Take a deep dive into sexed semen technology with SS Lab Manager, Cole Wagner. Cole started working in Trans Ova’s sexed semen lab almost 16 years ago. Here we discuss commonly asked questions and get into the fine details of the process.
What is the process of sexed semen from thaw to sort?
Reverse sorting means taking conventional semen, thawing it, then sorting it.
Our lab will thaw those units out when it is the appropriate time to meet the maturation window of the oocytes. When we evaluate the motility of those samples, we want a minimum of 25%. So, by large, that’s not a great semen sample. Most bull studs probably have QC parameters 35% or higher, but if we see 25%, we usually feel comfortable trying to proceed with sorting those. We can get by with lower quality simply because through the IVF process, we don’t need to fertilize with millions of cells. Some of the donors, we might only be fertilizing with five-six hundred thousand cells, so we don’t need as much sperm because we’re going to mix them in the dish directly with the oocytes.
If we see 25% motility, we’ll go ahead and continue processing those samples. The process is relatively simple, but at the same time serves specific purposes. Sperm are frozen in different extenders – like milk or egg yolk or some of the soy-based animal protein free extenders. Those can inhibit the staining process where we differentiate the males from the females. Even though they’re good for the sperm during freezing, they’re not good for when we go to sort. So, we will go ahead and dilute those with a clear buffered solution. Then, we centrifuge them to help wash the extender. After that centrifugation process, we’ll be able to reconstitute that sperm pellet that forms in the bottom of the tube.
From there, we’ll do a count to determine the number of actual sperm cells that we have. We then plug it into some formulas, and it tells us how much of the fluorescent dye to add to the sample. The fluorescent dye is what’s necessary for the sperm cells to be differentiated from male and female. In bovine, female sperm cells have roughly 4% more DNA than the male sperm cells. So those female cells then absorb more dye than the male cells. After we have that sample counted and add the fluorescent dye, we’re going to incubate it for 45 minutes for the dye to fully bind the DNA inside the cells.
Following that, we’re going to add a second extender to those samples. The second extender differentiates the live cells from the dead cells – that way when we put the samples on the sorter, we have hopefully some clearly defined male/female populations fluorescing in a different area, away from all the dead cells. Then, we can set what we call our “sort gate” around only the females or the males from the live population. From there the machine is able to charge only the cells that we tell it to sort using that sort gate, and for us to be able to collect only those desired cells as it flows through the machine.
How accurate is the sexed semen process?
The process works in such a way that we can’t tell the machine to sort for a specific accuracy or a specific purity as we call it. It’s basically up to the training and the experience of the technician to know which of the cells that are fluorescing within the given regions would be desired and safe cells for us to sort. We can’t guarantee specific accuracy or purity at all – but what we’re shooting for when we do the gating and the sorting is about 90% of the correct gender. You might see a little bit lower purity on some of the male sorts, and that’s just due to the inherent way that the staining process works. Theoretically, a male cell should never absorb too much dye to fluoresce too high, but a female might not absorb enough, thus fluorescing lower in a region, it appears as a male cell. Therefore, male purities might be a little bit under 90% – maybe 87%-88%.
But historically speaking, when we’ve looked at thousands of pregnancies created using reverse sort embryos coming through recipients in the Iowa recipient program, we see greater than 90% females and male purities are right around that 89 to 90% range.
How is reverse sorted semen different from a straw of conventional sexed semen?
When it comes to an accuracy or a purity standpoint, we traditionally are better on the reverse sort than historically sexed frozen have performed in our system. Some of that is due to when we’re doing a reverse sort, we’re targeting a certain number of cells for the fertilization. However, when you’re trying to make the sexed frozen units, you’re trying to maximize how many units you can make. I think we have the advantage of sorting more conservatively to keep that purity high. Within a given sort, we might only need six, seven or eight hundred thousand cells – we’re not trying to capture every million cells that we can to freeze into straws. So, historically we have a little bit higher purity on those reverse sorts compared to sexed frozen.
How long does it take to get a sample of sexed semen to the fertilization team?
From thaw until it’s finished, a fast one is probably about an hour and a half. Depending on the number of oocytes, the number of donors we need to sort and of course the quality of the semen, some of them might approach two to two and a half hours. As the number of oocytes increases, the number of cells we need to sort increases.
Semen quality plays a big factor in how fast those bulls sort for us. If we have a sample that has a significantly higher dead rate, the number of cells per second that the machine can collect can fall really, really low. If it’s a high dead rate sample and we have to sort for a high number of oocytes, those two things kind of make that problem a little bit worse.
What makes a bull good for sorting? Does a good sort always correlate to good embryo production?
We obviously want to see samples with the highest emission motility possible. High initial motility should hopefully tell us when we have some strong sperm because they survived the freezing process well. That also means when we put them on a sorter, we should see a high percentage of live cells, thus being able to sort faster. So, the motility and the percentage of sperm that are live versus dead on the machine will dictate how fast it sorts.
