specialty count when it comes to that.

. As I say, we tend to use the 50 mL po and chew, but again, and then you resuspend it in your extender that you've got and you dilate it out and then share it out and ship it out. Concentrations we talked about concentrations with 50 to 100 miles and where the boxes pan that we're gonna over here, yeah, so with the chilled scene. Here's one we prepared earlier.

So when you're shipping shipping it out, you would have done your concentration of maybe 50 or 100 million out. You, diluted the semen out. You wanna make sure you've got the right the stallion's name is clearly labelled on this, and again, if you're receiving samples, you wanna make sure that it's, put the stallion's name on it and everything and the date.

It's got a syringe cap on it, and it's got, it's called a two part syringe, so you don't want to have any rubber bum on these syringes at all, so they're called two part as opposed to 3 part in there. And It's got to have the necessary paperwork, so it's really important when we're, sending out that the clients know exactly what they're getting. So it's got to have the stallion's name is absolutely paramount.

We've got to have the stallion owner, the mayor that it's going to. Making sure that styling's been properly health tested. Right, so, tested for EVA and CEM we actually tested for EIA as well.

So that you need to know this information prior to inseminating that mare. You need to know the health status of that stallion. And massive but not least, you need to know if you can see that, what the quality of the semen is.

So 10 mLs of semen, 230 million per meal, they added 30 mLs to dilute it out. The progressive was 70, sorry, the total motility was 70, the progressives was 70, and it's got 1.6 billion sperm in there.

We usually recommend for chilled semen about a billion sperm cells. Because we think half of this could die out, so we really want about 500 million at point of insemination. Some people go lower, to, down to 300 million, but those are the sort of rough figures that you're looking for.

I think if you're playing it safe, you will have 500 million, but if, the stan's got really good fertility, you can go a little bit lower than that. So that's the information we want to do. We want to put it all in a sealed bag.

And then that, and when you're putting the cement in here, these ice packs go in here. They want to go in at the same time as the semen. What we don't want to be doing is putting the, the semen cold shocking into a, a box, so this box is what will be cooling down with the semen at the slowest rate possible.

Semen does not want to be cooling down too quickly, and then we just put the name, obviously where it's going to, and we usually put the name of a stallion a male in the outside box just for ease of use or all that. All right. Right, so we've come on to frozen semen now.

We got some floors over the next one. OK. Just, remember with the, with the chill cement, there's can be quite often issues with weekends, delivery and things like that, so you have to be prepping your mare and trying to get her to maybe ovulate on a Tuesday, Wednesday, Thursday or Friday, or sometimes you can get hold of her on a Saturday.

It depends whether it's coming from abroad or not. So the, the chill timings can also sometimes be a little bit tricky. So the time of the AI, but usually, you know, if they're inseminated, usually, about 12, sometimes 24 hours prior to ovulation, that, that's perfectly acceptable, but.

Usually you're putting it in, say, on a Wednesday, and the mayor's gonna ovulate sometime during that day on Wednesday is the ideal time for you'll see, because remember, it doesn't really want to ovulate 2 or 3 days later because that seems already 24 hours old when it, when it's arrived with you, so the clock is ticking, and every single stallion is different. Some stallions are chilled a lot better than others, so, on, on that side of it. So, frozen semen, right.

Here's one we say. So we'll go through some other tanks maybe in a minute, but when the frozen semen arrives with you, it quite often can arrive in a flask like this. This is called a dry shipper, and.

These br shippers. For health and safety, they're designed to ship the semen with no liquid in there at all, so, they should be just in the baker. So what we have to do is we have to fill them up with liquid nitrogen minus 196, cool it down.

It's got like a sponge in there, and before we ship it out to yourself, you tip it all out. And then, it should last about at least 7 to 14 days, but we say a minimum of 7 days, or sorry, a minimum of 7 days. So that's where we usually say, we sort of guarantee up to 7 days these flats, but they're really, really good, you've got paperwork in so in here it should be.

The paperwork. So in there you always want to look at the paperwork first before we go too far, you should have the name of the stallion, how many doses, numbers of straws per dose, and what the colour of the straw is. Doses is always an interesting one is how many, how many straws should there be in a dose.

It can comprise anything from one straw, sometimes up to 10 or 12 straws. If you to look at averages, I would say it's between 4 and 6 straws normally per dose, but this is dependent on the stallion who froze it, the fertility results for that stallion, and so on and so on and so on. So, .

With that side, there should be thaw and use instructions to how you thaw this semen out because different places might have different ways of doing it. Again, a bit like a chilled see there should be an inventory in there and how to show the flashbacks and and also about insemination form as well and the signs been tested for EVA, EIA and CEM as well, so that's. She'll be in there Now if the semens come from abroad, this is a another total new minefield, and we've had people just speak all day about this, but, it should have health papers that come with the semen.

