I was at your fine seminar in Witchita KS a few months back, It was a great experience. My first season is kicking in, and I'm trying to muddle through some of this. Collection is not going smooth, I believe mostly, I get presemenal fluid with live sperm, but I don't believe I'v gotten a full ejackulet yet. This afternoon, I got about 20 mls of fluid, with minimal gel in the filter. Under the microscope, there appears to be plenty of live sperm. But when I mix that tiny unopete with what seems like a huge volume of liquid in the resivior, then put it on the Hemo, I only count 12 sperm cells on the center square. Jos, Am I doing this right? Does 12 cells on the center square mean I only have 12 million cells per ml.? THere really is not a good page in the book that details the counting process. Could you help me? Thanks Rusty
If you've got sperm, you've probably gotten a complete ejaculate. While partial ejaculations do occur, you can generally get a pretty good idea simply by your stallion's behavior as to whether or not everything was to his liking.
With that said, when using Unopettes, they can be a bit tricky. First, you want the pipette (the little tube) to fill completely. It fills with capillary action, so it may take a little bit of time and "tapping" to get it completely full, but because you are dealing with such a minute amount, it is essential that it is completely full. Before you insert it into the opening of the resevoir, SQUEEZE the resevoir container slightly. Insert the tube while the resevoir is depressed and release. It will then suck the semen into the resevoir. Do this a couple times to be sure that you've cleared the pipette completely.
You then draw up the diluted semen and fill your hemacytometer. When looking at the grid pattern on the hemacytometer, there are a total of 25 completely gridded squares on each side of the "ramps" for a total of 50 gridded squares. To get the most accurate reading, count all 50 and divide by 2. That total will give you the total number of sperm in millions per ml. For example, if you counted all 50 squares and ended up with a final number of 352, divide that by two and your final concentration would be 176 million sperm per ml. Make sense?
If you're not convinced that the Unopettes are working for you, you can dilute the semen manually using a 10 ml syringe and a 1/2 ml insulin/tuberculin syringe. Fill the 10 ml syringe with 10 mls of Formalin. Draw back the plunger slightly so that there is a pocket of air. Draw up semen in the insulin syringe and put .10 mls of semen into the Formalin in the 10 ml syringe. Mix the solution well by gently rocking the syringe back and forth. Load the hemayctometer and start counting.
While it is possible that your stallion has a low concentration, at 12 million sperm per ml, I'm guessing you're not counting correctly . Hope that helps!
Wow Kathy that was really the information I needed. That kind of detailed info is just not availible out there, And I called everyone I could find. OK, My stud has quit ejaculating. I have a home made AV made from a 20 inch peice of 4 inch pvc pipe, and a latex bladder from Exodus. I've tried various Temps, from 113 to 105f. If I take out too much water, he doesn't flair at all, and if I leave too much in, He seems visibly uncomfortable after flairing, and comes down. I am using live mares in heat, So he's really excited. I've made over a dozen attemps in the last few days, with no ejaculation. I even brought the vet out once. He really had no ideas. He's trying really hard, and is probbaly getting really frustrated. He gets no other chance to breed. It's either my equipment, or my technique. I can either buy a professional AV, or I can bring out a local breeding vet (they'll cost about the same) Before I make that decision, I thought I would try and get some more of your excellent advice. I have shipments going out later this month (but no pressure) Thanks Rusty
Rusty, Rusty, Rusty...<shaking> <smile>...it's all there in your course notes. First, shorten your AV...Remember what we said about an AV that's too long? The semen runs over that hot liner and your chance of temperature shock to the sperm cells is significantly increased. The glans of the stallion's penis should be at the end of the AV when he's ejaculating.
Secondly, you've got the temperature WAYYYYyyyyy too cold... The temperature of the AV should be 113 to 130 degrees fahrenheit and with a young stallion or a shy stallion, on the hotter end is better.
As you know, we recommend the French AV and as you've now discovered, making one isn't always the best route to go. With a Colorado style AV, part of the reason it works is that it offers additionally glans stimulation due to the length. With the French, it's shorter and the rubber latex hood offers the additional stimulation that stallions seem to really like....
Dear Kathy. My Baby bottle run'th over. I can't believe I-mis read that chapter so badly. I was actually waiting in the barn for the temp to go down!! Once I hit him with full tempreture he about blew the baby botle right off the AV (too much information? sorry) anyway he had quite a build up going. I'll try again today, to see if I can can be consistant. Too say I am very greatful is probably an understatement. If all goes well today, I'll get busy in the lab, and see if I can complete the entire process. I have my trusty book, and I'll follow the notes carefully. Thank you Kathy Rusty
Dear Kathy. It seams after all the bragging I've done on my boy, his numbers are not very good. He seems to run about 60 million per Ml. He has strong motility, (over 75 % and 55% after 24 hours). Without using a certrifuge, The only way I can pack close to 1 billion sperm at 4 to 1 (any lower, his motility goes down) is to pack 80 Mls of fluid at 12 million per ML. How do you feel about that. I realize it's not Ideal, but the book says thats the limit. Thanks for youe input Rusty
Tease less for starters and you may be able to get a higher concentration. You can also try fractionating his ejaculate. While you are collecting, as he begins ejaculating, count the spurts. After the first three spurts, twist off the liner and do not allow the final spurts into what you have. You're eliminating much of the secondary sex gland secretions and hopefully, just capturing the sperm rich fraction of the ejaculate.
With regards to extending the semen at 4:1, you're extending him too much... Sperm do best at a final concentration of 25 to 50 million sperm per ml. Extending more or less compromises the conditions and will often result in poor longevity of the sperm. As you noted, our course notes state that an extension ratio of 4:1 should be the absolute minimum semen should be shipped at. There is an extender that is now available that you can actually extend at considerably lower. It is INRA 96. Pick some up either from IMV International or Exodus Breeders supply and try extending him in that. You can extend as low as 1:1 using INRA. Do a trial run with your stallion and see if there's a possibility it would work for you.
I would try to fractionate him first, however. If none of the above works, you may very well have to resort to centrifugation. good luck
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