Concentration of the sample or of the straws can also be important. For example, some dairy bulls traditionally have a lower concentration, so sometimes you might need more units to try to sort the same number of cells. Sometimes a higher concentration makes it easier to use a single unit, or it can also just make the sample stain a little bit better. Sometimes units with low concentrations do not stain as well as units with high concentrations, so for lower concentrations we will use multiple units to kick up the concentration for a better stain.
A good sample with good separation and differentiation, we might take 32-33% of the desired cells. If you have a sample that you can’t clearly see where that differentiation is, you have to go more conservative and sometimes might only take 20%. As the percentage that you’re able to sort or gate from decreases, your sort speed can drastically decrease as well.
To answer the second part, what we see and works the best for us to physically sort does not always correlate to how well that semen performs when it comes time for cleavage and embryo development. Motility is the easiest parameter for people to evaluate when they’re looking at semen, but it’s not always the strongest correlator to fertility. We might produce a sample that has a very high number of live cells after sorting, but if there are other inherent things wrong with those sperm cells, we just can’t add more sperm for better results. It’s usually a good indicator for a high chance of success if there are many live, healthy sperm, but there are a lot of variables within sperm cells that could affect quality. Also, by the time you consider all the factors on the donor side, there’s just a lot of factors that make it difficult to correlate one or two semen parameters to fertility.
What factors impact how many units of semen are needed for a sort?
That ties back into what the overall number of oocytes and the number of donors. If you’ve got one donor with a hundred oocytes, we might have to sort more cells than if you had five donors that were running on a no-stim protocol and combined only kicked out 50. The number of cells we need to fertilize with changes based off that. If you’ve got a high concentration unit but it has a high dead rate, sometimes we still need multiple units because we just can’t physically collect enough cells from that sample.
So, how well they stain, concentration of the straws and how many oocytes dictates how many straws that we might need from each of those bulls.
We try to work with clients as much as possible too. There’s some really rare and expensive genetics out there, so if clients want us to limit the number of units we use, we just try to approach it as clearly and effectively as possible.
What is the process of selecting oocytes used for rare and expensive semen?
Depending on the concentration of those cells, we usually can make one unit cover more than one donor. It’s a great way to use some of those rare genetics; we’re able to spread them out further. We’ve done some expensive bulls that we can use one unit across four or five different donors throughout the sorted process.
Compared to conventional, the advantage grows even more. We’ve done some really expensive, rare bulls with up to 13, 14, 15 donors before and been able to cover all of them with one conventional unit. That just speaks to the power of IVF and the ability to use those rare genetics and just spread them out really far.
When in the sorting process do you evaluate the semen?
The first time would be just that initial evaluation of the cells. If we thaw those and they don’t have that 25% motility, we will go ahead and call that sample a “rejected sample.” At that point, the client could still supply a different bull for us to try reverse sorting, and we can still usually meet the maturation window to fertilize those oocytes. They also have the ability the unit of semen that’s already been thawed and rejected, to switch and use it conventionally so we don’t waste it. We have clients that it might be 50/50, some people want to reverse sort something different, whereas other people want to just use the rejected sample conventionally.
Outside of that, the biggest thing comes when we go to the sorters: what’s the quality of that sample on the machine? If we put a sample on that has a percent dead rate that’s let’s say 75% or higher, those samples just sort really, really slow. The whole process takes a long time so that by the time it’s done sorting, even the sperm that were alive don’t have enough vigor or life to them to perform as effectively.
So, we might reject it on the sorter if the percent dead sperm is too high. The negative part about doing it at that point is we usually don’t have time to do a backup sort to try to cover those oocytes to a different bull. In that situation, we just try to work with clients as much as we can. We want to try to meet their needs be as flexible as we can.
What does “dead rate” mean for sexed semen?
With the second dye that we add in the processing portion, the machine can differentiate live sperm from dead sperm for us. It can tell because dead sperm are more permeable. The second media that we add to those samples penetrates those cells and dulls the fluorescence from first dye we added. The sorter will calculate the percentage of cells that are fluorescing within what we call our “dead population,” because they fluoresce so much lower than the live cells.
It’s correlated to the motility of the sample. If we saw a sample that has only 25- 30% motility, when we thaw those samples, they will probably have 65-70% dead rate on the sorter.
If a bull has a high dead rate and gets rejected, why can’t you thaw more semen and sort it longer?
It just goes back to some of that efficiency and what type of product we want to supply. We’ve seen where bulls with that high dead rate produce a subpar product. So we think, “well let’s just add more.” Then the sort takes twice as long, and the sperm that were already sorted continue to use up their energy and start dying off, etc. The timing is also particular, and we don’t want to continue to sort poor quality samples that would affect the timing of everybody else’s fertilizations as well. So, we just try to set some clear expectations and guidelines about what we think will sort and what we think won’t sort.
What are the advantages and disadvantages of sorting semen?
Within Trans Ova system, we do usually see a little bit higher cleavage and a little bit higher embryo development with reverse sorts. (Cleavage refers to the percentage that were fertilized and the embryo started cleaving and developing. Embryo development referring to the number of embryos that were grown that we could then transfer or freeze.) However, it is different for each bull.