We must have health papers with the semen, so. And this should have where it's come from, the, the consignor and the consignee where it's going to as well. It should have the signer's name, and that's usually at the bottom here.

So page they updated in January. Alright, that to keep you up to date, but it should have the date on here as well, that can signs go back to that actual, stallion as well. And then it's got all the health status in here, and it's really important to go through these, and it shows whether it's got EBA, EIA, and it's been tested, as well.

So it, it's got lots of different pages on here, you can see them all done there and then it should have an official stamp and a signature by the veterin there. These are the health tests here. So rather than just saying yes it's got health papers, I think it's just worth having 5, 10 minutes just having a quick look over because sometimes these aren't always right and as we know biosecurity and it's really, really important and obviously we want to be careful that we don't .

That, that we don't, you know, make sure that we, you know, there's no chance that any disease is coming because it can travel obviously with pro. Right, that's the paperwork, the boring bit over and done with, so we'll open up these, these. So in here, Whenever you're dealing with it, you must wear goggles and glasses for this, .

Sit down and some of these straws can really pop out and they can see might come a little bit closer when we're doing this. See the mist coming out. No, that's OK, you can see the mist is coming out here, .

Whenever you're handling. With these snores. Some gloves that you.

Where gold it's really wear glasses because they can, they can literally break up they can actually take your eye off. You wanna always be looking for straws below the neckline. So when we're looking at these straws initially is you can actually go in a pair of tweezers.

And you can have a look, got the right standard. What you don't, what, what you don't want to do is lift this straw up for any period of time cos that straw is already starting to thaw out. So you've got to be very careful of that.

So we'll be looking at the semen. Away from here is a hazardous goods and just look, yes, it's got the right style. Right.

The next stage, if we want to draw, draw the semen out. And we need a water bath at 37 degrees and we want to be thawing this semen out for 30 seconds. So the beauty about this, if you were to leave it in the water bath for 40 or 50 seconds, it's not going to do it any harm at all.

You want to be taking one straw out at a time. I'll try and demonstrate. If it's, if you take 2 out, what actually happens cause you want to go out as quick as you can.

So we're gonna go in. Wow. Yeah 2.

That's good. And they stick together when they thaw out and you're not gonna get a good thawing rate, that's why you want to do one at a time, so we pop them in. And OK, was it?

And it's 30 seconds. From when the last draw went in, not the first straw. Right now.

I wait. No. And you can just use your hands for for this bit it doesn't really matter too much.

You can actually get the straws. Out of the water bath and the water is a real contaminant. So, We've got 6 floors here.

And that's only 3 mLs and you can maybe get, if you don't wipe these straws off, you might be able to get half a m of water in that, and that would kill all the semen. So you wanna make sure you dry them off, use your 2 bits of towel. I'm up pretty well.

I'm gonna use the second piece. Dry them off, and it's at this stage, you want to actually look at these straws and double check again that you've got the right stallion, because it might say on the paperwork that they were white straws, but they might have another stallion that are obviously also white straws that could be in there, so you check they've got the right ones. Can you get us a clear straw.

You want to tap them all that you want, you can see the crimped end by coming close. This is the end that we want to be cutting, and you want to cut it as close to the end as possible, and I'll show you why in a second. I'll tap them down there.

Now I have to cut this one right, just a little bit lower. I'll show you the problems it can be and just so you see the straws. They're crimped at one end, on the other end you have this, this, cotton bung either sides PBA powder, and that should be sealed with, that's when the seal hits it it hardens seals.

So that's what I'm just showing you with these clear ones. You can get these other ones that are 5 mil macro tubes, so there's various ones on there. Right, so I'll bring these.

Here we go. It is that. We just got a quicker that we can a bit closer.

You can just push these out. Now this one, we cut a little bit lower down. Now, I don't know if you can see there, see the cotton bun that we saw on the other, this cotton bun, if we cut it too low, will actually come out and that will go into your semen.

And if you're using an AI gun, this can actually sometimes block the AI gun up, so you have to be very, very careful obviously on that side of it. So empty the straws out into there, and then obviously you can go and seminate your ma, OK. Right, we'll have a look at this, Steven.

And which one it is, but hopefully there's something moving. I know. One so that one she said.

I change this to 10. With a bit of mix. Yeah On their own Have a look.

Wow. So if you go up there, we can see, see the sea, it's amazing that semen. 0, 10 minutes ago was at -196 degrees, and then it sort of brought back to life, which is quite incredible.

The other thing I've done them, the other thing that we want to be able to look at is when we analyse the semen is to make sure not just the motility's OK, but the, the viability of the cell membrane structure's OK. And there's different ways you can do this. You can use something called niggles and ears and stain.

We've got something called a nuclear cell counter, which you can. Just need my packages time please. And.

