We are standing a 3 y.o. stallion. He went to the breeding clinic earlier this year to have his semen evaluated. He had a little difficulty at first but then his numbers came up. He is now home. We have live covered one mare (do not yet know if she is pregnant) and we have collected him three times. All three times we had no semen, just a lot of seminal fluid. It also appears that the filter seems to be trapping a lot of "stuff" at the bottom. I say "stuff" because I am not sure what it is. I have tried to use the hemocytometer, but I cannot even find the grid. So, two questions, 1. where is his semen? 2. what magnification should I use for the hemocytometer? Help!
Firstly, "semen" includes sperm and seminal plasma, so to be technically correct, your stallion appears not to be lacking semen, but rather sperm.
If you are not able to focus on the hemacytometer grid, then there is a good chance that you are not going to be able to identify sperm through the microscope either... You need 100x magnification initially for bot hemacyotometer and sperm, and then increase it to 400x. These magnifications are achieved typically with an eyepiece at 10x and objectives at 10x and 40x.
Are you washing the stallion's penis prior to collection? If not, the "stuff" may be skin cells and dirt.
Without seeing the whole situation it is very difficult to to be able to comment with any degree of accuracy beyond the above suggestions.
Yes, we are cleaning him. When he was at the clinic his concentration was good and his motility was 76%. But here, we have no sperm. He is a little different as far as preparing his semen to ship...and this is how I know we have no sperm even without the hemocytometer. After collecting him, he is extended and then centrifuged for ten min. After centrifuging we have had no seperation, which we should if he had sperm. As far as the "stuff", my sneaking suspicion is that is the sperm, but I cannot say for sure. Today, we are collecting him without a filter. Since we have to centrifuge him, all the dirt goes to the bottom and we can draw the seminal fluid and the semen off.
I was hoping someone else has had this problem and would tell me that since he is young and new to this, it may take a few collections, before we actually get our semen. At the clinic they used a phantom, and here we are using a jump mare. Maybe that is the issue. Could the temp of the AV also be of some issue? We think he likes it about 130 degrees. Last collection actually seemed textbook. He jumped, we slipped on the AV, he thrust many times, he pumped his tail three-five times, he waited a few seconds and then gently slid off the mare and out of the AV. He was limp and done. We had plenty of lfuid, it was opaque in color..so I kinda new there would be no sperm. I need swimmers!
There is no reason related to young age as to why one would require several attempts get a collection if he is well phantom trained - and in fact using a jump mare offers more stimulation than the use of a breeding mount.
A gel filter should not (in fact cannot) filter out sperm unless there is a specific problem with the sperm. Under normal circumstances, the sperm are considerably smaller than the holes in the filter. The one exception would be if the stallion is an "accumulator" stallion - i.e. he does not shed dead sperm in his urine on a daily basis and sheds them only when he ejaculates. In that instance, there may be clumps of dead sperm that could be filtered out - but you would still see plenty of sperm in the liquid portion of the ejaculate that had passed through the filter (although there may be a poor percentage motile).
AV temperature is definitely an issue to be aware of. If it is not to his liking - and pressure is another issue - he will not ejaculate. Indeed, if the AV is being handled incorrectly (not enough movement, too much movement etc.) that can have a negative impact as well.
What do you mean by "We had plenty of fluid, it was opaque in color..so I kinda new there would be no sperm? Opacity of the fluid usually is indicative of sperm presence! In fact, the more opaque it is, the more concentrated the sample usually is.
I can't help wondering - especially in light of all the other factors that you indicate - if your microscope is not set up incorrectly and not in focus. At the courses we give, we not infrequently have people telling us that they cannot see the sperm and we then find that they have the microscope completely out of focus, or have managed to get semen on the lens. In view of the fact that you previously indicated that you couldn't see the hemacytometer grid lines through your microscope, I think you should go back to your microscope and check that before doing anything else...
I guess what I meant to say is it was not milky looking, you could kind of see through it. Also, as I said, after we collect and exend, then we have to centrifuge him. When we do that, the sperm should be at the bottom and the seminal plasma on top. However, we are not getting any seperation. We sent what we collected to the repro vet and he verified that there was no sperm in it whatsoever. Also, as I said before, when he was at the clinic he had a contration of like 4 billion, so I know he has them. I think he is just not really ejeculating....maybe we need to allow him to jump again, if we get the results we have been getting.
I did get a different miscroscope today and hopefully it will work better.
Jodi Sounds like your getting just fluid and not getting the real collection. once you think he has collected, look at it,if it looks like just water then you may have got the pre-ejaculate and need to mount him again.
Also as Jos said AV is very important, we use 122 degrees on our stallion and just up to the wrist for pressure (using the french AV).
BTW how many mills is he giving in one collection? Also is it foamy looking? what kind of microsope sre you using? I gave up using the hemacytometer snd purchasrd a electric semen counter by nrxt generation Quick Check.
Before using drug therapy, I would encourage you to play around further with AV temperature and pressure, as well as the handling of the AV. The internal temperature at the time of collection should be between 113° and 130° F (45-54° C). In order to achieve that temperature, the water needs to be quite a bit hotter going in to the AV (it's going to cool down significantly as you fill the AV except in exceptionally hot weather).
Some stallions like it tight, others loose. Play with the pressure.
Some stallions like movement of the AV by the handler during collection, some don't. Some stallions like hand pressure on the glans of the penis during collection, some don't.
These are all areas that can make a significant difference between success and failure...
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