We have some pretty decent side-by-side data about using reverse sorts on a bull and then comparing that to the sexed frozen that’s available on the commercial market from the same bull. Most of the time we’re able to be a little bit higher on those, but other bulls may be flip flopped. So, it’s not always a consistent advantage, but we do have some strong data that suggests throughout our system, reverse sorts historically perform higher from that cleavage and EDR (embryo development rate) standpoint than sexed frozen do.
The advantage to a sexed frozen unit would maybe be able to spread it out across more donors. But it just depends on the quality of the sexed frozen unit. There’s also a price difference between the two: sexed frozen derived embryos are cheaper for the client compared to reverse sorted.
What is the correlation between a stress test and embryo development rate?
If a client wants to supply an extra unit of semen, we can just do a simple stress test evaluation. We’ll look at the motility of that sample upon thawing, then we’ll do either a two hour or three hour stress test motility assessment. So, we’ll thaw the semen, look at the motility, immediately incubate it for two to three more hours and then look at the motility of the sample. We can do that for clients free of charge, pretty much at any given time.
We also can do what we call a test sort. If a client is willing to burn a unit of semen, we can mimic the whole sort process but then not actually fertilize any oocytes. That process will give clients an idea of the quality of that sample as it goes through the sort. It’s not a guarantee of anything, but it is a guarantee in terms of peace of mind. That test sort we do have a charge for because we are taking some time, utilizing materials and utilizing the sort.
In terms of correlation, we haven’t done enough test sorts to have sufficient data in terms of direct IVF results using the units that we test sorted. Given the number of variables, the correlation probably isn’t super direct. You’ve got an entire oocyte population that could impact the results as well. I just look at the test sort about just taking it for what it is: an evaluation of how well that sample will look and sort on the machine. That tells the semen labs, “I think I can get this many cells off of this straw so it can cover this many donors.” Or we even have the ability sometimes if the sample doesn’t stain as effectively as we would like, it can tell us that the next time to alter that dye level a little bit to see if we can make that better.
Do you provide feedback when bulls don’t sort? How do you handle that information?
Absolutely. Recently a client had some questions: “Hey, I had really low cleavage or EDR on this, did you guys see anything?” We provide them with whatever information that we can in terms of what the semen looked like when we thawed it, what it looked like in the sorter and what it looked like during our post-sort evaluation. Throughout that process, I look for red flags. If we end up with low performance, if it was borderline at thaw or if it was borderline rejectable on the sorter, you put those red flags together and those kind of make sense.
It’s trickier when the semen looked good all throughout the process, but just didn’t make any embryos. Those conversations are more difficult, but we have them.
I try to frame conversations around what we need for our process. So, trying not to make blanket statements about semen quality and categorizing as that straw not being good enough to sort. We just stick to the facts and provide information about exactly what we’re seeing.
Is there some semen that works well for AI and not IVF? Or vice versa?
Yes. Some of that goes back to the process of sorting the sperm. It’s stressful for them. For some bulls, the motility of the sample is really high and the dead rate on the machine is really low, but by the time we get done sorting those samples they just look sluggish and really slow. Those bulls tend to not freeze well either. So, I think some bulls just inherently don’t respond well to all the stresses that are put on them throughout the sorting process.
How can I ensure more successfully sorted semen?
Once the oocytes are collected, we’re not generally able to improve the quality. It’s the same with semen. If you’re working with a sample that has a low motility percentage already, I can’t just make those dead sperm alive again. It becomes just trying to mitigate and deciding how to sustain and keep good cells that you do have. From there it falls on the lab and ensuring we use the best practices to ensure success for our clients.
What are your thoughts on HEIFERPLUS and BULLPLUS? How do they fit into the IVF system?
I’m not an expert by any means, and I don’t understand a lot of the science behind it. However, what I understand is they’re trying to add something to semen that basically slows down the sex you don’t want. So, in HEIFERPLUS semen, they’re adding something to those samples that slows down the motility of male sperms, and vice versa. We haven’t done much research in terms of how effective that is in our system.
In IVF, the fertilization process happens relatively quickly compared to AI. Fertilization occurs within four to five hours in an IVF plate, but it takes much longer for the sperm to swim through the cow’s repro tract to fertilize that egg. If that mechanism does work, we don’t suspect it’s working fast enough in an IVF system for us to see a purity advantage to using HEIFERPLUS versus a conventional unit.
Can you cut a straw in half and use it for reverse sort or IVF?
There’s already so few sperm in those straws that you just limit how much you can do with that unit when you cut it. We have cut sexed frozen units in half before, but it just simply covers fewer donors.
For a reverse sort it gets more complicated. The concentrations of all those straws are so different, so we’ve not tried sorting any units that we’ve cut in half. Some of those units might only have 10-12 million sperm in them to begin with. By the time you cut it in half, there’s just not enough sperm in that product left for us to sort.
For right now, we just don’t cut units in half for sorting. If it were a rare, expensive case, we’re not opposed to cutting it in half trying to see what happens, but it just becomes a risk versus reward. It’s not something we encourage on the reverse sorting side at all.
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