You get a certain amount of measure amount here. I'll get this cuvets, and this is a way that we can tell. What first of all, how concentrated those sperm cells are.

And then we test it again, we can look at the viability. By putting a different stain in there. So what it's quite clever at doing is one will tell us what the total concentration is per meal.

And this will tell us on straws we have written. On here, I don't know where you can read that, it will say it, we have the date and it'll say 200 million or 200 times 10 to 6, it should be in this draw. And I'm here, we've just done a concentration reading.

And it's come up 205, you see that there? So I don't know where you can zoom in up, maybe not, don't worry, but that says 2005, so that's pretty accurate. So that shows how accurate.

So it's telling us 205. A million sperm per mil in there. So in other words, if there's 3 mils, that's 600 million sperm cells that we have in there.

The next, it will tell us, it will just look at the viability, it will tell us all the ones that have got a membrane that's not intact, and it'll cross reference the two and then tell us what is viable, and that's quite a key point.motility is only one tool when it comes to fertility when when we're doing this. So, we have some straws that we print off in the second.

Double checking And this this is a very accurate way, so motility just isn't always. Fantastic indicator of fertility. It's one of the ways you want stuff moving, but it's not, doesn't mean it's definitely just because it's moving, it's gonna get mas involved, but it's viable as well.

We want to see how viable it is. Viability is really, really key to things, so. If we have a look here.

We should hopefully give us a figure, you can see all these tiny little dots here and here that says 205 million sperm per meal, and each dot is the head of a sperm cell, so that's telling us what the concentration is. It's coming back now, it come in again and it's told us that there's 86 non-viable, which gives us a 56% viability. Really.

Anything with frozen semen, over 40% we count as OK. If it's a fresh sample, we really want it above 50 and above, but this is a frozen sample, so this is actually exceptionally good, being 56% viable on that site. Right, so we've only got, a little bit, further, further to go.

I think we've looked at fresh, sample going straight to me. We've looked at chilled semen on that side of it, and we looked at frozen semen as well. The thing with frozen semen, just remember it's all down to the di there are different mediums out there, different cry protectants.

Some are DMF. Which stands for dimethyl, formaldehyde and the and some glycerol, these are the ones that permeate that cell membrane when you freeze steam, it's a bit like your frozen pipes, you know in the winter time, how they, frozen pipes when they freeze, they rupture and break because it expands. It does exactly the same in a sperm cell, but if we can have a cryoprotectant we can dehydrate that cell.

And when we thaw it out like you just saw a minute ago, we can rehydrate it and it swims away. It's quite incredible now what we can do with cryoprotectants so it's like an antifreeze of sperm, so to speak, so it works very very well, right, we'll have a quick look here at printing our straws, and then we'll go over the tank room and have one last look in the tank room and then so we've slightly done this the other this is a, it's a printing machine. Mhm.

What year? Receive your straws, in theory. Oh, very good.

So I don't know you can read that. It says the webinar vet on there, it might be difficult to read. So in other words, the name of your stallion, say the date that we collected and what the concentration is, and then it'll say our details stallion AI.

Is that possible to read? Oh right, cool, well done. Right, so we're just move to the end room now, so we'll just come this, come this way.

It's, sorry, it's red meat, yeah. So this is where the storage side. Now, you might have most people with storage tanks, these are different types of storage tanks, it's all different types of work on the market, but what I can say when you get those tanks with the the dry shippers that you can see here, these are only shipping tanks, remember, they're not holding tanks.

As soon as they arrive in these, these tanks that you saw us getting one I thought, you wanna transfer it to your holding tank straight, straight away. So, right, if you wanna come round here, eat the big holding house talk to a lot of things come up here at least don't let my phone call in the internet. So you can stand on there, and you just have to push it forward the other way, that's it.

And in here you can see. Incredible, there is nearly quarter of a million straws in this. On tank suspended in animation at minus 196, it will last 1020 or 1000 years' time, it would be very little difference from the day it was frozen.

It's quite incredible how with freezing semen, how we can, what we can do. So. So I think that's, we're nearly about, we're nearly about there now, so we've gone through the whole process.

it's a bit of an overview again on any one of these we could spend quite a bit of time on any one particular subject, but that's showing you a little bit how to analyse semen. You know, a few tips and tricks on that, making sure you've got your warm stage, make sure you've got 10 microliters, make sure you've got the right cover slip and slide. When you're centrifuging, make sure you centrifuges about 600g for 10 minutes for chilled team.

Make sure you pick the right extender, really important on that one because every standing and test those standings every year as well. I don't think just because one year it works the next year, it's gonna be the same. It will not, won't necessarily be that, and, .

With frozen semen, obviously, when you get it in these tanks, put it into your storage tank and then it'll sit there quite happily as long as you keep those tanks topped up. So, right, well I hope you enjoy this webinar and er very much looking forward to seeing you next time. Thank you.